Blue Light Green World
-
Upload
edward-a-quinto -
Category
Documents
-
view
124 -
download
0
description
Transcript of Blue Light Green World
1
BBlluuee LLiigghhtt GGrreeeenn WWoorrlldd ______________________________________
A splendid chronicle of life, light, and vision on microbial bioluminescence and how we can use this good blue light to
keep ourselves healthy, our food and water safe to eat and drink and our world clean
Edward A. Quinto (CoolBioluminescence) Copyright November 2013, Manila; The Philippines
2
Specks of Life create Light
Light flashes a new Vision
Vision of Life United and One
3
Dedicated to the
Grand Mentors and Academic Pillars
of Philippine Microbiology:
Ma’am Lydia Joson
Sir Ireneo Dogma
Ma’am Priscilla Sanchez
Ma’am Asuncion Raymundo
for their selfless devotion to excellence in teaching and research
which served as a “Beacon of Distinction” that steered me and
countless others on our rewarding expedition of a lifetime to
discover, explore and enjoy the
Wonderful World of the Microbes
4
Prologue
In the spring of 93, my life changed after witnessing an awe-inspiring first
sight of cool bioluminescence in Germany. This unique encounter has since
fascinated me to a wonderful microbe and its redeeming heavenly message.
Thus in the summer of 2005, several months before the movie “An
Inconvenient Truth” was shown in Philippine cinema, I started writing a book
narrating up, close, and personal a distinctive encounter that I had with the
“Microbes of Light”. Bioluminescence, the good blue light produced by the
marine luminous microbes, can be harnessed to the fullest to make our
world greener by providing safe water, clean foods, and a healthy
environment. Having written only technical articles for science journals in the
past has made it difficult for me to reprogram my brain to compose
something far less esoteric and pleasurable to read. I really felt like a fish out
of water writing this book. As challenging as it was with the occasional bouts
of doldrums, I confronted my limitations and finally completed this book with
much elation and pride. This book’s tripartite prose dealt on an enriching
interaction of life, light and vision distilling everything I have learned, loved,
and cherished about the marine luminous microbes. The first chapter - of
Life, is a brief memoir of my first encounter with the marine luminous
microbes, recounting the wonderful beginning in the Saarland in Germany. It
5
also enumerates the blessings that many and I received in the course of my
scientific inquiry on this special group of microbes. The next chapter - of
Light, is a simplified discourse based on existing literatures on the what,
where, how and why of microbial bioluminescence. Lastly, of Vision
describes how our dark-adapted eyes can discern the luminous message
that the microbes live to tell us when we used them as biological sensing
tool for toxicity testing. The good blue light can uplift the quality of life by
ensuring safe drinking water and a clean environment. Complementing the
content of this book are pictures of the marine luminous microbes in the
dark. Using long exposure digital photography, I was able to capture the
apparitional manifestation of the “living light”, their “life energy”, or their
“aura” so to speak. Through this book, I want to introduce a new world
viewed in the different light of bioluminescence. Today as global warming
looms forebodingly on the horizon, bioluminescence – the good blue light
can embody our concerted effort to fight it as well as to empower us by
using it for our greener day-to-day living. Bioluminescence in addition to
solar, geothermal, wind, wave and natural gas can usher in a new green and
healthy way of living for all of us. So please join me now, in my personal
advocacy to start a “bioluminescence revolution” in the world. The task
ahead is so overwhelming and daunting but nothing should deter us in
6
upholding the public’s health and to fight global warming. In my own little
way, this book is it! Lastly, I am encouraging our eager beavers to embark
on their own journey of discovery and exploration. To motivate the younger
generations to become men and women of science is to find fulfillment in
life, knowing that I have taken part in nurturing and cultivating the seeds of
science. These precious seeds will germinate someday into the next batch
of scientists, which will carry on with the noblest of quests of moving the
frontier of knowledge forward.
7
Table of Contents
Acknowledgments
Prologue
Chapter I - Of Life
The light-bearer in the world of the microbes
From the very small comes the high and mighty
“Ich bin ein Berliner”
Having known John, a Rockefeller, in Freiburg
The Saarland, a small jewel nestled in the Franco-German border
Winter wonderland in the Saarland
EQ in Paris
Up, close and personal with the marine luminous microbes
An archipelago of the marine luminous microbes
Blessings through the microbes of light
Winning the gold in the Asia-pacific Young Inventors Award (YIA)
The awarding ceremony at the top of the world
PIBiT – the invention
The winners of the YIA through the years
Go west where the skies are blue
Sojourn in the famous Silicon Valley
Meeting the winners of the 2001 U.S. Collegiate Inventors Contest
The Hewlett Packard experience
Bioluminescence projects shining brightly in science fair contests
Winning in the Intel science and engineering fairs
8
Chapter II - Of Light
The nature and habitats of the marine luminous microbes
How does Vibrio fischeri look like?
Where is Vibrio fischeri found?
Cultivation of the luminous microbes on solid culture medium
Cultivation of the luminous microbes in liquid culture medium
Microbes that can talk with one another
The identification of the marine luminous microbes
The chemistry behind bioluminescence
Chapter III - Of Vision
The Eye
The standard microbial bioluminescence toxicity test
The bioluminescence oxygen demand (BiOD) for water pollution
The tube bioluminescence extinction technology (TuBET)
Preparation of small paper-discs
How to make the paper-discs bioluminous
Tales of light from the bioluminous paper-discs
Background information behind bioluminosity and immobilization
Paper-disc Immobilized Bioluminescence Technology (PIBiT)
Clean and safe drinking water
PIBiT’s applications and its resource sustainability
EQ phone NASA
Future plans for PIBiT
Appendix
About the Author
9
Acknowledgment
Tendering credit for an achievement gained is like sailing in one’s boat of
inadequacy but buoyed up by the great ocean of recognition and
appreciation. Indeed, my own personal limitations have become amazing
capabilities by the assistance of others. Without these people and
institutions, my fortuitous encounter with the “Microbes of Light” will not have
happened; the wonderful opportunity to contribute to a greener world will
have been missed, and this book will not have been printed. In all candors, I
am extending my sincere appreciation to the following whose exceptional
influence and distinctive inspiration left me permanently honed to the service
of science particularly in microbiology for the common good.
The Helmholtz Institute (HI) formerly known as the Gesellschaft fur
Biotechnologische Forschung (GBF) in Braunschweig, Germany, for
my first wonderful experience of a sophisticated and international
microbiology.
The venerable Deutscher Akademischer Austausch Dienst (DAAD) or
the German Academic Exchange Service for the superb and enriching
personal experience of a postgraduate study.
10
To Prof. Dr. Paul Mueller, Dr. Schaefer (Dozent), Prof. Dr. Heinrich
Kaltwasser and Dr. Christoph Siersdorfer for making it possible for me
to study in the University of Saarland.
Lastly, to the gracious and good people of Germany and particularly to
Frau Walburga Westphal of GBF, Frau Heidi Schlenther and her family
and Prof. Dr. Karsten Krohn and his wife Odille whose memories of
friendship, camaraderie, generosity, and kindness I will never forget
and to which I will be eternally grateful..
11
Chapter I: Of Life
Du bist das Licht in meinem Leben…und die Welt ist wunderschön wenn wir zusammen
sind (You are the light in my life…and the world is wonderful when we are together)
- Starlight Express in Bochum, Deustche Aufnahme)
The light-bearer in the world of the microbes Do we humans want this planet to be exclusively ours? The emphatic
and resounding answer I am sure is a Big No. It will be a place quite dreary
to live in if we humans are planet alone. A world that lacks biodiversity will
be big, boring, and barren. We love this world the way it has always been,
teeming with so much life of which are own is just one of the multitudes of
species of animals, plants, and microbes. A plethora of life forms nurtured
and sustained by a blue planet we call mother earth.
Lichens: a symbiotic partnership between a fungus and an alga
12
Surely, something binds all living organisms together on this planet into a
single super family. Among all of the hundreds of millions of known species
of living organisms existing today, only a few have been endeared and
cherished by man. These much loved species are the higher plants and
animals. Our overbearing birthright of dominion over all creations resulted in
the domestication of many plants and animals. Chosen members of kingdom
Animalia, the realm of those that can move, were held in captivity for
livelihood, companionship, entertainment, worship, and friendship. This
seemingly inadvertent intrusion into the process of natural selection has
forever change the course of the evolution of the wild. Many plants and
animals even suffered the tragic fate of extinction from our gung-ho
incursion into nature. On the opposite end of this multifaceted relationship, is
our subservience to animals to the point of venerating them as gods. This
practice reached its peak in the ancient civilizations of Egypt, Babylon, and
Persia where dogs, cats, cattle, birds, and snakes were accorded the status
of deity and received acts of worship. However, the rise to power of the
monotheistic religions put an end to this practice of animal worship.
Nevertheless, our fascination with animals continues unabated even to this
day. Walt Disney’s enduring success, and worldwide popularity,
transcending cultures and races, are proofs of our undying love for animals.
13
Disney’s wonderful and colorful world of animation created the amiable
characters of Mickey, a mouse; Donald, a duck; Bambi, a deer; Dumbo, an
elephant and Goofy, a dog. My generation grew up so fixated with the
lovable animal cartoons of Disney. Although we are now wiser, many
including myself still watch the movies of Disney through a child’s eye as
something truly magical. For a long time now, dogs got the esteemed title as
man’s best friend, but cats recently stole the limelight in Broadway. The
common household cat: Felix domestica, received widespread accolade on
both sides of the Atlantic from Andrew Lloyd Webber’s musical “Cats”. Cats,
a deeply lovable music theater production with its origins in London,
portrayed so perfectly the exquisite charming attributes and the humorous
mundane lifestyles of the different breeds of the domesticated cat. Sly and
stealthy feline characters; slithery and beguiling huggable furry mammals
with whiskers and paws; acted out their unique roles and sing their lives in
songs so emotive. The overall effect is an artistic work of melodies and
music woven together into a seamless tale of the anguish and joy of our
inherent human nature. “Cats” captured the minds and hearts of millions and
went on purring its way into historical success as it broke box office records
in major cities all over the world.
14
Fishes and honeybees are members of Kingdom Animalia
However, to some people, the members of Kingdom Plantae, the realm
of the green, like algae, ferns, roses, bromeliads, orchids, and trees,
afforded the kind of personal relationship that gave lasting fulfillment in life.
Indeed, many poets were inspired to immortalized flowers and trees in their
poetry. The sight and distinct smell of plants with their colorful flowers
unloads us of our anxieties and provides welcome relief from the stresses of
everyday life. Beautiful flowers that come in a wide variety of forms and
colors instill in us feelings of euphoria as well as impart deeper meaning to
many of our life’s special events. In fact, love is express so naturally with
flowers. In addition, fragrant essential oils produced by plants were used
since ancient times to bring about the scents and aroma needed to set the
right mood to our social occasions. Today, the cosmetics and food industries
15
have commercialize these special plant extracts into the multi-billion dollar
business of flavors and fragrances.
The members of Kingdom Plantae found in Caleruega, Batangas
My case is so completely different. A personal encounter with an
unusual organism in the spring of 1993 in Germany made an overwhelming
difference in my life. Surprisingly, the organism was neither an animal nor a
plant; it was a lowly microbe, a speck of life. Microbes belong to the kingdom
16
Eubacteria, the realm of those that are too small to be seen. Although it may
at first seemed so trivial since the organism was a tiny and humble microbe,
it was however, a one of a kind microbe endowed with a beautiful celestial
gift. A microbe is a one-celled organism not visible to the human eye and
considered primitive by biologists in terms of its cellular structure and
function. You will need a good microscope to be able to see them one by
one. Indeed multitudes of these imperceptible creatures have already taken
permanent residence in our mouth, tummy, and respiratory tract as well as
on our teeth and skin without us ever knowing it. They are so tiny that
millions of them can easily be crammed together in a space the size of a
pinhead. But what makes this microbe that I met in the spring of 93 special
and distinct from the other species of its kind I met previously, is it’s innate
ability of eerily generating light in the dark with a cool delightfully bluish-
green hue. In fact, this tiny microbe has something in common with giant
celestial bodies called stars. This microbe and stars produce light copiously.
However, stars generate heat while the bioluminescent microbes do not. In
the realm of the microscopic, this light-bearing microbe must have been truly
heaven sent. Not only is it heavenly, it is also smart; a microbe that knows
how to talk to one another and to us through the cool light that it makes.
Biological illumination for this microbe is achieve only by relating itself in
17
unison with others of its kind and collectively as a whole attains bright
luminosity. It is a perfect example of what we call “togetherness” or
“cooperation”. It was from the study of this clever microbe that
microbiologists learned that they though considered the lowest forms of life
do actually talk to one another. Unlike what happened to the Tower of Babel,
microbes because of this exceptional communication skill can initially build
small communities composed of streets that gradually develop into colossal
mega cities with skyscrapers and freeways. In microbiology, these microbial
mega cities are simply called “Biofilm”.
Stacked plate cultures of the luminous microbes shining with a cool
bluish-green light in darkness
My chanced encounter with this intelligent luminous microbe must have
been made in heaven because it lit up my life and changed it so wondrously.
18
It also made a lasting impact in the lives of many of the younger generations
of Filipino scientists. Hopefully, it can also make a lasting global impact in
safeguarding our health and that of our beloved planet.
From the very small comes the strong and mighty
Microorganisms or simply microbes are one of the least understood
groups of living organisms existing today. Primarily because of their
miniscule size we know little of them. Most of us have not yet even seen a
microbe in our lifetime. Since “size does matter”, it is indeed not often that
we see them everyday as plants and animals. Even Disney, Columbia,
Pixar, Warner Brothers, and the other movie studios have yet to venture into
animating microbes into lovable characters that can be popularized into
famous characters like Mickey, Nemo, and Chicken Little. And why not? The
lowly microbes have so much to offer to keep us incessantly amused and
entertained. With their diverse and amazing ways of life, extraordinary
qualities and bizarre attributes, new and inspiring movies full of enduring
virtues can be produced with microbes as protagonist. Theirs is a completely
new universe to explore and discover that will surely broaden our culture of
science and knowledge of microbiology. To know them well is to give us a
19
fair and a better understanding of this much misunderstood and maligned
group of living organism. The popular media’s perception of microbes as
harbingers of sickness and pestilence worsened even further our ignorance
of these infinitesimal organisms. This unfounded fear of the invisible must
have imprinted in our psyche the various waves of deadly plagues that
scourged our forefathers since time immemorial. Indeed infectious diseases
like cholera, dysentery, tuberculosis and pneumonia continue to claim
millions of lives today. However, to microbiologists, this is a grave
misconception of the microbes since the disease-causing microbes, the bad
microbes, are just a minority in the realm of the small. The vast majority of
microbes, the good microbes, live freely in the soil, on our skin, inside our
gut, in seawater, and other habitats where they carry out the tasks meant for
them by nature. Microbes are actively involved in the building up and
breaking down of substances around them. They play a major role in the
recycling of the various essential chemical substances that make up the
biosphere. In short, they keep our planet clean and healthy. Microbial
species though miniscule in size have in eons past, acting ever so slowly but
surely, achieved something enormous. Microbial activity is solely credited for
transforming our atmosphere into what it has become today. From an
anaerobic and reducing atmosphere in the earliest periods of earth’s history
20
to what it has recently turned into: aerobic and oxidizing! An oxidizing
atmosphere is rich in oxygen causing iron to rust rapidly. Unfortunately, for
higher life forms it also speeds up a biological process called “ageing”.
However, an oxygen-rich atmosphere creates and sustains fire allowing its
manifestation on earth as the process of burning. Burning is a chemical
reaction in which elements like carbon combines with oxygen to produce
radiance and heat. In the absence of the diligently working microbes and
their pervasive and collective activities on this planet, transforming earth’s
atmosphere from zero percent oxygen in the past to twenty-one percent
oxygen in the present must have never happened. Moreover, without a
substantial amount of oxygen in the atmosphere, the emergence and
existence of plants, animals, and humans would not have been possible.
Cyanobacteria like this Oscillatoria gave earth an oxygen-rich atmosphere (1,000X)
21
Many of these microbes possess strange and amazing characteristics.
For example, a microbe called Thiobacillus thiooxidans produces highly
corrosive sulfuric acid in practically the same manner as we humans make
and excrete urine. Another microbe called Aquaspirillum magnetotacticum
swims along defined paths in its watery world guided by the earth’s lines of
magnetic field. It is able to do this because of a string of spherical iron oxide
magnets found within its elongated spiral body. Moreover, there is a very
ancient microbe called Pyrolobus fumarii thriving in its oxygen-free world at
temperatures way above the boiling point of water; hot and anaerobic
conditions believed to be prevalent in the early periods of earth’s history. It
surely is not cook at this high a temperature. Instead, it even grows at its
best making the rest of its own kind to populate and prosper in their nasty
niche! For many years now, microbes in the group called Actinomycetes
have been seek out out by scientists in many soil samples collected all over
the world. This undertaking that gained massive support from the large
pharmaceutical companies led to the discovery and subsequent industrial
production of wonder drugs called antibiotics. An example is the multi-billion
dollar annually sold antibiotic known as erythromycin produced by the
microbe Saccharopolyspora erythraea formerly called Streptomyces
erythaeus. This microbe was isolated from a soil sample in the Philippines
22
by an Eli Lily chemist to produce their company’s banner antibiotic product.
The use of many antibiotics produced by the members of the group
Streptomyces ushered in the golden era of chemotherapy in medicine.
Photomicrograph (1000 X) of an indigenous streptomycete with its long filamentous cellular structure. Most commercial antibiotics come from this group of soil microbes.
Antibiotics saved millions of lives throughout the world from the ravages of
infectious diseases. My first ever research project dealt with the cultivation of
Streptomyces levoris and the production of its antibiotic: Levorin using
indigenous waste agricultural substances. I was under the able guidance of
the late Dr. Patrocinio Santos, famed pharmacist, microbiologist and NRCP
Achievement Awardee in the Philippines. Aside from the streptomyces, there
is also Lactobacillus acidophilus that produces lactic acid and other edible
23
products derived from the fermentation of milk and other foods. This microbe
is just one of many lactic acid microbes like Lactobacillus casei and
Lactobacillus plantarum employed as probiotics or good microbes in many
functional foods.
Photomicrograph of a Lactobacillus acidophilus USTCMS strain (1000X)
showing long rod-shaped cells
They are responsible for the traditional production of fermented dairy
products like cheese, butter, yoghurt, and sour milk as well as fermented
vegetables like suaerkraut and pickles. Incidentally, my work on the
antimicrobial activities of the fermented medium of Lactobacillus acidophilus,
a brief foray from my main work on the marine luminous microbes, earned
me recently the Tom Bergan Memorial Award. The International Society for
24
Chemotherapy (ISC) based in London gave me this award on its 24th
Biennial International Chemotherapy Congress held in Manila in June 2004.
The 2005 Tom Bergan Memorial Award
Finally we now come to my favorite and friend, a smart and heavenly
microbe scientifically named Vibrio fischeri, which like the angel Lucifer
when he was still in God’s favor was the bearer of light among the angels.
The creator has indeed endowed Vibrio fischeri with a celestial gift of being
able to produce bright bluish light on its own initiative. Bioluminescence is a
biological phenomenon in which living organisms produce cold light from
25
their own life processes. Light production from inanimate systems like bulbs
and incandescent lamps heats up after some time but this is not so with
bioluminescence. Among the bioluminescent organisms like fireflies, lantern
fishes, glowworms, dinoflagellates and the luminous microbes, almost all of
the allotted biochemical fuel found inside their cells intended for
bioluminescence is transformed almost entirely into light energy. Only a
small fraction of the transformed energy, when converted from chemical to
radiant, is wasted as heat. The conversion ratio is high that it renders
bioluminescence amazingly cold to the senses. The bluish-green hue of the
luminous microbes is much like the beautifully shining green beads of glow-
in-the-dark rosaries and wristwatch hands.
“Ich bin ein Berliner”
Ever since I was held spellbound by my first sight of brightly shining
liquids of luminous microbes in a dark laboratory years ago in Germany, my
devotion to these microbes has never waned and has even become an
integral part of my academic life. It was candidly “true love” at first sight! So
awe-inspiring and thrilling indeed were my visions of the luminous microbes
that it reminded me of the time back in high school when I first laid
telescopic eyes on the moons of Jupiter and the rings of Saturn. I was in
ecstasy to see them real; it was just awesome. Indeed, all budding scientists
26
are astronomers and space travelers at heart. Today, as someone who
finished a career in chemistry and microbiology and has become an honest
to goodness servant of the life science, I affectionately describe
bioluminescence as the “Light of Life” or simply to coin a new word
“Lifelight”.
My unexpected encounter with the wonderful world of the marine
luminous microbes started in 1992. It was an event that I believed was
meant to be. In that year, the venerable DAAD (Deutscher Akademischer
Austausch Dienst) or the German Academic Exchange Service gave me a
one and a half-year scholarship grant. The grant was for a one-year
certificate course in biogeography and environmental assessment. DAAD
has been very generous to the higher educational system of the Philippines
and to other developing countries. DAAD offered numerous training courses
as well as opportunities to pursue research and postgraduate studies in
German academic and research institutions. Countless Filipinos were
recipients of DAAD scholarships and have now come back to make their
outstanding marks and significant contributions in the local academe,
industry and various government agencies. My scholarship grant initially
provides for a six-month German language course in Freiburg im Breisgau,
a charming city nestled in the nurturing embrace of the enchanting “Black
27
Forest”. A German language course is required of the one-year certificate
course in the university to enable one to live an independent life in Germany.
The course on Biogeography and Environmental Studies is tenable at the
University of Saarland’s Department of Social and Environmental Studies.
Prof. Dr. Paul Mueller, an intelligent, well respected, highly admirable, and
able-bodied German ecologist and educator, who can easily climb several
hills as if it were just a light stroll in the park, headed the department. It was
actually my third time to be in Germany, a land I consider my second home
and a country that I always regard with deep affection and joyful yearning.
This nation, an economic power located in the very heart of Europe, is the
great and venerable homeland of Johann Sebastian Bach, Ludwig van
Beethoven, Johannes Brahms, Albert Einstein, Pope Benedict XVI, Willy
Brandt, Helmut Kohl, Steffi Scholl, Steffi Graf, and other famous persons
who change the direction of world affair. Germany is known, recognized, and
respected far and wide for his eminent position in philosophy, the arts,
sciences, and technology. For me, Germany is “God’s own country” of heart
captivating landscapes painted in living green and freezing white; peaceful
and idyllic pastoral villages; impressive and mighty cities and most of all the
country of a good, noble, and lovely people. I first visited Germany for six
weeks in 1988 as a participant to the international training program (ITP) in
28
biotechnology. Germany’s prestigious and venerable National Center for
Biotechnology sponsored and held this training course. To many who went
through the portals of this venerable research institution, it is simply and
amiably called “GBF”. GBF stands for “Gesellschaft für Biotechnologische
Forschung”. Recently, GBF has been renamed as the “Helmholtz Zentrum
für Infektionsforschung” translated literally as “Helmholtz Center for Infection
Research”. GBF has mow become HZ. The change highlights a redirection
of Germany’s biotechnological research thrust into a multi-disciplinary fight
against infectious diseases. Visiting Germany for the first time in 1988 was
like venturing into another world of endless wonder. The view, the ambiance,
the places, the people, the climate, the culture, and the very air that I
breathe were altogether such a fantastic first-time experience. I found it so
thrilling talking a lot and exhaling my lungs out because of the thick vapor
that comes out of my mouth. It happened to be my first foreign travel; an
exciting journey that took me through the charming German cities of
Braunschweig, Hannover, and Berlin. It was an unforgettable six-week
sojourn in the summer of 88 that I consider the best thing that ever
happened in my life. Having visited divided Berlin in 1988 and unified Berlin
in 1990 gave me a deep and profound perspective of Germany’s identity,
history, and role in the modern world. Remember it was just in the last
29
century when Germany went through periods of revolutions, redemption,
and resurrection. The mighty German Empire ruled by Prussia disintegrated
after the First World War. A fleetingly bright moment followed the collapse of
the empire with the birth of the “Weimar Republic” in 1919. This beautiful
republic, bearing the promise of freedom, peace, and prosperity for all
Germans, unfortunately died in its infancy. What followed was the darkest
and most chilling period in European history: the rise to influence and power
of Hiltler’s National Socialist Party. Because of the NAZI, millions perished in
Europe from the war and from the gas chambers. Germany suffered
widespread destruction and almost complete annihilation. Though it seemed
that everything was lost, Germany made contrite reparations for his mortal
misgivings and underwent an immensely remarkable transformation marked
by sincere reconciliation and rapid industrial development. The Fatherland
rebuilds himself from the smoldering black cauldron of the last world war to
rise meteorically into a proud global economic power as a free, united, and
flourishing German nation.
Germany’s international capital, the vibrant city Berlin, once divided into
two rival philosophies, now breathes a sophisticated culture and a vagabond
urbanity. When Pres. John F. Kennedy bravely visited Berlin and spoke the
famous words “Ich bin ein Berliner”, a city instantly captured the hearts and
30
minds of all people on earth. Suddenly everyone was a Berliner who knew
fully well to which side of the divided city he belongs. It highlighted the great
divide of a world partitioned by the iron curtain; of “us against them” of the
free against the bound. Everyone in the 60s lived precariously on the angst
of a third world war kept by détente of the superpowers. When I visited
historical Berlin for the first time in 1988, I cannot help but savor and marvel
at the ambiance of an occupied city; a city torn between west and east,
democracy and communism, freedom and tyranny, heaven and hell. A city
hallowed by the Nordic gods of Valhalla with beauty, power, and nobility.
Conspicuously flying high above the city is the golden statue of the “Angel of
Victory” symbolizing for the Berliners the blessings of success and
achievement. In 1988, my first time to be in Berlin, the massive wall of
shame stood arrogantly between the famed “Gate of Brandenburg” and me.
All I can do was to gaze at it from afar inside the comfort and security of
West Berlin. However, in 1990, my second time to be in Berlin, I was able to
walk freely through the famed Brandenburg gate passing by the monumental
colonnade of the chariot-triumphant goddess of dawn - Aurora. I entered the
gate with overwhelming sentiments of harmony, happiness and prayers of
thanksgiving. Indeed, the mighty “Westerly Winds of Freedom” knocked
down decisively the infamous Wall of Berlin and blew on farther east
31
destroying the fortress of the once powerful Union of Soviet Socialist
Republic (USSR). What was once thought impossible, the unification of two
German nations put asunder by conflicting global powers, happened
overnight so fortuitously and miraculously? A divine will must have
intervened, for the special turn of events leading to the fall of communism
took us all by surprise. At first, events seemed poised to trigger the third
world war but instead took a complete turnaround and ended so
diplomatically for the US and Russia. Indeed, we are forever grateful to the
foresight of Pres. Ronald Reagan, to Pres. Mikhail Gorbachev’s Perestroika
and most of all to the guidance of the beloved “Man of the Twentieth
Century” his holiness Pope John Paul II. His papacy steered the world so
peacefully during the dying days of soviet communism as we move on into a
new order. With enthusiastic and happy feet, I tread through the popular and
spacious boulevard called the “Unter den Linden Strasse” extending behind
the Brandenburg Gate right into the very heart of East Berlin. My strides, full
of fervor, took me all the way to the imposing monumental bronze statue of
Emperor Frederick the Great. I enjoyed every moment of it as I glanced all
around me and savored every beautiful feelings in that historical afternoon in
East Berlin in September of 1990. Soon it was darkening and I have to go
back to my hotel. Tony Bennet left his heart in San Francisco but in Berlin I
32
left not only my heart but my soul as well. It was indeed so great to be young
then in a cosmopolitan Germany caught up in such a momentous event in
world history. Coming from a developing country, the experience totally
changed my outlook in life. It gave me a genuine appreciation and open-
mindedness of other people’s way of life. That my way of living and beliefs
are not necessarily the best but there are other beliefs and ways of life
equally deserving as mine. In the 1988 training course on industrial
biotechnology, there were 20 participants coming from countries in South
America, Africa, and Asia. My second trip to Germany in 1990 was made
possible by a two-week Carl Duisberg Gesellschaft (CDG) travel grant. This
travel grant enabled me to attend the Biotechnica 1990 convention in
Hannover, a study visit to the GBF for one week and the second memorable
visit to Berlin. Hannover is just a stone’s throw away from Braunschweig
where GBF is located. The BIOTECHNICA is a biennial international
showcase highlighting all the recent breakthroughs and up-dates in the field
of biotechnology. It was so far the best international symposium, convention,
and trade fair all rolled into one that I have ever attended.
On my third journey in 1992, I embarked on my longest sojourn of one
and a half-year in Germany. I and Sunday Duque a friend of mine from the
Department of Biological Science’s of the University of Santo Tomas (UST)
33
arrived in the charming city of Freiburg im Breisgau for the language course
at the Goethe Institut in April of 1992. DAAD awarded us both the same
scholarship program in January of 1992. When we arrived in Germany,
beautiful spring was in the air as it began to lay hold of the seasons. It was
still indeed quite cold for us, the people from the tropics but numerous
juvenile leaves were beginning to sprout and cover the bald brown branches
of trees. Bright yellow Dandelions were popping up everywhere on the bare
lifeless ground around us. The sleepy hills and the groggy mountains of
southern Germany were waking up from a deep winter slumber by donning
various shades of green. Ah yes! I said to myself, Greek mythological
goddess Proserpina has once again arrived in Europe.
Having known John, a Rockefeller, in Freiburg
John, a Rockefeller, also made my stay in Freiburg unforgettable. In the
second cycle of my language course, during the blue summer skies of June
and July, two graduates from the prestigious Yale University came to study
German at the Goethe Institut in Freiburg. Both were assigned to the
boarding house where I was staying. It was located in the outskirts of
tranquil Freiburg around 15 to 20 minutes bus ride away from the city center.
34
The new kids on the block were Ching Chiu, a Korean-American and of
course a John D. Rockefeller V, first-born son of Sen. John D. Rockefeller IV
(Democrat – West Virginia). A cause that bound the three of us neatly was
our desire to read the “International Herald Tribune” (IHT). To realize this
need we would contribute one German Mark each to enable us to buy the
IHT. I had been reading the Tribune since I first arrived in Freiburg in April. It
kept me busy while waiting in the bus stop for my ride home after my
German class. The IHT fulfilled a yearning in me to read something
international and American and in some instances snippets of news about
the Philippines. John and Ching Chiu saw me carrying the IHT one sunny
afternoon on my way home as I entered our boarding house and asked me
where I bought the papers. That question started a fruitful conversation and
a friendship in the duration of the language course in Freiburg. For the next
two months, I shared the cost of the IHT with John and Ching Chiu and most
of all a wonderful company. We would usually go together to a nearby large
mall whenever we ran out of foods and other provisions. I even remembered
teaching these Yale Graduates how to cook rice properly by simply using the
top line of the middle finger to measure the required level of water. Ching
Chiu and I saw an impressive production of the opera “Carmen” in the
Freiburg Theater one evening. The night was beautifully mild and warm as
35
we traveled back home by bus. Though John is a Rockefeller, he did not
show it in his ways. He is far from being a very spoiled brat. One time John
and his friend Charles stayed over in my place for another week when his
rent ended in July. My place, which is the most expensive in the boarding
house for being the biggest happened to have a large ante room adjoining a
bedroom. Ching Chiu left ahead of John for Berlin. Staying with me, John
was able to save a week’s rent of pay from the owner of our boarding house:
the Webbers. John is a tall and a delightful all American guy, very down to
earth, so unassuming and charmingly friendly with always the best of
intentions. Several times a week I would catch him talking by phone to his
parents and friends since our phone is located in the corridor at the corner of
the stairway leading down and out of the house. He is ever ready to defend
the “American Way” of life. I remembered amusingly, John getting into an
intense argument one early morning with a German while we were waiting
for our bus ride to the Goethe Institut. John was complaining loudly not
knowing that the only other person with us in the bus stop, a German,
understands and speaks good English. Their argument centered on why the
shopping malls and stores in Germany are closed on Saturdays and
Sundays while in the U.S., and in the Philippines they are open. After the
first week of August, John left and traveled to his next destination. Before
36
John left for Bamberg, he wrote me a short funny note on a bond paper. He
left it so conspicuously on my big old wooden table, which I managed to
keep to this very day. The following months of August and September, the
third and last cycle of my language course, were personally difficult as I went
through one of the most trying times of my life. A new student from Nepal
called Kedar moved into our boarding house. Kedar was a nice and friendly
guy who always carried a plastic bag full of curry powder during meals. He
liked to garnish all his dishes and foods with curry. It was I believe typical of
the people in his homeland to eat foods containing curry much like some
Filipinos’ use of patis (fish sauce) in every meal. After six months of stay in
the city of the enchanted Black Forest, at the end of September, the three
cycles of the German language course at the Goethe Institut were finally
over. Sunday and I once again packed up our things, bade Freiburg farewell
and traveled by train to our next destination: Saarbrücken, the heart of the
Saarland.
The Saarland, a small jewel nestled in the Franco-German border
The green summer in Freiburg had given way to the orange and colder
season of autumn in Saarbrücken, the Saarland’s capital city. Sunday and I
traveled to Saarbrücken in early October of 1992 to start our two semester’s
37
course in biogeography and environmental studies at the University of
Saarland. My recollections of the long train journey from Freiburg to
Saarbrücken as I gazed out were picturesque fleeting views of a lovely
emerald countryside bathing intermittently in rain and sunshine. When we
arrived, Sunday and I were assigned to different accommodations located
far apart from each other. The university’s “Auslandsamt” (Office for
International Students) is in-charge of housing the international students.
Once again, I was placed in a big room in the 2nd floor of a large two-storey
boarding house with eight rooms and a cold basement located in the
suburbs of Saarbrücken called Dudweiler. The charming house stood alone
besides a big forest but it also stood strategically along a major highway with
bus stops nearby. Outside this big house is a connected wide spaced area
with ceilings that served as party area for the student occupants. I shared
the big house with Erasmus European students studying in the university.
However, the “Auslandsamt” assigned Sunday in a big student dormitory
located close to the city center. There were seven of us DAAD scholars
pursuing the biogeography program in the University of Saarland: two from
Nigeria, one from Senegal, one from Indonesia, one from Pakistan and
Sunday and I from the Philippines.
38
Winter wonderland in the Saarland
My December in Saarbrücken was marked by a very memorable short-
lived white Christmas. The winter was unusually mild in 1992, which was
probably good for my nasopharynx and upper respiratory system. I
experienced only a week of a half foot-deep snow-covered streets. The night
the white flakes of snow first fell, I recalled dashing out of our boarding
house to savor the cool, nice sensation of the slowly falling ice particles
melting on my face and hands. I got so excited that evening that I aroused
the curiosity of all my friends: Cyrill from France, Pawel from Poland,
Marianne and Helena from Denmark, Sabina from Spain, Althea from
Canada and Kirstie and Anthony from England. They too went out as well of
our boarding house and shared in my excitement and my innocence of
everything that is winter. There was a time one late evening when I had a
hard time entering our boarding house. From the main road, the door is
some way off around the house with the pathway sloping up a little. The
pathway was covered by ice and so to prevent me from slipping down I have
to hold on tightly to the walls and windows of the house to get to the door. In
a blink of an eye, the never-ending renewal of the seasons has brought back
spring. Sunday and I had spent a year in Germany.
39
At the start of our last semester at the University of Saarland, we were
required to complete an “Aufbaustudium” which is very similar to an
American version of a thesis. I had always wanted then to do a thesis in the
field of environmental microbiology in the Department of Applied
Microbiology. So one morning, claiming only to a brief stint at the GBF way
back in 1988, I went directly to the office of Prof. Dr. Heinrich Kaltwasser
and applied personally for an Aufbaustudium in environmental microbiology.
Prof. Dr. Kaltwasser, a soft-spoken, kind, and learned microbiologist, was
the head of the Department of Applied Microbiology at the University of
Saarland. Without any hesitation, Prof. Dr. Kaltwasser accepted me
immediately and assigned me to the supervision of Herrn Christoph
Siersdorfer. Christoph Siersdorfer was at that time a Ph.D. student doing his
dissertation on the use of different microbial systems for the toxicity testing
of the treated wastewaters of the coal processing plants in the industrial
region of the Saar. I believed that my training at the GBF was my magic
ticket in getting into the department of applied microbiology so fast and
without hassles. Prof. Dr. Paul Mueller gave me his permission to do my
“Aufbaustudium” in another department. Sunday remained and did her
Aufbaustudium in the department of social and environmental studies.
40
EQ in Paris
In June of 1993, a student club in the University organized an excursion
to Paris, France. Oh my God, Paris! The trip was simply too precious for us
to passed up and so we hurriedly secure a visa to join the trip. I vividly
remember waking up very early and while it was still quite dark trekked all
the way to the campus. We got up to a beautiful big tourist bus and before
we knew it, we were on our way and crossed the border. For all I know, our
bus made a beeline to Paris from Saarbruecken. After awhile, artwork of
geometric figures began appearing along the road. This got us all so excited
for we knew we are approaching the great city of the arts and fashion. As we
raced to Paris, we soon entered into a long tunnel lined up with flashing
visions of ceiling lights and columns that eventually opened up to a grand
view of the city of Paris. The bus soon stopped near the city center and we
got out of the bus at around 10:00 or 11:00 am. All the students broke up
into small groups so that no one will be left behind. The organizer told us
that the return trip to Saarbruecken would be at around midnight. That
leaved us with just 12 hours to see all the major tourist spots in Paris! How
the heck do you get to know Paris in 12 hours but we have to do it. Our
group composed of Sunday and I (Phil), Bob (U.S), Vitz (Czech), Gulfraz
41
(Pakistan) and Fagamou (Senegal) were at a loss as to how we will do it. All
I can remember was when we took the subway train in a hurry.
From left to right, Bob, Sunday and Vitz at the Sacre Coure.
As I recall, we definitely started our city tour at the “Sacre Coure” (Sacred
Heart) and then on to the Parisian business District where the Arch De La
Defense, Arche Triomphe, the Panthenon, Notre Dame, the Eifel Tower and
lastly Champ Elysee are located. It was a whirlwind sojourn in Paris that was
filled with so much fun and amazement as we crisscrossed the city to see
the famous places in half a day’s time. By the end of our stay in the city, we
42
were back in the bus to begin our trip back home. Though exhausted it was
well worth the trip. We will always remember the summer of 93 in Paris
where fond memories were formed and friendships strengthened. It only
occurred to me now that I was wearing a green polo shirt and a green
“United Colors” of Benetton wristwatch. It was a memorable gift given to
me by Benjie Jornales, a close friend going back to my college days in UST,
who has now settled in Melbourne, Australia. Back then, I never knew that I
will be promoting a green world through the intervention of a blue light. Only
the meteorologists and geologists probably knew of climate change and
global warming in the early 90s. Global warming was definitely not the issue
back then. It was mainly through the movie “An Inconvenient Truth” by
former Vice Pres. Al Gore that the world had known the perils of global
warming and climate change. After that movie, global warming was
transported out of the journals and conferences of scientists and has moved
to the center stage of the world news. Now that we are all beginning to feel
and see the ill effects of a changing climate, everyone has finally awaken
and taken a pro-active stance against global warming.
43
Paris in the summer of 93, a gem in my treasure trove of memories
Up, close and personal with the marine luminous microbes
Working on his dissertation, Christoph used several microbial test
systems to evaluate the toxicity of various untreated and treated wastewater
44
samples. The toxicity tests he employed included the bioluminescence
toxicity test, a standard bioassay using the marine luminous microbes.
Bioluminescence from these organisms responds sensitively to the presence
of minute amounts of toxicants in the water samples where they are added
and suspended. Christoph Siersdorfer is a true Saarlander, he was born and
raised in this small, beautiful and richly blessed land sandwiched between
France and Germany’s state of Rheinland Palatinate. He is a nice, blonde,
gray-eyed, and handsome chap. He is very friendly, always ready to give
assistance and easily accommodate requests from others students in the
laboratory. Christoph is strongly committed to his profession in microbiology.
He directly supervised me with my aufbaustudium on the utilization of freshly
cultivated and harvested marine luminous microbes for the bioluminescence
toxicity testing of wastewater samples from the coal processing plants in the
Saarland.
Giant industries and the coal processing plants in the Saarland generate
large volumes of toxic wastewater. Ammonia, sulfides, cyanides, and the
polycyclic aromatic hydrocarbons (PAH) are just some of the harmful and
poisonous chemicals found in the wastewater. Before this toxic wastewater
is released into rivers and lakes, it must first undergo a detoxification
process through series of physical and chemical treatments. The wastewater
45
treatment processes, undertaken by each industrial plant, should remove
most of the dissolved toxicants and improve the physico-chemical quality of
the wastewater. Though the physical and chemical treatment employed by
industry to clean up their own wastewater is expensive, the people of
Germany willingly shouldered the cost to live in a healthy and clean
environment. Germans now enjoy longer and quality life, the product of a
healthy living and a clean environment free of harmful pollutants. Each
industrial company has the responsibility to transform its own dirty
wastewater into clean treated water; from a wastewater that is heavily
polluted to a non-polluted water that can be released safely into rivers and
lakes. Protection of the environment and ecological awareness through the
populace’s religious practice of reducing, re-using and recycling have made
Germany one of the cleanest if not the cleanest country in the world. In the
Philippines, it is so sad that our neglect of the environment has caused the
demise of the once beautiful and viable Pasig River of lore. The river has
now become for all practical purposes dead due to so much pollution. For
the river to once again sustain plant and animal life is now just probably a
thing of the past. To bring Pasig River back to its former beauty and pristine
life will require a gargantuan and disciplined effort on the part of industries
46
located along its banks as well as from the residents of Metro Manila not to
pollute it.
Bioluminescent flask and plate side by side
Christoph employed the microbial bioluminescence toxicity test based on a
method from the German Standards for Water Toxicity Measurements or
DIN. The standard method requires the use of a complex and expensive
light-measuring instrument known as a luminometer. The luminometer
measures the intensity of the light output or luminance produced by the
luminous microbes when they are added to water samples, which may or
may not contain toxic agents. Since the effect of toxicants on the luminance
47
of the treated luminous microbes is measurable using a luminometer, it has
made the process of water toxicity detection quantitative. The
bioluminescence toxicity test using a microbe is just one of several other
toxicity tests utilizing lower animals like worms, insects, and fishes. These
lower animal-based tests can also determine the degree of toxicity of the
treated industrial wastewater after it has gone through several physical and
chemical processes. Once the wastewater is rendered clean and non-toxic
by the wastewater treatment processes as evaluated by the
bioluminescence toxicity test and the other animal-based toxicity tests it can
then be released safely into natural bodies of water. This treated wastewater
is so clean that it does not affect or alter the state of the natural
environment. The protection of the environment is of paramount importance
in Germany and industrial companies strictly observe its proper
implementation
In addition, the bioluminescence toxicity test is one of two tests required
by the German government to check the drinking water samples for
potability to complement the standard coliform test. The Coliform test
detects the presence of disease-causing microbes in water while the
bioluminescence toxicity test using the luminous microbes detects the
presence of harmful chemicals in drinking water. A water sample that
48
passes both tests is surely clean and safe to drink; it is free of harmful
microbes and free of toxic chemicals. By the way, my second award from
the professional category of the 1oth PCHRD – DOST annual scientific
poster competition was on the development of a simple “do-it-yourself” way
of detecting the presence of the fecal coliform bacterium: Escherichia coli in
water samples. My research poster bore the title “ A Simple (Do It Yourself)
and Rapid Culture Media Color Reaction Test for the Detection of Fecal and
Non-fecal Coliform Bacteria in Various Types of Water Sample”.
My PCHRD – DOST award winning invention called “EcoliTest”. The picture to the left is
before the addition of the water sample to be tested and the right is the result of a contaminated water sample containing fecal coliform bacteria.
In the applied microbiology laboratory, two kinds of luminous microbe
were used for the toxicity testing: Vibrio fischeri from Germany’s Dr. Lange
Company and Photobacterium phosphoreum from the United States’
Microbics Company. A test kit purchased from either Dr. Lange or Microbics
49
contains the luminous microbe supplied as freeze-dried preparation
contained in small test tubes. Special solution included in the test kit is
added to the freeze-dried luminous microbes to bring the frozen cells back to
life again. Once revived, the cell suspension begins to shine bluish-green in
the dark. The luminous liquid suspension, teeming with millions of shining
microbial cells, is mix with a water sample whose toxicity is to be tested.
Mixing is done in a small glass test tube, which is then inserted into an
equipment called a luminometer. The luminometer measures the light output
of the interaction between the mixed luminous microbial suspension and the
water sample tested. Two results are possible, either the luminance of the
mixture is sustained brightly over time or it fades out rapidly. If the bright
luminance of the mixture is sustained, the water sample does not contain
anything toxic. However, if the luminance fades out completely after a
period, let us say half to an hour, then something toxic is present in the
water. If the water sample tested is to be used domestically, then the water
sample is not safe and therefore should not be used at all. The luminous
microbes act as a biological sensing device whose luminance determines
the presence or absence of toxic chemicals in the water samples. Since
these microbes are of marine origin, the toxicity test has to be conducted in
water samples that have been made saline through the addition of table salt
50
(sodium chloride). In the bioluminescence toxicity test, the luminous
microbes, smart as they are, are talking to us by sending light signals; telling
us if the water contains something toxic or if the water is clean and safe.
That was how I got introduced and trained in the nooks and corners of the
proper handling and cultivation of the marine luminous microbes for the
bioluminescence toxicity test. Day in, day out, I would grow and cultivate
these microbes in nutrient broths or soups contained in glass flasks and in
solid nutrient agar contained in plastic plates called Petri dish. In the broth,
the liquid inside the flask glows brightly bluish-green specially when shaken
due to the aeration effect on the large numbers of swimming, shining, and
healthy cells. However, in nutrient agar plates the luminous microbes give a
different appearance. They produce small, numerous, round objects known
as colonies that shine brightly bluish-green in the dark. Colonies are large
masses of growing microbial cells piled together and rendered visible to the
unaided human eye appearing on the surface of the solid nutrient agar
medium.
They say that familiarity breeds contempt. However, with the marine
luminous microbes my familiarity with them bred a deeper understanding
and respect for this humble speck of life. Whenever, I am alone with these
luminous microbes in the dark holding them in flasks and Petri plates with
51
my bare hands I can feel their life energy flowing to me through the cold light
that they produce. It still kept me enthralled in awe and wonder whenever I
am alone with them in the dark. There is something in the cool bluish-green
light emanating from them that pierces the darkness so beautifully. It is a
living light shining so brightly in the surrounding darkness. These microbes
can actually talk to one another and to us as well! When they are happy,
enjoying a state of well being, they shine long and bright but when they are
sad due to harms inflicted to them by, something toxic in their watery
environment their light dims rapidly and in some instances fades out
completely. The luminous cells will “black out” instantly when they are killed
by exposure to intense heat, strong acidity, toxic chemicals, and potent
disinfectants. This innate ability to communicate using light is so common
with many organisms found and living in the sea. Deep in the dark vastness
and great depths of the oceans of the world, inhabitants great and small
have evolve the means to use bioluminescence to communicate with and
attract other creatures. Others have used the bright radiance of
bioluminescence to evade their predators by temporarily blinding them with
a squirt of brightly shining fluid containing millions of luminous microbial
cells. Set against a pitch-black world squirts of bioluminescence generate
52
brief moments of intense luminosity that must have been briefly blinding to
killer eyes.
My “Home Laboratory” in the university with a great view of the campus
At the end of the second semester, I wrote a paper on the results of my
research work with the marine luminous microbes and completed my
“Aufbaustudium” with the highest grade of “Sehr Gut” given by Prof. Dr.
Kaltwasser. I sincerely thanked and bid Prof. Dr. Kaltwasser and Christoph
farewell. On the day of my departure, Christoph fetch me at my boarding
house in Dudweiler located at the outskirts of Saarbrücken. He took me to
the Main Train Station in the center of the city. From Saarbrücken I was to
53
travel to Frankfurt to take my flight back home to Manila. He said good-bye,
gave me a bear hug, and walked away. That was the last time I saw
Christoph. Christoph and I remained in contact for two years by mail and
email after I arrived back in the Philippines in1993. It was at the University of
Saarland that Sunday and I was introduced to the earliest semblance of
what was to become the hallmark of the computer age: electronic mail and
the internet. We log on to the internet then using a black and white monitor
mainly to write letters to our friends the U.S. using their own university
computers. Yahoo and Google were nowhere to be found in 1993. Now that
the internet has evolved into far more than just sending electronic mails; it
has come to symbolize everything that is modern in the advancement of
information storage, retrieval and exchange. After a year, I was even able to
send Christoph a box full of various kinds of Philippine shells, which he
enjoyed very much. He told me that he was even proud to show it off to his
friends. The last time I heard from him was when he sent me a post card
from Ireland, the emerald isle, where he and his girlfriend Miriam were
spending a wonderful vacation together. It would surely be nice to see him
again someday to thanked and tell him of all the unimaginable nice things
that happened to me for having learned from him the wonders of microbial
bioluminescence.
54
They say that all good things must end, and so, in September of 1993, when
the red and orange autumn leaves have once again reappeared and
blanketed the trees in Saarbrücken, Sunday and I bade Germany
aufwiedersehen. I flew eastward directly back to the Philippines while my
friend Sunday flew westward and stayed in Texas for a month before
returning to the Philippines. With much trepidation, I recalled trying
desperately to secure a visa at the U.S. embassy in Bonn several days
before I was to depart for Manila. My friends doing their graduate studies at
the Southern Illinois University in Carbondale located close to St. Louis
invited me to stay with them for a month. Unfortunately, my visa application
in Bonn was denied and I left the American embassy so broken hearted. It
was so unforgettable that I still vividly remember the afternoon I left the
embassy. It was dark and gloomy; indeed the rainy weather was with me,
empathizing in my hours of unhappiness. To comfort myself, I just
entertained the thought that it was not yet probably the time for me to visit
the U.S. and wished profoundly that someday I will get there too. While in
Bonn, I really cannot miss paying my homage to Germany’s towering figure
of classical music – Ludwig van Beethoven. I stood for a few minutes in front
of his bronze statue in the city square. In my heart, I am profoundly thankful
55
to him for his musical compositions that evoke timeless elegance and power.
Even today, Beethoven’s music continues to stir up raw human emotions of
love and hate, fear and anger and above all the right for boundless joy. It is
innate joy to be experienced in life even in the midst of adversity and
sufferings.
On my way back to Manila, flying via Lufthansa, I remembered hearing
with much nostalgia the song “Top of the World” by the Carpenters for that
was where I surely was then way above in the sky. Peeking through the
window, I saw giant columns of bright white clouds billowing so majestically
below. Back in the Philippines in November of 1993, Sunday and I resumed
our academic responsibilities in teaching, research, and community service
at the University of Santo Tomas (UST) with much vigor and with so much to
share on what we learned in Germany. Every now and then, I still get
deeply nostalgic about my German experience bringing back fond memories
of sights and sounds as well as feelings and even of smell. Whenever I hear
over the radio the song “Fields of Gold” by Sting, it summons up sentiments
of pleasure of a sunny late afternoon bus ride through the streets of
Saarbrücken. While, Vanessa Williams’ “Save the Best for Last” conjures
sensation of cool balmy and lingering summer dusks in Freiburg. Sunday
became the chairperson of the Department of Biological Sciences at the
56
University of Santo Tomas. Soon after that, she married Robert Phillips an
American whom she met in her Sunday service congregation in Germany.
Sunday introduced me to Bob in Paris during a brief trip to the “City of
Lights” organized by a student club of the University of Saarland. Rob is a
good-looking guy, very friendly, and articulate. Sunday got a marriage
proposal from Rob when she was already back in Manila. Rob must have
really missed Sunday very much. They soon got married in Manila and they
have now settled permanently in Texas, Bob’s home state, with their three
pretty daughters.
An archipelago of the marine luminous microbes
Back in UST while planning for my research proposal, I became restless
as a fruit fly. I wanted to work once again with the marine luminous
microbes. So many possibilities can be achieved in improving the quality of
life of many people with the use of the marine luminous microbes in a
developing country like the Philippines. Besides, no one was doing any
research in the Philippines on the marine luminous microbes at that time and
to start such an endeavor was indeed a pioneering undertaking. To start my
research by buying the strains of luminous microbes from foreign companies
57
is simply out of the question due to the high cost involved. In UST, we work
on a limited research grant of only 600 US Dollars per annum.
Microbiology tells me that these marine luminous microbes should occur
abundantly in the Philippines, which is blessedly rich with so much natural
resources. The country ranks at the top for biodiversity in the world. Equip
with my years of training in microbiology, I embark on an exploratory work to
isolate the indigenous, Philippine strains, our very own species of marine
luminous microbes from many local sources. At first, I thought it would be
difficult to find and isolate them but I soon found out that it was actually as
easy as ABC. Jumpin Jellyfish! To my surprise, I found out that our local wet
markets are gold mines as sources of the marine luminous microbes. The
luminous microbes are ubiquitous occurring in squids, all saltwater fishes,
crustaceans, shellfishes and even in seawater itself. Before I knew it, I had
achieved the isolation of many species of marine luminous microbes that I
stored in a laboratory in UST. Various strains of luminous microbes, isolated
from the fresh surfaces and juices of Pusit (squid) and from the intestinal
contents of saltwater fishes known locally as Sapsap, Galunggong, Espada,
Besugo, Matang Baka, etc. and also from the seawater of Manila Bay, were
maintained and preserved in a special ultra-low freezer located in the third
floor of the Thomas Aquinas Research Complex (TARC). The ultra-low
58
freezer facilitated and made possible the long-term preservation of these
microbes.
Fishes, squids, crustaceans, mollusks and even seawater are good sources of luminous
microbes.
In 1995, when I beheld and held once again in my hands the brightly
shining nutrient agar plates of my luminous microbes in a dark room in the
UST Charity Hospital, feelings of euphoria overwhelmed me; it was like
having found a long lost and cherished friend. It was really so good to see
them again, strongly shining bluish-green in a dark room in UST’s old
research center with strongly sustained radiance like the ones I worked with
in Germany. On many occasions, while I am all alone with them in a dark
room in the process of adapting my vision to the dark for several minutes. I
would talk to them in my mind, asking them as if they were my friends to be
kind to me by revealing to me their life’s secrets. “Lessons in Green” that will
59
help me better understand them so that I can employ the light they
generously produce to safeguard the water we drink and to protect the
environment we live in. In all candors, my “Microbes of Light” did not leave
me wanting. The secrets they revealed brought numerous blessings to me
and to many others. It has inspired the younger generation of Filipino
students to investigate problems in biology using microbial bioluminescence.
Teachers from high schools, colleges and even graduate schools have used
them to dazzle their students with awe and bewilderment. Happy of my
achievement for having isolated Philippine strains of marine luminous
microbes, I began showing them to my friends, to my students and
colleagues in UST and they all marveled at what they saw. Very soon I was
using and demonstrating the marine luminous microbes in science seminar-
workshops for secondary and tertiary level teachers conducted at the Far
Eastern University, Philippine Normal University, Technological University of
the Philippines, Ateneo de Manila University, Manila Central University, La
Consolacion College and many times in UST. I also presented my study on
the marine luminous microbes in the annual conventions of the Biology
Teachers’ Association (BIOTA), Philippine Society for Microbiology (PSM)
and the Natural Products Society of the Philippines (NPSP) as well as in the
60
Philippine Association of American Scientists and Engineers (PAASE) and
the Asian Association for Biology Education (AABE).
With much pride, I can also say that in 2003, the luminous microbial
species that I had isolated and collected served as the initial microbial
holdings that subsequently evolved into an important repository of
microorganisms in UST called the University of Santo Tomas Collection of
Microbial Strains (USTCMS). Like a devoted microbiologist fresh from my
success with the marine luminous microbes, I also undertook the isolation of
other indigenous species of bacteria, yeasts and fungi that were deposited in
the USTCMS. The USTCMS served to provide microbes like bacteria,
yeasts and filamentous fungi to students and faculty members of UST for
their research and in their teaching of general microbiology and other
microbiology subjects. Students and teachers from the neighboring
universities and schools availed of this service.
Blessings through the microbes of light
Indeed, what followed were highly rewarding and exciting years of study
with my luminous microbes that went far, far beyond my wildest
61
expectations! My life with the “Microbes of Light” as I often call them now
has showered me with so many blessings. Foremost in my list of blessings
was finishing my Mater of Science degree in Microbiology at the UST
Graduate School in 1998. My masteral thesis dealt with the isolation and
identification of indigenous marine luminous microbes from various marine
animals and seawater, which were then individually screened for their
sensitivity to various toxic chemicals using a luminometer. The most toxicant
sensitive strain of luminous microbe was selected and tested against
standard toxicants like phenol, cyanide, mercury, and the detergent: sodium
dodecylsulfate. This toxicant sensitive strain was identified as Vibrio fischeri
with the assigned accession number of 1063 in the USTCMS. The output of
my thesis work, aside from a paper presentation and a published paper in
the proceedings of the Philippine Society for Microbiology (PSM), were the
various species of marine luminous microbes like Vibrio fischeri,
Photobacterium leiognathi, and Vibrio harveyi deposited and preserved in
the USTCMS.
In addition, I also received national and international awards. I was able
to publish articles in local and international journals. I was also able to
actively participate in numerous poster and paper presentations in local and
international science conventions and symposia. I received speaking
62
engagements in scientific gatherings and would you believe appearances in
TV (ABS-CBN) and Isla Cable TV and also radio interviews. The Philippine
Charity Sweepstakes (PCSO) announced my achievement over the radio. I
also got news clippings in all the major Philippine broad sheets from the
Manila Bulletin to the Inquirer and from Philippine Star to the Manila Times
and also a substantial amount of entries in the internet’s major search
engines. All of these venues were used to the best of my ability to spread
the knowledge I obtained with microbial bioluminescence. The clean and
useful radiant biological energy produced by these uniquely smart microbes
that can be harnessed for the promotion of the well being of Filipinos by
safeguarding drinking water’s cleanliness and protecting the environment
from pollution. In addition, it has also inspired students from near and far to
pursue careers in science as well as pioneered new methods of teaching
biological concepts.
My first national award on the use of my luminous microbe came in July
of 1997. Dr. Fortunato Sevilla, my masteral thesis adviser, and I presented a
research poster in the professional category of the eight annual scientific
research poster competition sponsored by the Department of Science and
Technology’s (DOST) agency the Philippine Council for Health Research
and Development (PCHRD). The poster entitled: “The Development of a
63
Biosensor for the Rapid Measurement of Water Toxicity Based on
Indigenous Marine Luminous Bacteria” won grand slam for the first time in
the annually held competition of the PCHRD by bagging the first, second
and third prizes of the contest. My adviser and I won a total cash price of
45,000 pesos. I was so happy to have won in the DOST – PCHRD contest
for it was the first time that I won something big, prestigious and of national
in scope in my budding professional career. Dr. Fortunato Sevilla attended
the awarding ceremonies for the annual competition and received the cash
award. We decided to split the cash prize at 25,000 for me and 20,000 for
Sir Fortune Sevilla. During the contest, I remembered standing with the
other contestants in front of our posters in the lobby of the Philippine
General Hospital explaining to the five judges as well as to the interested
public and onlookers what our entries were all about.
In school, I served as the undergraduate thesis adviser of students
majoring in microbiology and biology whose research studies dealt with the
use of microbial bioluminescence. There is a wealth of problems in biology
and chemistry that students can study using bioluminescence as a tool in
shedding light to their investigative problem. Many groups of students from
the BS Microbiology program and one group from the BS Biology
Accelerated Program worked on microbial bioluminescence research. One
64
group from the BS Microbiology won first place in the Best Poster Contest of
the College of Science’s Annual Science Fair and Exhibits. In addition, a
research study on the luminous microbes also won first prize in a poster
competition from a group of students supervised by Mr. Arthur Alipao of UST
for the analytical chemistry division in the 2001 Philippine Chemistry
Congress. In 2002, something GREAT happened that was way beyond my
wildest expectations. It really swept me off my feet into what I consider as
the second best thing that ever happened to me in my life.
Winning the gold in the Asia-pacific Young Inventors Award (YIA)
If the year 1992 was for Her Majesty Queen Elizabeth II an “annus
horribilis” or horrible year, the year 2002 will go down for me as my “annus
mirabilis” or my wonderful year. What transpired was truly a shining moment
in my career and the opportunity of a lifetime. In that year, UST and I got the
coveted recognition of winning the Gold in the 2001 Young Inventors Awards
(YIA) sponsored by the prestigious Far Eastern Economic Review (FEER) of
Dow Jones Company and Hewlett Packard (HP) Invent Asia Pacific. This
prestigious international contest for innovation ran for five straight years with
the objective of uplifting the quality of life for many people in the region
65
through original ideas hatched by students from the Asia-Pacific institutions
of higher learning. The awarding ceremony was held in Hong Kong in the
years 2000 to 2002 while in the years 2003 and 2004 it was held in
Singapore.
It is with the fondest of memory that I will always looked back to the
beautiful sunny and cool morning of January 18, 2002 at the Department of
Biological Sciences in UST. While UST’s Varsitarian science news reporter
Stephen Rojas-Chua was eagerly interviewing me on what it is like to be one
of the finalists in the Young Inventors Awards, I received the most
memorable phone call of my life! It was Dada from the Dean’s Office of the
College of Science informing me with much elation that HP Philippines in
Makati just disclosed the news that I won the Gold in the 2001 Asia-Pacific
Young Inventors Award Competition. I was speechless and petrified holding
the phone! My weeks of eager anticipation were finally over and what a
magnificent ending it truly was. After regaining my senses, I immediately
conveyed the good news to my friends and colleagues in the department
and it opened up the floodgate of congratulatory remarks from so many
people coming from near and far; and from the past and present. Fr. Victor
Badillo, S.J. former President of the Philippine Astronomical Society (PAS)
and former director of Ateneo’s Manila Observatory also congratulated me. I
66
was really so happy to hear from Fr. Badillo, a Jesuit mentor and close
friend whom I have known way back when I was still a member of the
Philippine Astronomical Society (PAS) during my late high school and
college days. Fr. Badillo an NRCP achievement awardee in Physics together
with Engineer Jose Caburian of Marsman Company are the “Fathers of
Philippine Amateur Astronomy”. They had inspired several generations of
Filipinos to become dedicated amateur astronomers like my friends Edwin
Aguirre and Imelda Joson, famous amateur astronomers, now based in the
U.S. Edwin and Imelda became very popular recently when a new comet
they discovered, Edwelda, was named after them. Many of us including my
brother Johnny and friend Michael Nealega have been in many ways
influenced by Fr. Badillo and Engineer Caburian. Prof. Fabian Dayrit, Ph.D.,
Dean of Ateneo’s School of Science and Engineering, a highly esteemed
professor and a towering figure in Philippine chemistry education and
research also congratulated me by email. I also received notes of
congratulations from Filipinos living in Singapore and Australia. My winning
the contest also got featured in a news-magazine devoted to Filipino
activities and concerns based in Singapore.
67
Winning the gold was for me truly unbelievable due to the sheer chance
of winning first prize in a contest of 220 entries from different universities all
over the Asia Pacific region. Universities from Pakistan to New Zealand and
from Japan to Australia joined the contest in 2001. The entries were
received and organized by the Far Eastern Economic Review in 2001, were
subsequently narrowed down to fourteen finalists. My entry was short listed
as one of the finalists and all were featured in the December 17, 2001 issue
of the Far Eastern Economic Review magazine. I told myself then that I
would be very happy even if I just get one of the two slots for honorable
mention. It was indeed humbling to compete against such high Asiaweek
ranking universities as the National University of Singapore (NUS), the Hong
Kong University of Science and Technology (HKUST), South Korea’s
Pohang University, New Zealand’s Massey University, Taiwan’s National
Cheung Kung University and other venerable institutions of higher learning.
In fact among the fourteen finalists no university is ranked lower than 32 in
the Asiaweek 2000 survey of Best Universities in Asia and the pacific except
for UST, which was ranked 74. But hope indeed springs eternal and my
pioneering work on the marine luminous microbes in the Philippines has
once again brought with it a brilliant outcome.
68
The December 2001 issue of FEER featuring the Asia-Pacific Young Inventors Awards
The awarding ceremony at the top of the world
The weeks that followed were very stressful, as I have to make haste to
secure a new passport to enable me to fly to Hong Kong. My missing
69
passport had to be replaced as soon as possible. I got my new passport in
the “nick of time” so to speak a mere three days just before I was to depart
for Hong Kong to attend the awarding ceremony. I finally arrived on an all
expense paid trip to Hong Kong in the afternoon of February 27, 2002 and I
was amazed at the level of advancement and prosperity that this small
former British colony has achieved since gaining independence. Indeed the
influence of the British in the prosperity of Hong Kong, Malaysia, and
Singapore is something that these countries can be proud of. Hong Kong’s
large spacious airport built on an isolated island is truly a national pride and
an infrastructure par excellence. The awarding ceremony was held at the
Apex of the posh Central Plaza, Hong Kong’s then tallest building. The Apex
is truly a marvel of modern architectural design. To reach it from Central
Plaza’s impressive spacious lobby with long crisscrossing seemingly aerially
suspended escalators you have to go through three changes of elevator ride
walking passed by glistening floors with huge artistic pots containing
beautiful plants and colorful flowers of different kinds. The last elevator trip
opens up to the “Apex” a marvelous and opulent all glass and steel hall
perched on the seventy-fourth floor and boasting of a 360 degrees
panoramic view of the whole of Hong Kong. The blue surrounding sea seem
to stretch infinitely far out into the edge of the hazy gray horizon. What an
70
awesome breath-taking sight it was! Being in the Apex was certainly the
closest place to heaven that I can ever get to here on earth. A long spire
protrudes high into the great blue yonder from the center of the Apex.
Central Plaza towering majestically for years against the Victoria Harbor
skyline is an icon of prosperity and cosmopolitanism that was Hong Kong.
The towering and posh Central Plaza set against the glittering Hong Kong Skyline and
the historical Victoria Harbor
The silver awardee Bini Thumbarathy, an Indian national, studying at
the National University of Singapore and Chen-chi, the bronze awardee from
Taiwan’s National Cheung Kung University were already in the Apex when I
71
arrived. We were all very happy and pleased to have finally met one
another. Both were Ph.D. students who were so nice, very friendly and so
unassuming. The famous cable TV business newscaster Bernie Lo of CNBC
interviewed us and we spent the whole afternoon with him going through the
hectic rehearsals for the evening’s awarding gala ceremony. We shared
ideas and insights and Bernie gave us a brief history of Hong Kong and its
famous airport. Bernie noticed the clothes I wore and he said that he saw
President Joseph Estrada wearing something similar as well. I explained to
Bernie that our beloved President was wearing the Philippines’ national
costume for Filipino men, called the “Barong Tagalog” and we wear it for
very special occasions. We then took our position before our posters after
the rehearsals to explain, discuss and answer queries from the visitors as to
what our inventions were simply all about. Students from the prestigious and
venerable Hong Kong University of Science and Technology (HKUST) were
also around for winning the honorable mentions of the 2001 Young
Inventors’ Awards.
72
A page inside the Far Eastern Economic Review (FEER)
The VIPs of the prestigious Dow Jones Company arrived from their
various offices located in the same building below; the men were in their
73
formal black suits and the women in their cocktail dresses. Mr. Phil Revzin
publisher of the FEER, his personal guest Mr. Paul Saffo, headed the Dow
Jones contingent. Mr. Paul Saffo is the director of the “Institute for the
Future” based in California. Mr. Michael Vatikiotis, chief editor of the FEER
and their associate editors and reporters also arrived. Dow Jones Company,
a name synonymous with the best in global business news, is the publisher
of the popular Wall Street Journal in the US, the Asian Wall Street Journal
and the Far Eastern Economic Review. The representatives of Hewlett-
Packard (HP) Invent Asia-Pacific, Ms. Cecilia Pang, and Ms. Yee Foong,
advertising managers of HP based in Singapore and Mr. Raymond del Val
president of HP Philippines were also in attendance. The representatives
from Polycom donor of the VIACOM videoconferencing units were also
present to demonstrate their latest high-tech gadget. Guest of Honor was
Mr. Chau Tak Hay, secretary of Commerce & Industry of the Government of
Hong Kong Semi Autonomous Region (HKSAR). Deans and academic
officials from various Hong Kong universities were likewise in attendance, as
well as the consul general of the Philippines to Hong Kong Mrs. Zenaida
Angara-Collinson, and her British husband. Mang Nards of the Philippines’
Department of Trade and Industry (DTI) also graced the occasions. I am so
happy to meet in person the associate editors of the FEER: Helen
74
Pryzidowski whom I got to know very well through our several email
correspondences during the submission of my entry and in the screening of
the finalists. Helen was the one who gave me that seemingly far away
glimmer of hope of ever winning in this contest when she informed me in
October 2001 that my entry has been shortlisted as one of the finalists.
Special mention also goes to Sofia McFarland, the freelance Swedish
reporter who grilled me for several days in UST on my invention. She was
the one who wrote the beautiful article on “A Luminous Vision” found in the
January 18, 2002 issue of the FEER and in its website. Sofia is a lovely,
soft-spoken, well-organized person and a freelance writer par excellence.
She is a mild mannered writer who is strongly devoted to her profession.
She lives permanently in New York with her children and husband - Jeffrey.
Jeffrey, a tall, handsome, self-assured, and intelligent physician and
epidemiologist works at the World Health Organization (WHO) for the pacific
region based in Manila.
To cap it all up, I was so happy almost at the verge of tears to see the
arrival in the Apex of Asst. Prof. Milagros del Callar whom we fondly call
Ma’am del. She is the cherished chairperson and endeared academic
mother of UST’s Department of Biological Sciences. Her husband the
esteemed Dr. Achilles del Callar, professor of mathematics and physics at
75
UST’s Graduate School, accompanied Ma’am del in their trip to Hong Kong.
At last, I said to myself, I have someone really close to me from the
Philippines with whom I can share my happiness and this momentous event
with.
It was truly unfortunate that the UST administration was not able to
send any representative to graze the occasion and to receive the beautiful
heavy trophy and symbolic check of 7,500 US Dollars from the FEER.
Evening finally came and Hong Kong unfurled a spectacular view! The city
of Hong Kong literally exploded with lights of various colors as seen from our
vantage point high up in the Apex with all the neighboring skyscrapers
towering brightly and magnificently around us. I am simply at a loss with
words to describe the sophisticated ambiance, the terrific place, and the
gathering of notable academics and business people at the Apex of the
Central Plaza that evening. The event was so magical and memorable
rendering it almost dream-like; it was the kind of stylish sights and sounds
that I had only seen in Hollywood movies.
The awarding ceremony started at 6 pm with a snappy video
presentation of what the Young Inventors Awards is. I remember being
seated between Mr. Raymond del Val of HP Philippines and Chen-chi.
Chen-chi from Taiwan’s National Cheng Kung University was the first to be
76
called up the stage by Bernie to receive the bronze award and the trophy
and computer prizes from HP. Bini Thumbarathy from the National
University of Singapore was next to receive the silver award and prizes . My
turn finally came to receive the gold award. I felt so happy to go up on stage
and beaming with pride I delivered a brief and concise answer to Bernie’s
question. His honor Mr. Chau Tak Hay gave me the heavy trophy and then
posed with him for the picture taking. I went back to my seat carrying the
heavy blue and yellow trophy of the gold award for UST.
Icon of the young inventors’ awards (YIA)
Lastly, it was Ma’am Del’s turn to represent the UST administration. She
went up on the stage and stood proudly during the climax of the program.
77
With much applause, Ma’am del received the large symbolic check of 7,500
U.S. Dollars in-behalf of UST although she and her husband went to Hong
Kong on their own private initiative. The ceremony took place on a formal
candle-lit dinner, drapes of red and white cloths covered the long tables,
catered with sumptuous foods and lavish drinks, and of course, to cap it all
up, the charming and articulate Bernard Lo acted as master of ceremonies.
The awarding ceremony went like clockwork; so smoothly and precisely as
we had rehearsed it in the previous afternoon. At 8:00 pm the awarding
ceremony full of wonderful moments finally came to pass. We all went down
the Central Plaza building in the elevator in batches. The attendees all
received souvenir items and a small handsome powerful binocular from HP
contained in a small black leather bag. Some of us walked back to the Luk
Kwok Hotel where the other winners and I were booked. The hotel is just a
few blocks away from the base of Central Plaza. Somewhat tired but still
very much in high spirits, I carried with me the heavy trophy for UST and of
course my HP Omnibook 6100 laptop. The Omnibook 6100 is a top of the
line multi-functional computer product of HP, which has been of much use to
me in my teaching and research work in UST. In fact, I used this laptop, still
functioning perfectly after 6 years, writing this book. Ma’am Del and her
husband Dr. Achilles del Callar took the impressive and clean subway of
78
Hong Kong to travel back to a hotel in Kowloon where they stayed. I am truly
grateful to Anna Lam, a pretty and petite woman and Jonathan Hardy both
of the FEER for having managed and attended to our needs during the
rehearsals and the awarding ceremonies.
In the Luk Kwok hotel in Wanchai as I reflected back on the exceptional
events that transpired, I cannot help but wonder and be amazed at the
seemingly meant-to-be turn of events. It is indeed true that it is better to
have tried and failed than never to have tried at all. What started out for me
as just a late evening curious internet visit to the website: www.feer.com
using an old computer with a black and white monitor in the UST Research
Center in the charity hospital turned out to be the chance of a lifetime. My
research work on the light generating indigenous marine microbes has been
for me a very rewarding endeavour ever since I learned of it in my
“aufbaustudium” at the University of Saarland in Germany in 1993. I surely
will never forget the awarding ceremonies in Hong Kong for it was for me the
closest ever of winning a major science award like perhaps a Nobel Prize!
Winning the Gold is indeed a fitting tribute to start-off the university’s
countdown towards its Quadricentennial founding celebration in the year
2011. Someday people will look back at the list of universities, which have
made their mark in this prestigious competition of innovation in the Asia-
79
pacific region. Thomasians will surely take pride in knowing that in the
second year of the “Young Inventors Awards” (YIA) sponsored by FEER and
HP that ran into a full five- year course, UST took the highest honors. It is
the first for a Philippine university and the first for a developing country like
the Philippines to be in the list of winners.
Back in UST, Ma’am Del and I paid a courtesy call to the Father Rector,
Very Rev. Fr. Tamerlane Lana, O.P., in the imposing Rector’s Office and
gave him personally the Gold winning cheque worth 7,500 USD. The
Polycom videoconferencing units were delivered to me in Manila. One unit
was for UST and one for me, which Ma’am del and I decided to donate to
UST so that they can have a pair of it. The cash prize was converted to
384,000 pesos, which was fully utilized to purchase much needed laboratory
equipment for the Pure and Applied Microbiology Lab. of the Research
Center for the Natural Sciences as well as for the Department of Biological
Sciences of the College of Science. Digital balances, distillation apparatus,
magnetic stirrers, refrigerators, and microwave ovens were bought using the
cash prize. New computer units were also made available to the Biology and
Microbiology Student Organizations of the College of Science. The
laboratory equipment and computers purchased were thought out carefully
to maximize the benefits to the biggest number of graduate and
80
undergraduate students, faculty members, and researchers alike. Indeed the
equipment purchased continues to serve the needs of students,
researchers, and faculty members up to now. The distillation unit still
supplies distilled water to four laboratories in the 3rd floor of the Thomas
Aquinas Research Complex.
The last time I saw the FEER-HP trophy for the university was during
the birthday of the Vice Rector Fr. Juan Ponce, O.P. in 2003 when the
academic community paid him a courtesy call in his office. His office
connects to the Rector’s Office in the second floor of the university’s
historical main building. The Rector’s office is the university’s “sanctum
sanctorum” and it is not often that one gets to enter it. So we took the rare
opportunity to enter the Rector’s Office and viewed a large table bearing the
memorials of success and victory that the university has earned through the
years. There I saw the trophy once again standing side by side with the
others. What immediately came to my mind was how heavy and solid it was!
Carrying it from the top of Hong Kong’s tallest building during the awarding
ceremonies back to UST in Manila was a big effort on my part but well worth
the trip. I remember so well, Bernie Lo jokingly told us winners that we can
use the heavy trophy as weights to keep us fit. Well he can surely say that
again. I will forever be grateful to the very Rev. Fr. Tamerlane Lana, O.P. for
81
it was during his rectorship that researches in the arts and sciences flourish
and peak in UST earning for the university numerous recognition at the
national and international arenas.
News from the Phil. Daily Inquirer. Many thanks to Prof. Dr. Abercio Rotor of the UST
Graduate School for framing this news clipping for me.
Some scientists in the U.S., England, Germany, Poland and Croatia
likewise took note of my novel method of immobilizing marine luminous
microbes in small paper discs through reprint request of my first international
article published in the Journal of Biological Education (JBE) in 2001. The
JBE, an Institute for Scientific Information (ISI) listed journal, is the official
publication of the Institute of Biology (IOB) the biggest organization of
82
biologists in the British Isles. My article’s title was used as well by the IOB in
its advertisement subscription form. The “JBE” has the biggest worldwide
circulation of a peer-reviewed journal devoted to biological education. Dr.
Baby Angtuaco of Ateneo’s Department of Biology, a professor active in
teaching and research, subscribed to the “JBE” and congratulated me when
she saw my article in the Spring 2001 issue of the journal. The acceptance
of my first article in an internationally ISI (Institute for Scientific Information)
listed journal and Expanded Science Citation Indexed journal based in
London has indeed given me the confidence to submit publications in other
journals devoted to microbiology, water and the environment. Like any other
scientist, I should be able to convey my research findings clearly to other
scientists in my field by publishing them in special science publications
called journals.
PIBiT – the invention
I look forward to the day when the vast potentials of this simple
bioluminescence invention, with the proposed name “Paper-disc
Immobilized Bioluminescence Technology” and having the acronym “PIBiT”,
will have been fully realized. It may take a decade, a lifetime, or even a
83
century but I am sure it will come. PIBiT’s main use is to safeguard our
drinking water from toxicants and protect the environment from pollution.
The more the world needs to reuse, recycle and reduced due to the threat of
global warming, the more this invention’s significance will be appreciated
and bring into practice in most of the developing countries of the world.
PIBiT holds the promise of being a multipurpose analytical process that is
inexpensive, user-friendly, and environment-friendly and exhibits almost no
energy consumption and zero emissions. PIBiT was featured in the
prestigious September 17 issue of the Sustainable Practices 2004:
Innovations, Technologies, and Products through Mr. David Schaller.
Sustainable Practices 2004 is compiled and provided by the EPA
(Environmental Protection Agency of the United States.) Region Eight’s
Sustainable Practices and State Partnerships Program. EPA’s Region Eight
serves Colorado, Montana, North Dakota, South Dakota, Utah and
Wyoming. Surely I could have made the invention complex and complicated
by introducing a light-measuring device instead of just using plain scotopic
or dark vision to observe the light signals of the luminous microbes. But it
defeats the purpose of making it readily available to the vast majority of the
poor people of the world. Digital Photography is a much simpler and far less
expensive alternative to the use and procurement of a luminometer although
84
its light measuring ability may not be as sensitive. I subscribe to the school
of thought known as KISS, which I first heard from an MIT professor giving a
lecture in the BIOTECHNICA 1988 in Hannover, Germany. He said that at
MIT, they keep everything including research output KISS which means
“Keep It Simple Stupid”. Hopefully, the day will come when the World Health
Organization (WHO) and UNDP and also UNESCO will have adopted this
simple invention in many of the developing countries of the world as a
simple and inexpensive means to guard the safety of their drinking water
and for the protection of their environment. I am sharing a simple note that I
sent to the people, I believed that I am indebted to most particularly to the
2001 YIA judges, the people at FEER and HP. It goes
My winning the Gold in the Asia-Pacific Young Inventors Award
Is very much like my culture of luminous microbes
One microbe alone cannot shine on its own
Much less attain a state of brilliant luminosity
But by being in the company of other microbes
Can one truly shine and together as a whole attain amazing luminosity
It is with joyous pride and profound gratitude that
I myself have likewise attained this state of brilliant luminosity
85
Only because, I have been in the company of the other 2001 brilliant
inventors.
Myself with Mr. Chau Tak Hay, secretary of commerce & industry of the government of Hong Kong Semi Autonomous Region (HKSAR) receiving the gold trophy of the 2001
Young Inventors Award
86
The winners of the YIA through the years
Year Gold Silver Bronze
[2004]
Wang Qijie China
Nanyang Technological University
Randall Law Singapore
National University of Singapore
Liang Xiaojun, Sun Yi, &Zhang
Xuming China
Nanyang Technological University
[2003]
Ryuji Inai Japan
National University of Singapore
Material Advantage
Sangjin Han South Korea
Seoul National University
Fuel-Cell Thrift
Joanna Tan Hwa Lay
Singapore
Ngee Ann Polytechnic
Growth Market
[2002]
Anthony Samir Australia
University of Melbourne
Life-saving Precision
Robert Fearn Australia
University of New South Wales
Music to My Ears
Michael Zheng Zhongming
Singapore
National University of Singapore
The Fungus Among Us
[2001]
Edward Quinto Philippines
University of Santo Tomas
A Luminous Vision
T.B. Bini Singapore
National University of Singapore
Nerves of Steel
Yu Chen-Chi
R.O.C Taiwan
National Cheng Kung University
What a Catch
[2000]
Mulyoto Pangestu Australia
Monash University
BIOLOGY
Tse Kowk-Kuen China
HongKong
City University of Hong Kong
SOLAR ENERGY
Chong Wai Yin, Kelvin
Singapore
Ngee Ann Polytechnic
GENETICS
www.ntu.edu.sg/home/EXJLiang/YIA2004.htm
Go west, life is peaceful there; Go west, where the skies are blue To finally complete the prize of having won the Gold in the Asia Pacific
2001 Young Inventors Award, I had the rare privileged of being flown on an
all-expense paid trip to Palo Alto, California through the courtesy of Hewlett-
Packard Invent. The trip entailed visiting Stanford University and the
87
Hewlett-Packard Nanotech Laboratories and most of all to meet the winners
of the 2001 Collegiate Inventors Contest considered America’s most brilliant
young minds. I was supposed to fly to California with the two other winners
of the 2001 Young Inventors Award. But Bini Thumbarathy and Chen-chi
both cannot fly to the U.S. Bini was on her eight month of pregnancy and
Chen-chi had academic commitments and so I have to go to the U.S. alone.
The visit to the U.S. was something that I could not miss for the world. The
chance to visit the U.S. came to me once again and this time around, it was
offered to me as a gift for having won the Young Inventors’ Awards (YIA)!
Vividly, I recalled my sad experience in 1993 when my earnest desire to go
to the U.S. was squashed by the U.S. consul in Bonn by denying me a visa.
I left the embassy so broken hearted that day because I do not know when
the opportunity to visit the U.S. will ever knock again. Astonishingly in 2002,
it did knocked again and even opened up the door for me! It was so different
in 2002 than in 1993, so confident and proud am I of going to the U.S.
embassy in Manila for I know fully well that I will get my coveted visa this
time. The processing of my visa in the U.S. embassy was a breeze. With just
a question from the consul on what it is that I have invented that won me the
award and presenting my certificate of employment from UST and the official
letter of invitation from HP I finally got my precious six-month visa the
88
following day. I was so pleased with how I was treated by the woman consul
that I left her a copy of the FEER magazine containing a write-up of my
award winning entry. The saying that the best things in life are free meant so
much to me than ever before. So far, all of my foreign trips had been free of
charge. Recently, in 2007, I requested again for a U.S. visa to enable me to
attend a conference in Texas. Lo and behold, I was so pleased and thankful
that I was given a ten-year visa.
I woke up very early at around 2:00 am in the morning of 2001 Easter
Sunday in my rented place near UST. Immediately, I organized myself and
soon I was at the international airport at 4:00 am. From Manila, I first flew to
Hong Kong to get a connecting flight that will take me all the way to the City
by the Bay - San Francisco. The journey to California was quite long. I felt so
excited, somewhat daunted because it is going to be my first trip to the
United States. Surely I have made many long trips to Germany before,
always flying westward across the vast landmasses of Asia and Europe.
Now, I am flying eastwards across the vast expanse of the Pacific Ocean.
Flying via United Airlines, we cruised at an altitude of more than 30,000 ft.
Seated beside the window, I did not see the ocean below just a thick blanket
of white-grayish clouds extending all the way yonder.
89
Sojourn in the famous Silicon Valley
America here I finally come! I reached San Francisco in the early
morning of Easter Sunday after about fourteen hours of flight. It was a very
memorable 2002 Easter Sunday for me because I finally set foot on the land
of milk and honey. The land that many Filipinos will do everything possible to
enter, settle down and work. It was indeed a relief that I am standing once
again on firm American ground. I can hear in the back of my mind the
Mamas and the Papas singing “California Dreamin”, and I almost put some
flowers in my hair. The long trip made me feel so plane-sick that I felt sleepy
and groggy upon exiting the plane. Mustering enough energy, I began my
long trek in the imposing corridors of the magnificent airport moving towards
the area of the U.S. immigration officials. The cold early sunny Easter
morning was truly a welcome relief. The mere thought of being in the U.S.
revitalized me and gave me enough push to move on. It really felt so great to
be finally in the U.S. of A.
The first thing I noticed was the presence of so many Filipinos holding
various types of job in the airport. The immigration official that gave me a
six-month stay and the woman in charge of the money exchange looked like
Filipinos. Often one can hear Filipino being spoken in some quarters of the
90
Airport and in the streets of San Francisco. I guess that really makes San
Francisco a home away from home for many Filipinos. Unfortunately, I
missed the person who was supposed to fetch me at the airport and who will
bring me to the Sheraton Hotel in Palo Alto. It made me a bit nervous
because I don’t have enough money to take a taxi ride to the Sheraton. So, I
bravely went out of the Airport holding on to my luggage to explore the
public transportation system of San Francisco. What I did was to take the
free shuttle ride to Caltrain from where I hope to travel to Palo Alto. Since it
was a Sunday, the scheduled Caltrain trips were far apart in between. I
decided to return to the Airport and soon enough I was so happy to discover
a bus route that finally took me to the Shopping Mall in Palo Alto located
close to the Sheraton hotel.
It was a delightfully languid day. The weather had a welcome charm, it
was sunny and cool, and the sky was blue as cheery American eyes.
Alluring two-storey houses lined the suburban streets; showy and well-
maintained green lawns and a modern vibrant city were my first memorable
glimpses of San Francisco. Palo Alto is a captivating place with the widest of
flowing free ways and seemingly boundless parks and vast locales for the
multi-national business companies. Ah yes! It is the envy of many -
California’s ostentatious display of limitless open spaces. Stanford University
91
and Silicon Valley are the famous landmarks of this region nestled several
kilometers south of San Francisco. I reached the Sheraton Hotel at
noontime, some of my relatives visited me at mid noon, and at 6 pm I was
already in bed deeply asleep due to the long exhausting journey that
originated from the other side of the globe.
The following refreshing morning, in the Lobby of the Sheraton Hotel,
still suffering from an intense jet lag, I had the pleasure of being introduced
to the five winners of the 2001 U.S. Collegiate Inventors Contest. Steve
Anderson, Manager of HP Brand Sponsorship, a tall black American who is
an avid fan of the Giants and who likes Filipino dishes like noodles (pancit)
and spring rolls (lumpia), was our HP host. We toured the HP Main Office in
Palo Alto, the original offices of Dave Packard and Bill Hewlett where a
literal “open door” policy was implemented to all the employees of their
corporation. This policy surely made HP a very competitive corporation that
is able to continuously re-invent itself to meet the challenges and the needs
of the world.
92
Meeting the winners of the 2001 U.S. Collegiate Inventors Contest
Due to my jet lag, I almost fell from my seat while trying so hard to keep
myself awake listening to HP’s CTO Mr. Stephen Squires’ message. He is
the chief technical officer of HP who gave a short inspiring round-table
discussion on HP Invent and on innovation. During the light lunch tendered
by the HP administration in the scenic HP garden, I finally got the chance to
really know in person the winners of the U.S. Collegiate Inventors Contest.
Two of the winners, Michael Oddy and Daniel Fletcher recently earned their
Ph.D. in Engineering from the prestigious Stanford University. Would you
believe that the research endowment fund of Stanford University alone stood
at 7.6 Billion US Dollars, which was roughly the size of the Philippine
government national budget in 2001! Sascha Welz is also an Engineering
Ph.D. holder from the University of Illinois at Chicago. Sascha comes from
the Saar region in Germany. The Saarland for me is a region full of
wonderful memories and in whose university I finished a one-year
postgraduate certificate course in Biogeography and Environmental
Assessment way back in 1992. Small world isn’t it! Dana Perkins, the only
rose in the group, recently earned her Ph.D. in Pharmacology and
Experimental Therapy from the University of Maryland. Incidentally, there
93
was a big celebration that Monday evening among us because the
University of Maryland made history by winning for the first time in the NCAA
against Indiana University. Dana proudly said that she was born in the
Dracula-famed Transylvania region of Romania and ultimately migrated to
the US. Dhaval Doshi was an Indian national who like the others earned his
Ph.D. in engineering recently from the University of New Mexico. The sixth
winner who was not able to make it to Palo Alto was Xiangfeng Duan from
Harvard University.
What is it that they invented? Xiangfeng won by developing nanowire
building blocks. Michael who comes from Arizona invented a novel device
for the rapid stirring of micro and nano liter (extremely small) solutions for
molecular diagnostics with potential for aiding genetic and drug discovery
research. Daniel a true-blooded Californian invented a microsurgical tool
that provides more precise tissue dissection and drug injection capability
than is currently possible with existing procedures. Sascha invented a cost-
effective technique to produce dynamic seals coating for vehicle engines to
improve life and prevent failure. Dana successfully utilized a Herpes virus as
transfer agent to combat degenerative brain diseases like Alzheimer in
laboratory animals. She said that clinical trails would follow soon. Dhaval
94
developed a technique using UV light to alter a thin film’s pore size, optical
characteristics and other properties to improve microelectronics. Inventors
are not nerds, which is usually how Hollywood portrays them. Just like any
other young people, inventors know how to have a good time. They enjoy
going out in the company of friends to dine in good restaurants, drink beer,
laugh at good jokes, and watch basket ball games. Traits that unite them are
being open-minded and inquisitive, analytical in thinking and a strong
dedication to science. An inventor sees things not with his eyes but with a
mind that dares to confront the conventional and challenge mediocrity. They
are gifted with sparks of imagination that can make huge leaps and bounds
in technological development and scientific investigations. One simply
needs to look back at numerous events in history when the sons and
daughters of science initiated and led revolutions in ideas and ways of life.
They started a cascading reaction of events that created milestones and
breakthroughs in many areas of human endeavors leading to the twentieth-
century’s conception of gene-manipulation based Biotechnology and the
computer-based Information Technology. In today’s world of modern living,
every aspect of the level of progress and the high quality of life that we now
enjoy so much can be traced to the ideas of great minds. The discovery of
antibiotics, structure of proteins, functions of nucleic acids and other
95
biomolecules, and the invention of the printing press, gyroscope, plastics,
jet-propulsion, robotics, photography, DNA recombinant technology,
polymerase chain reaction (PCR), computerization, LCD, LED, and
nanotechnolgy has brought all aspects of human living well beyond the
space age”.
The Hewlett Packard experience
We also visited the HP Archives, the HP Cooltown Experience which
was really so “cool”, the Computer Museum in Mountain View and the
Explotatorium in the Bay Area. The vicinity of the Exploratorium with its tall
huge roman columns is memorable for I have seen it several times in the
movies. The Pacific Bell Park home of the San Francisco Giants is very
impressive and modern. We took our meals in some of the famous
restaurants in the Bay Area and we visited the famed HP Laboratories
where research in Nanotechnology, the current stuff of science fiction, was
conducted. Imagine developing a chip the size of your thumb that can hold
several terabytes of stored information as well as creating machines the size
of molecules. We also visited the famous Garage, birthplace of the famed
Silicon Valley, where HP had its origin. Dave and Bill started their business
96
of inventing electronic gadgets like oscilloscopes and calculators and
transformed it into the multi Billion-dollar computer and information
technology company known today with pride as HP. It is also amazing to
know that Dave Packard worked as an engineer for General Electric (GE)
during the great depression only to resign and to put up a company that now
rivals General Electric itself. Even though other companies like Dell have
over taken HP in terms of personal computer sale, it still is one of the best
brands to consider when buying computers, computer accessories, and
computer services. In fact, my second more modern laptop is an HP
Compaq Pressario.
The afternoon of my last day in San Francisco was truly hectic. I got to
see some of my relatives, which brought me to Daly City where they lived. A
visit to San Francisco will not be complete without the trip to the Golden
Gate Bridge. Uncle Jesse and I made a rapid buying spree for gifts to be
brought back home. My relatives, some of which I saw for the first time, were
all so generous to give me whatever dollar bills they had and I got to be
sixty-four dollars richer than when I entered the U.S. carrying a mere 100
dollar bill. When I finally got back home in Manila in the wee hours of the
next Sunday morning after almost a week of a whirlwind visit in San
97
Francisco, I switched on my FM stereo to help me unwind and reflect on my
memorable sojourn in San Francisco. The very first song that I heard,
unbelievably, was the song “I left my Heart in San Francisco” by Tony
Bennett! Absolutely true, I swear.
It is my wish to visit San Francisco again someday. San Francisco is a
welcoming, wonderful, and energetic city with so much to offer to the unwary
visitor for him to stay on for good. It is a metropolis for the world’s dreamers
and visionaries where they create the future of technology. Silicon Valley
has pioneered many technological innovations whose impact on modern
living continues to affect the way we live and view the world today. The
experience of having won the Gold in the 2001 Asia Pacific Young Inventors
Award is really the chance of a lifetime that opened up new doors of
opportunity for me. I wish all our budding scientists and inventors across this
vast archipelago all the best in joining the various science contests that are
held annually in the Philippines. I am hopeful indeed that just like Germany
and the U.S. we can finally move our country forward into the globalized
world of Biotechnology and Information Technology through innovative
ideas.
98
Bioluminescence projects shining brightly in science fair contests
After going through years of conducting demonstrations on microbial
bioluminescence in numerous lectures, seminar-workshops, training courses
and in annual conventions held in UST and in other universities, microbial
bioluminescence has finally aroused the curiosity of the country’s education
sector. High school students came to UST motivated by their desire to apply
bioluminescence as a tool to study problems in biology for their science fair
projects.
Shining colonies of Vibrio fischeri in the dark
99
In 1998, Dean Emeritus Carmen G. Kanapi of UST’s College of Science
introduced me to a group of four high school students from the Assumption
in Makati. One of the girls was the niece of Dean Kanapi. The Assumption
girls were pretty and zesty, so confident of themselves and so
enthusiastically dedicated to their science project. These girls were definitely
far from being the picture of spoiled rich girls that most ordinary people have
of the “Assumptionistas”. They worked hard for three weeks in the
university’s old research center located in the charity hospital until the early
hours of evening and were so ecstatic to see the beauty of bioluminescence.
They were holding brightly glowing flasks of bioluminescence high up in the
air and swaying as if they we were in a rock music concert. Suffice it to say,
they won first place in their high school’s science fair contest.
In 1999, a lone female high school student from the UST Pay High
School reached the national level contest of the Intel Science and
Engineering Fair with her study on the use of bioluminescence for the
ecotoxicological monitoring of rivers in Bulacan. Many rivers in Bulacan are
contaminated with heavy metals due to the heavy presence of the tanning
industry. The Tanning industry throws out wastewater containing high
concentration of chromium. Her entry won first prize for the UST Pay High
100
School in the Intel Science and Engineering Fair Contest for the National
Capital Region (NCR). The NCR is roughly all of the Metropolitan Manila
area. She went on to compete in the Intel National Level Contest and won
second place. But only the winner of the first prize goes on to the US to
represent the Philippines in the International Intel Science and Engineering
Fair.
It has been said that when it rains, it pours. In 2004, after five years,
high school students composed of an individual from Davao’s Assumption
High School and a group of three students from the Quezon City Science
High School had once again worked on a project dealing with the marine
luminous microbes.
Winning in the Intel science and engineering fairs
The first group of students that I took in who did their project in UST’s
new Thomas Aquinas Research Complex (TARC) was from the Quezon City
Science High School. One of my favorite strains of luminous microbes was
Vibrio fischeri USTCMS 1063, a brightly shining luminous microbe that I
isolated from the fresh juices of squids. Vibrio fischeri is one of the safest of
microbes that one can work with for a science project according to the
101
Chicago Science Fair Board. When the students from the Quezon City
Science High School composed of a boy and two girls named Jayson
Reggie Obos, Melanie C. Melchor and Trina G. Napasindayao came to me
one afternoon they wanted to work on disinfectants. It was indeed timely to
work on disinfectants because SARS had just then ravaged Canada and
Hong Kong. Using disinfectants should prevent the spread of infectious
diseases like SARS and flu. At first, I turned down their request of
supervising them simply because I find the project on disinfectants too
common something that lacks novelty. Nevertheless, Jasyon simply will not
give up and their collective persuasion and perseverance finally paid off. I
finally took them in and they were granted permission to do their project in
the Pure and Applied Microbiology Lab. However, instead of working with
disinfectants, I gave them a different science project something that involves
bioluminescence. A year before, I had worked with the use of microbial
bioluminescence to measure the dissolved oxygen content or the degree of
pollution of water and wastewater samples. In a screw-capped tube, a
suspension of luminous microbes is mixed with the water sample to be
tested. Once mixed by shaking and swirling, the liquid inside the tube shines
uniformly with bluish-green hue in the dark. This novel method should
enable bioluminescence to measure the degree of pollution in water
102
samples. My findings have shown that the longer the luminance of the water
sample inside the tube persist, the more dissolved oxygen there is in the
water and therefore the less polluted it is. Polluted water contains small
amounts of dissolved oxygen thereby sustaining the period of luminance,
which needs oxygen, at a much shorter period. In non-polluted water where
the amount of dissolved oxygen is high, the period of luminance of the liquid
inside the tube should persist longer. I gave this project to the “QueSci”
students, a nickname that they fondly call their school. The QueSci students
were bright, articulate, and assertive enabling them to explain their work so
extemporaneously. Surely, they have what it takes to win. They had full
confidence and they were able to organize their thoughts rather quickly and
speak it out with coherence and clarity. Rarely have I seen such qualities in
students not even with most of my college students. Thus, this group from
the Quezon City Science High School was given Vibrio fischeri USTCMS
1063 for their science project. It dealt with the simple and rapid
measurement of dissolved oxygen (DO) in wastewater samples based on
the length of time it takes for the luminance of the liquid inside the tube to
black out. They collected water and wastewater samples from different
areas in Manila and evaluated their degree of pollution using the method,
which I called “Tube Luminance Extinction”. Using a calculator and their
103
knowledge of basic statistics, they processed their data into a beautiful
equation showing inverse relationship between the length of time of
extinction of the luminance inside the tube and the degree of pollution in the
water samples.
What a Grand Slam this project brought them. In a year they won
several prizes in various science contests held in the Philippines. Their
science project entry earned the QueSci students, the following national and
international awards:
Best Science Project sponsored by the College of Engineering of UP
Diliman for the country’s science high schools
Best Science Project in the First National Science Fair Contest (2004)
sponsored by the Department of Education (DECS) in Sta. Cruz, Laguna
First Place in the Physical/Applied Sciences category at the Intel 2004
Regional Level Contest
First Place in the Intel 2004 National Level Contest held at the Bahay ng
Alumni, University of the Philippines at Diliman
Intel 2004 Best Science Project
Intel 2004 Excellence in Environmental Science Award
104
Cash Price from the US based Science News Magazine
Fourth Place in the 2004 Grand Awards of the Intel International Science
and Engineering Fair held in Portland, Oregon; USA.
Another group of three girls from La Consolacion High School in Manila
used Vibrio fischeri USTCMS 1063 to determine the presence of residual
chlorine in various tap water samples collected all over Metro Manila.
Residual chlorine in tap water is an effective shield against the spread of
water-borne diseases like typhoid fever, cholera and dysentery. Indeed
using bioluminescence, they found out that residual chlorine is no longer
present in almost all water samples collected in various places in Metro
Manila. Water samples from Tondo had practically no detectable residual
chlorine, which explains several outbreaks of water-borne diseases in that
area. Several people died from these outbreaks caused by illegal water
connections and leaking pipes. The La Consolacion students won first place
in their school’s contest and won as well in the National Capital Region
(NCR) Division of the Intel Science and Engineering Fair Contest.
Unfortunately, they failed to reach the Intel National Level Competition. I
really thought that this project owing to its novelty and public health
significance should have won in the national level. Probably the girls were
105
simply not able to impress the judges with the impact of their research
project.
A third group of four students from the Parañaque Science High School
completed a project on the use of Vibrio fischeri for the rapid, simple and
inexpensive way of screening our indigenous medicinal plants for the
presence of antimicrobial compounds. The luminous microbes have been
found from previous studies to be very sensitive to the action of antimicrobial
agents in plant extracts. Indeed a less than one-percent garlic extract in
water was found to be most potent in exhibiting antimicrobial activity when
compared with several plants that have been tested. Onion, ginger,
Malunggay and Lagundi were just some of the plants they found to possess
significant antimicrobial activities based on the length of time it would take
for the plant extract to extinguish the luminance of the microbes. The shorter
the period of time for the luminance of the plant extract treated luminous
microbes to fade out, the potent the antimicrobial activity of the plant extract
is. They won prizes in the Intel regional science fair contest held in Alabang
as well as in the first nationwide Science Fair Contest held in Sta. Cruz,
Laguna sponsored by the Philippines’ Department of Education. Lastly, a girl
from the Assumption High School in Davao made used of the marine
106
luminous microbes to determine the water pollution level of environmental
water samples. She won first place in the Intel regional level and went on to
represent her region at the Intel national level competition.
One evening in 2005, while I was all alone in Microbiology Laboratory,
the students from QueSci dropped by for a surprise visit to thank me for all
the assistance. As a token of their appreciation, they left me their ISEF
(International Science and Engineering Fair) pins from the contest that they
just attended in Portland, Oregon. They relayed to me with so much
excitement and happiness the wonderful experience of having been given
the opportunity to represent the country in the Intel “International Science
and Engineering Fair” (ISEF). It was remarkable for them to be in the U.S.
at a very young age. What they had achieved was the kind of fairy tale
contest adventure that millions of Filipino high school students can only
dream of achieving. To have been given such honors and the privilege of
representing their country in an international competition in America is for
them the chance of a lifetime. Meeting thousands of other young participants
in the contest from countries all over the world must have enriched and
changed their outlook and ambition in life. Two of them went on to De La
Salle University to pursue a degree in engineering and one went on to the
107
Ateneo de Manila University to pursue a career in mathematics. They are
really the future scientists and engineers who will someday take their control
post and chart the course of science and engineering in this country and
surely its direction into socio-economic prosperity. When they left me that
evening, it was for me deja vecu! All of a sudden, I was transported back to
the year 2002 my “annus mirabilis” and relived once more in my thoughts
the thrill of victory, of what it felt like to win the Gold in the Young Inventors
Awards. The fond memories of receiving my award at the top of Hong
Kong’s tallest building “The Central Plaza” and the trip to “Silicon Valley” in
California to visit the HP’s headquarter and the Nanotech Laboratories came
back to find me wistfully longing. The year 2004 was truly for these QueSci
students what 2002 was for me. It was a year that they and their school will
always look back to with so much nostalgia and pride; a year that they will
always reminisce with so much happiness in their lifetime. Through it all, my
research work on the marine luminous microbes have been a great blessing
to me and to many of the younger generation of Filipino scientists. Every
now and then, I still received queries on bioluminescence from students
coming from the Philippine Science High School, the Manila Science High
School and from the other science high schools. I still get a feeling of
amazement and gratitude when I am alone with my luminous microbes in
108
the dark. They can surely heighten one’s awareness of what it is like to be
alive. Seeing and feeling the cool bright bluish-green light that they generate
is also their way of telling me how happy they are to be alive as well. A
speck of life has indeed made contact with another more advanced life form
through the gift of light and the complexity of vision. Through it all, I am
confident and secured in the notion that whatever the future holds, my
“Microbes of Light” will continue to shine a path that will guide me to new
and rewarding endeavors. Bioluminescence, the light of life, coming from
whatever organism, be it a protist, a copepod, a firefly or even a fish, will
continue to inspire our country’s young brilliant minds to be analytical and
innovative; bold and daring and to explore the great and the small! Indeed,
Vibrio fischeri is teaching us “lessons in green” that can be used to
safeguard our, the environment’s and the planet’s health. Very recently, I
supervised a group of three boys from España’s Ramon Magsaysay High
School on their own original special project. They worked on the effect of
different wavelengths in the drying of guava leaves on its antibacterial
activity. I am glad that they won in the NCR and went on to the National
Level. Recently, a group of students from the Manila Science High School
visited me and told me the good news that their science project won several
awards in the 6th Regional Congress: Search for SEAMEO Young Scientists
109
(SSYS) Awards held last 3 – 6 March 2008 at the SEAMEO Recsam,
Penang Malaysia. Emmanuel D. Delocado, Justine Timothy P. Cruz, Jose
Noel Gamba, Edilberto Barcelona and Mr. Jonathan P. Derez their adviser
won 2nd place in the Science Category 1 for Best Project Exhibit and 3rd
Place for Science Category 3 for Best Presentation for their project entitled
“Bioluminescent Bacteria (V.fischeri and V. phosphoreum) To Direct A Killing
Mechanism on Leukemia Cells”. The group of Emmanuel Delocado was
indeed so generous to give me a cake, a key chain from Malaysia and the
souvenir program of the 6th SEAMEO Regional Congress.
The key chain from Malaysia given to me by Emmanuel Delocado
110
When Emmanuel Delocado inquired on Bioluminescence a year earlier
he already had in mind a clear idea as to what he wants to do. He wanted to
use the energetic blue-green light to kill cancer cells. Indeed, I was skeptical
of the project’s success. I provided him the luminous microbes and he used
it the way he intended to. The rest was history. He came back to me with
several awards to be proud of. His group also won 2nd place in the Intel
Science and Engineering Fair at the National Level held at UP Diliman. They
went on to pursue biology at UP Diliman and Ateneo de Manila University.
The contest where the group of Emmanuel Delocado from the Manila Science High
School won with their project on the use of Vibrio fischeri
111
Participants’ pins for the Intel Science and Engineering Fair 2004 held in Portland,
Oregon given to me by the students of QueSci
A photograph taken in the dark of luminous agar plates of Vibrio fischeri USTCMS 1063 as single letter/number proclaiming the Quadricentennial celebration of UST in the year
2011. Picture was taken during the day of the launching of the University of Santo Tomas Collection of Microbial Strains (USTCMS) in the Thomas Aquinas Research Complex.
112
From all the years I had serving as supervising scientists to students coming
from various high schools , I can reckon that 9 groups in one way or another
were granted the privilege to work in the Microbiology Laboratory of the
Research Center for the Natural Sciences (RCNS). Seven made it to the
regional level of the Intel Science and Engineering Fair competition. Five
made it to the national level held for many years now at the Bahay ng
Alumni in UP Diliman and one made it to the US and won a grand award.
Below is a picture of the famous HP Garage bearing a historical marker with the inscription “Birthplace of “Silicon Valley” and meeting the winners of the prestigious US “Collegiate Inventors Contest”. From left D. Doshi (Univ. New Mexico), D. Fletcher (Stanford), S. Welz (Univ. Chicago), D. Perkins (Univ. Maryland), myself and M. Oddy (Stanford)
113
Chapter II: Of Light
A light shines in the darkness and the darkness overcame it not – New Testament
The nature and habitats of the marine luminous microbes
Bioluminescence is an awe-inspiring attribute manifested by a wide
array of terrestrial and marine organisms like fishes, squids, insects,
crustaceans, fungi, and microorganisms. It is more prevalent among the
marine organisms were it is employed to either attract mates and potential
preys or distract enemies and their predators. The species of the marine
luminous microbes are ubiquitously found in seawater as well as in
association with many of its inhabitants: plants and animals, of this vast
realm known as the hydrosphere. Indeed fishes, squids, crustaceans,
shellfishes, as well as seaweeds have been known to harbor luminous
microbes. The marine luminous species are classified into three microbial
groupings or genera: vibrio, photobacterium and alteromonas. However,
another microbial group Xenorhabdus, a genus whose member species is
Xenorhabdus luminescens, is not marine but terrestrial in nature. Terrestrial
means that the member species are found on land and infects a specific
type of nematode (worm). Strains that I have isolated and preserved in the
USTCMS are identified as species of Vibrio harveyi, Vibrio fischeri and
photobacterium leiognathi. Since these microbes are of marine origin, their
114
growth medium whether in solid or in liquid form must contain salt at a
concentration of three percent by weight of salt per volume of water. This
three percent concentration of salt required by the marine luminous
microbes simulates the salt content of seawater their main habitat. Without
the required amount of salt in their liquid world, these marine microbes will
die instantly. Plain water exerts a phenomenon known in biology as
hypoosmosis to the cells of these halophilic or salt-loving microbes.
Hypoosmosis simply means that there are more dissolved substances in the
liquid inside the cells than in the liquid outside the cells. This imbalance in
concentration of dissolved substance with more inside and less outside the
cell will cause water outside the cells to exert a net uni-directionally
movement into the cell causing it to swell gradually and subsequently to
burst and die. However, when salt is dissolve at a final concentration of
three percent in the liquid medium outside the cells where they are
suspended they neither swell nor shrink in size. This biological phenomenon
is known as isoosmosis indicating an equality in the amount of dissolved
substances found inside and outside the cell. Since the concentration is
equal, the amount of water entering the cell is equal to the amount of water
leaving the cell thereby achieving a state of structural equilibrium and viable
integrity for the cells. The net movement of water molecules into and out of
115
the cell is zero in isoosmosis. Cells in isoosmotic liquid medium live and
grow normally. The strict dependence on salt and the non-disease causing
nature of these luminous microbes, particularly Vibrio fischeri and
Photobacterium phosphoreum and Photobacterium leiognathi on humans
make them suitable and safe test microbes for many biological experiments
that can be developed into sound and original science projects. Washing
these microbes from the tap is enough to kill them thereby precluding their
ability to spread and multiply in the sewer system. Employing sound
microbiological pure culture techniques, it will be easy to isolate these
marine luminous microbes from squids and all salt-water fishes. The
brownish fecal contents of the intestines of fishes that can be squeezed out
easily by the fingers, the juice that readily drips out of the bodies of squids
and the sticky and slippery bodily surfaces of saltwater fishes are good and
rich sources of the luminous Vibrio and Photobacterium species.
How does Vibrio fischeri look like?
Vibrio fischeri and their kinds, the marine microbes, were most likely
one of the very first living organisms to debut on earth more than three and
half billion years ago. To get a very good picture of this enormously
116
incomprehensible time scale, I used the one-year analogy that I first learned
from Carl Sagan in his TV show: Cosmos. Let us compress the billions of
years that the microbes have been on earth in a span of one year and set
the time of the microbes’ emergence on earth at 12 o’clock AM of January
first New Year’s day. In this geological time-scale, the emergence of humans
is registered at 3 o’clock PM in the afternoon of December 31, on the last
day of the year. Microbes have been on earth from January to December
while humans have been on earth only in the last 9 hours of the year. In
short, based on this one-year scale, the microbes have been on earth much
longer than we humans have been on earth. Vibrio fischeri, like any other
microbe, leads a very simple life lacking all the complexities associated with
the structure and functions of higher plants and animals. Because of their
life’s simplicity, microbes are robust survivors. They have lived through
several waves of major extinctions and global destruction that struck planet
earth in its long history of evolutionary development. These cataclysmic
events wiped out ecosystems and communities of living organisms forever in
the past. The Vibrios are roughly short and bent rod-shaped single celled
organism with an outer envelope composed of fat-carbohydrate-protein
mixture. Vibrio fischeri has three to seven whip-like structures conspicuously
protruding out from one end of its body. These whip-like structures are
117
called “Flagella” which they use for moving rapidly like a torpedo in their
watery world. The rigid flagella rotate and they get propelled. Because of
these flagella Vibrio fischeri is one of the fastest swimming microbe known.
Inside their plump rod bag-shaped bodies are complex interacting biological
structures and many biomolecules that sustain life and subsequently
produces bright light. The whole cell converts large amount of biochemical
energy into radiant energy, causing it to light up like an incandescent bulb
whenever they acquire the critical population needed to express
bioluminescence. Bioluminescent cells consume a large percentage of its
ATP (Adenosine Triphosphate), an energy chemical currency, into radiant
energy. Hereditary units called genes are found inside their cells carrying the
genetic information coding for the very essence of their own biological
identity and the capability to reproduce and propagate their kind. This
biomolecule functioning as carrier and repository of genetic information is
called Deoxyribonucleic Acid or simply DNA. DNA is a very long fibrous
polymer made up of billions of building blocks called nucleotide. Genes or
coding portions of DNA are attached together in tandems separated by
segments of non-coding regions of DNA in highly coiled and tangled fibers
called chromosome. In addition to chromosomes found inside the cells,
there are other numerous specialized biomolecules called enzymes.
118
Enzymes perform specific functions like making DNA and proteins essential
for the maintenance of life. Enzymes are the protein engineers and builders
of the cell; they speed up the building up and breaking down of biological
structures. All the chemical reactions that take place inside the cell are
referred to as metabolism. Some chemical reactions are involved in the
breakdown of chemical compounds like simple sugars and fats to provide
energy. For instance, glucose is broken down in the presence of oxygen
primarily via a gradual combustion process to produce carbon dioxide,
water, and energy. The oxygen requiring process of the breakdown of
chemicals like sugars inside the cell is accompanied by the release of a
tremendous amount of energy. This breaking down of chemical substances
into simpler substances that usually provide energy is called catabolism
specifically respiration. On the other hand, some reactions that lead to the
building up of large substances like cellular proteins, nucleic acids,
carbohydrates, and fats comprising the very structure of the cell itself require
energy. This building up process that requires energy is called anabolism.
Catabolic reactions provide energy that fuels anabolic reactions to proceed
to completion. A tough external covering called the cell wall encloses
microbial cells. It protects and maintains the shape and integrity of the cell.
Located beneath this tough outer covering is a more fragile cell film called
119
the membrane. It defines and delineates the cell into the intra from the extra-
cellular region and is responsible for transporting inwardly the dissolved
nutrients like sugars and peptides that it meets in their liquid vicinity.
Microbial cells consume food for growth and reproduction and excrete out
waste products like organic acids and carbon dioxide. Organic acids and
carbon dioxide are the products of their metabolism.
Each cell shown below by a simple drawing reproduces by dividing into
two daughter cells after a time interval through a process called “binary
fission”. Each of the two daughter cells ultimately become parent cells
themselves dividing further again to give rise to four daughter cells and then
to eight and so on. The length of time required for a Vibrio fischeri cell to
reproduce itself, known as the “generation time”, is roughly twenty minutes
long. Before one knows it, a single cell will have given rise to more than a
million progeny cells after one day or twenty-four hours. It is vital that every
cell of Vibrio fischeri need to be placed in a three-percent saline to live well
and happily. This is due to the marine home of these microbes. If they are
suspended in water without substantial amount of table salt like tap water or
distilled water, they will die instantly. Without salt, water will swiftly enter
120
their cellular body causing it to swell like an inflating balloon and ultimately
bursting.
A Single Cell of Vibrio fischeri can have 3 to 7 prominent whip-like appendages called flagella used for locomotion
A photograph of a 1,000 times microscopic picture of the cells of Vibrio fischeri USTCMS
1063 shown as small blue oval-shaped structures occurring singly and some in pairs
121
Where is Vibrio fischeri found? The marine luminous microbes particularly Vibrio fischeri are
universally found in seawater where they exist in planktonic and free-living
forms. Planktonic means that the individual cells of the luminous microbes
move with the flow of the sea current and free-living means being able to live
on their own initiatives independent of the intervention of other organism.
Vibrio fischeri from its Binomial Latin name describes vibrio, a bent, short,
and rod-shaped microbe found on the external surfaces and in the intestines
of saltwater fishes, squids, and crustaceans. Thus, the second name fischeri
referring to the saltwater fishes. Even Hollywood’s Nemo should harbor
luminous microbes on its body and in its tummy. The marine luminous
microbes can establish various types of biological relationships with many
marine animals like mutualism, commensal, and parasitism.
Where my Vibrio fischeri USTCMS 1063 was isolated. Inky black liquids from squids
is a rich source of marine luminous microbes
122
Some luminous microbes have evolved to become the component of
the light organs of fishes and squids exhibiting shared benefits between fish
and microbe called mutualism. I remember a news article several years ago
in the Philippines reporting a group of fishermen from Negros Island seeing
large bright illuminations deep in the sea at night. Many of the fishermen
believed then that these undersea rapidly moving illuminations are UFOs or
more appropriately USOs (Unidentified Swimming Object). Only to be
explained rationally by marine biologists from the prestigious Silliman
University that these undersea illuminations seen at night are large schools
of lantern fishes. This fish possesses light organs scattered all over its body
and swimming together in large numbers called schools can give them
collective brightness in the sea. At night a school can shine brightly together
thereby producing large moving undersea illuminations.
American microbiologists discovered an astounding symbiotic
relationship between the luminous microbe Vibrio fischeri and the small
bobtail squid found in the Hawaiian seas. The luminous microbes get food
and shelter from the squid. On the other hand, the small and cute squid is
rendered invisible by its luminous microbial symbiont thus preventing it from
ending up in the belly of some huge hungry fishes. In the sea, the Bobtail
123
squid uses the symbiotic luminous microbes to illuminate its body when the
moon above is also bright. This ingeniously evasive action by the Bobtail
squid erases or cancels its own shadow completely and like Harry Potter
with his magical invisible cloak disappears from the sight of voracious
predators that may be swimming beneath it. Isn’t that neat; a squid that can
make itself invisible to hungry eyes! However, in the abysmal pitch-black
depths of the oceans of the world where the sperm whale battles the giant
squid the opposite effect occurs. National Geographic tells us that at this
depth where no light from the sun much less the moon ever reaches, the
location of the giant squid is rendered visible to the sperm whale by the light
that the squid emits through the associated marine luminous microbes.
Photo of the small and cute Hawaiian Bobtail Squid (From Internet:www.ou.edu.)
124
The luminous microbes are in significant quantity found as commensal
in the gills and on the skin of marine fishes where they become an integral
part of the existing surface layer of numerous microbial communities.
Commensalism is a relationship between two organisms in which one
derives advantages like food and shelter without harming or benefiting the
other. The marine luminous microbes are abundantly found in the intestinal
content of salt-water fishes comprising as much as 90% of the aerobic
microbial population. The microbes exist as commensals for the saltwater
fishes and other marine animals. This is exactly the main source of my
isolated luminous microbes when I first discovered them after I got back to
the Philippines from Germany. The juices, surface slime, and gut content of
saltwater fishes and squids are rich sources of the commensal marine
luminous microbes.
Some species of luminous microbes particularly Vibrio harveyi can be
parasitic on crustaceans. Parasitism is a relationship between two
organisms in which one gets the upper hand and benefits from it to the
detriment of the other. Vibrio harveyi, a cousin of Vibrio fischeri elaborates
chitinase a protein product that helps in attacking crustaceans like shrimps
and prawns. This serious disease inflicted on crustaceans is called luminous
125
vibriosis. Chitinase dissolves the hard and glistening chitinous outer
covering of crustaceans called exoskeleton allowing the microbe to gain
entry inside their body and start the process of infection. Luminous vibriosis
is a big problem for the prawn industry.
Marine animals like fishes, squids, and prawns are goldmine of the marine luminous microbes of which the Philippines and other coastal regions are abundantly blessed
Lastly, these microbes are also saprophytic meaning they clean up the
marine environment. Like many other microbes, they are involved in the
natural decomposition of organic matter and speed up the process of decay
thereby maintaining the cleanliness of the marine environment.
126
The Milky Sea
Seafarers of ancient times have reported rare phenomenon in which
large areas of the sea light up or glow brightly at night. Cruising through this
shining sea has been likened to being in the midst of a sea of white much
like milk that extends all the way to the horizons. The glow is actually blue
but may appear white to human eyes. Based on its whitish apparition, this
rare occurrence in the sea is known in scientific literature as the “Milky Sea”.
Marine luminous bacteria undergo a population explosion, a bloom, made
possible by still hitherto unknown physico-chemical conditions. When their
population becomes very dense, the individual cells shine by virtue of
“quorum sensing” and collectively the entire seawater as well. From a few
hundred cells per liter of non-milky seawater, the cell count can go up into
the trillions for the milky seawater. It must be a very exhilarating experience
to be inside a “Milky Sea” which even mentioned by Jules Verne in his
famous book: 20,000 Leagues Under the Sea.
Recently, Steven Miller of the U.S. Naval Research Laboratory was
able to confirm 1995 Satellite images taken by a merchant ship. U.S.
Defense Meteorological Satellite Program was that of a large milky area.
The Milky Sea, located off the coast of Somalia, covered an area of 15,400
square kilometer a little bigger than Samar Island. It brightly lit up this part of
127
the Arabian Sea for three consecutive nights in 25, 26 and 27 January of
1995. From my experience with luminous microbes, they will have to adhere
together on some solid surface. Immobilization causes them to shine much
longer than if they are suspended in seawater. But in all the times that I have
observed these luminous microbes in the dark, there was no instance when I
saw them shining white. All the luminous observations I made with them in
darkness even under scotopic vision they shined with a stable blue-green
light.
Cultivation of the luminous microbes on a solid culture medium
The use of a solid medium for the cultivation of microbes goes all the
way back to the pioneering effort of a famous German microbiologist and
Nobel prize winner named Robert Heinrich Koch in 1882. His techniques
and the lab glass wares he used are still very much utilized in microbiology
today. Koch’s significant contributions to microbiology and to modern
medicine have been recognized so well in Germany and in other countries
that streets, hospitals, and research institutions were named after him.
Microbes like humans need nutrients like soluble proteins and carbohydrates
to sustain growth and development. Growth for humans is associated with
128
an increase in size. Among microbes, it is manifested by an increase in
numbers or in population size over a period of time. Soups or broths are
prepared by boiling chunks of beef or pork or chicken in water resulting in a
liquid that is rich in soluble proteins, vitamins, and minerals. If soups or
broths are left unattended for long exposed to the air at room temperature
they will spoil rapidly. Spoilage indicates that microbes have gotten into the
soup from air-borne dusts or from the non-sterile bowl containing the soup
and have grown tremendously into the millions of microbial cells per drop of
the soup. The microbes have eaten up the nutrients in the soup and
subsequently secreted out waste substances into their surroundings. These
waste substances accumulate rapidly because of the booming population of
microbes causing the development of undesirable changes in the properties
of the soup. The soup after a day or two becomes turbid, smells foul, and
tastes bad, which are all signs of microbial contamination. In microbiology, a
solid medium is called “Nutrient Agar” and a liquid medium is called “Nutrient
Broth” which are commonly used for the cultivation of microbes. Nutrient
agar is composed of a broth and a solidifying agent, usually agar. Agar is a
complex carbohydrate extracted from the red marine seaweed that
possesses gelling and stabilizing property. It is employed in desserts to
prepare colorful and delicious solid matrices containing embedded pieces of
129
fruits. Nutrient agar is prepared as a hot yellowish liquid of broth that
hardens once it cools down. If marine luminous microbes are to be
cultivated, there is also a need to add sodium chloride at an amount of three
percent in the nutrient agar before they are allowed to solidify. Microbes
grow inside the nutrient agar or on the surface as visible round objects
called colonies. Traditionally soups or infusions of cow or pig’s meat, heart
and brain mixed with agar are the ones prepared as solid culture media
used for the cultivation of microorganisms. At present, microbes’ food has
become highly sophisticated and now comes in powdered dehydrated form
of many kinds for special purposes. The light yellowish powder of the
commercially prepared microbes’ food, looking much like infant formula,
must first be weighed, added to a specified volume of distilled water, heated
to dissolve and sterilized at high temperature. Sterilization kills
microorganisms that may have already been present in the nutrient medium
or may have entered it during the preparation method. After the sterilization
process, while still warm and in the liquid state, nutrient agar is poured into a
Petri dish. A Petri dish is an enclosing pair of glass dishes. The bottom
shallow glass dish is enclosed loosely by a cover glass dish. Once poured
inside the bottom glass dish, it is closed with the cover glass dish and the
culture medium inside is allowed to solidify as it cools down to room
130
temperature. The entire procedure in the preparation of culture media is
much like preparing fruit flavored gelatin. A fixed weight of the powdered
gelatin product contained in a foil sachet is added to a defined volume of
water; mixed by stirring; heated to boiling to dissolve the contents; poured
while still warm and liquid in specially shaped plastic containers called
moulds and allowed to solidify upon cooling.
Next step in the cultivation of microbes requires the use of a metal wire
called an “inoculating loop” or simply a “wire loop”. It is a nichrome or
platinum wire about a third of a foot long in which one end is closed into a
loop the size of the tip of a pen. From the opposite open end of the wire
loop, a metal rod is attached as a handle. This special device is used for
transferring microbes from their sources to any solid or liquid nutrient
medium. The “source” can be a culture medium already harboring trillions of
actively thriving microbes or it can be a soil suspension, saliva, mucus
secretions, or dirty water that is known to contain microbes by the millions.
For the marine luminous microbes, their “sources” are juices, the surface
slime, and the gut content of salt-water fishes.
A Wire Loop, with handle at its left end and a loop at its right end
131
While holding the handle of the wire loop much like holding a pen, the
other circular end called the “loop” is touched or dipped in materials called
“sources” deemed to contain large numbers of microbes. The loop now
containing millions of clinging microbial cells can be introduced inside a
nutrient agar plate. Removing the upper glass cover of the petri dish
exposes the solid nutrient agar inside the bottom glass dish. The loop by
now contains millions of clinging living microbial cells. It is then touch gently
on the surface of the exposed nutrient agar and at once streaked repeatedly
on the face or surface of the nutrient agar moving from side to side of the
glass dish until the entire surface has been streaked. After the streaking
procedure, the cover dish is used to close the petri dish. Covering the petri
dish ensures that unwanted microbial contaminant from the immediate
surroundings like air dusts does not get inside the petri dish where the
nutrient medium is found. Using the method above, called pure culture
technique, assures that only the microbes coming from the wire loop will be
the ones that will grow inside the petri dish.
Looking carefully on the nutrient agar inside the petri dish, no noticeable
change on the surface of the nutrient agar after the streaking procedure can
be seen. But there are actually several thousands of microbial cells
132
scattered on the surface of the nutrient agar that are simply too small to be
seen. But given the time for these single cells scattered on the nutrient
agar’s surface to grow and multiply to two cells and the two to four cells and
then to eight cells and so on. A single isolated cell, after a day or two, would
have given rise to millions of daughter cells all piled up together into a visible
mass called a “colony”. For the luminous microbes, each of the small round
colonies will be glowing bluish-green in the dark due to bioluminescence. A
period of one to two days is just right length of time for these colonies to
render themselves visible to the unaided human eye. All the millions of cells
found in a single colony are “clones”, “replicas”, or “mirror images” of the
very first single progenitor cell that gave rise to all of them by continuous and
rapid binary fissions. A cell undergoing binary fission enlarges and then split
into two cells. We really cannot view each cell of the millions of cells making
up a colony or a population called “culture” as truly distinct like each human
on this planet is unique. The individual identity of a single microbial cell
simply does not exist since the cells making up a population are all identical
to one another. It therefore makes the entire population act as one and
renders it as a single organismic entity. Most microbes are prolific; they can
grow very fast in a short period. Vibrio natriegens, a close non-luminous
cousin of Vibrio fischeri has a generation time or doubling time of only ten
133
minutes. Compare that to the human’s generation time or doubling time
measured in years. A microbe’s generation time is more than a million times
faster than human’s generation time. This short generation time allows them
to mutate or change themselves faster allowing them greater flexibility in
adapting to any alterations in their environmental conditions. And adapting
they did! Not only have microbes persisted for billions of years; they even
survived the cataclysmic events in earth’s history that have caused waves of
large-scale extinction of plant and animal life. Today, man’s activities have
caused the extinction of many animals like the Dodo and the Tasmanian
Tiger but has not yet caused the extinction of any living microbe. The world
would probably be a better place if the extinction of microbes like
Mycobacterium tuberculosis or Porphyromonas gingivalis can ever be
achieved.
Photograph of numerous brightly luminous colonies of Vibrio fischeri growing on
nutrient agar inside a Petri dish against the slit of a door
134
Cultivation of the luminous microbes in a liquid culture medium A liquid culture medium is a soup or broth that retains its fluidity even at
room temperature simply because no solidifying agent is added to it. The
wire loop having millions of clinging microbes coming from a “source” is
simply dip into the liquid culture medium contained in a glass vessel like a
test tube or a flask. Once dipped, the cells from the loop are dispersed and
suspended in the fresh liquid nutrient medium where they can grow and
multiply. Right after dipping the loop in the broth you will not see any
observable change in the liquid. But again given the time, the initially
introduced cells in the culture medium grow and multiply into the trillions.
After a day, the liquid will turn cloudy and turbid indicating the presence of
millions of microbes per drop of the liquid. Once again, if the cells are of the
marine luminous microbes, salt will have to be added to the medium.
Growing to large a population will ultimately cause the medium containing
the marine luminous microbes to instantly shine bluish-green in the dark
after several hours.
135
Microbes that can talk with one another
Individual cells of a population of Vibrio fischeri are able to communicate
with one another by using a chemical signal. Microbiologists have isolated
and identified the chemical signal that luminous microbes used for cell to cell
communication as “homoserine lactone”. This phenomenon is called
“Quorum Sensing”. Individual cells are able to know or sense how big their
population is; based on the amount of the homoserine lactone that is found
within their immediate vicinity. When the cells of luminous microbes are
introduced into a fresh nutrient medium, the individual cells that are found in
small number don’t light up just yet. The cells are all dim and are spaced far
apart from one another. But as the cells reproduce and the population
rapidly increases in number, a point is reached wherein all the cells light up
at the same time causing the population as a whole to shine brightly. It is
much like a star that explodes radiantly into a supernova outshining the
entire galaxy where it is located. It has been estimated by microbiologist that
this critical mass of cells that must be attained to make the entire population
shine brightly is about 50,000,000 cells per drop of the liquid containing
them. That is indeed an astronomical number!
136
For us humans we are able to know how many we are in a place by
talking to one another and doing a roll call. For the luminous microbes they
are able to know their population size or sense their quorum by talking to
one another through their unique chemical signal. The concentration of the
signal homoserine lactone in the liquid medium accumulates and goes up
when the population increases in size as well. The larger the population, the
more cells are present all producing and secreting the chemical signal into
their surrounding environment. The space between cells becomes smaller
as they increase in number and the concentration of homoserine lactone
goes up as well since more cells are making it and secreting it out of their
cellular bodies. The rising concentration of the chemical signal in the liquid
medium will soon bathe each of the millions of cells making up the
population, ultimately bringing about the “switching on” of special group of
genes in their chromosomes called “lux” found inside each cell. Lux is an
appropriate term since in Latin it means Light. The high concentration of
Homoserine Lactone in the medium will cause it to enter each cell in the
population at the same time and switch on the lux genes of individual cells
causing them to instantly shine brightly. It is much like switching on the lights
of the large and tall Christmas tree in the Big Apple, all the bulbs lighting up
137
at the same time once it gets plugged in. The lux genes found in each
luminous microbial cell govern the expression of the bioluminescence trait.
The identification of the marine luminous microbes
As part of my research work in UST, I am constantly isolating more
strains of marine luminous microbes. The luminous microbial holdings of the
USTCMS will continue to increase and indeed boasts of being the biggest in
the Philippines. Classification and identification of the marine luminous
microbes and other microbes require isolating them in pure form and
subjecting them through a series of scientific investigative process resulting
in the assignment of new binomial names to each of them. This investigative
process studies the characteristics or attributes of the microbial isolates in
terms of their microscopic appearance, appearance as colonies, kinds of
nutrient on which they can grow, physico-chemical conditions affecting their
growth, innate abilities to transform specific chemicals, information found in
their DNA and their habitats. Results for unknown microbial isolates are
compared with that of a known or a previously identified microbial species as
well as information contained in an authoritative book called the “Bergey’s
Manual of Determinative Bacteriology”. High degree of similarities in the
138
studied characteristics obtained for an unknown isolate with a known
microbial strain indicates that they are of the same species. However,
dissimilar characteristics between unknown and known microbial strains are
indicative that they are not the same and therefore belong to different
species. If an unknown isolate have characteristics that does not jibe with a
known strain that has been reported in scientific journals and technical
books then the unknown strain must be a new or a novel species. Finding a
novel species is actually like having discovered a new form of life much like
discovering a new plant or animal. In biology, the discoverer has the
privilege and rights to give the novel species its new name, a binomial name
composed of a genus and species by which it is made known to all. Relying
on the very rich diversity of marine living organisms found in our country, it is
indeed a matter of time before novel indigenous species of marine luminous
microbes will have been discovered. If I were to fine one, I will surely name it
Vibrio manilanensis or Vibrio philippinensis in honor of our country!
Application of a holistic approach on identification, like polyphasic
characterizations coupled with the information that can be derive from the
16S rDNA sequencing should facilitate the attainment of this noble goal.
Known luminous species in the genus vibrio are Vibrio fischeri, Vibrio
139
harveyi,Vibrio logei and Vibrio vulnificus. From the genus photobacterium,
Photobacterium phosphoreum and Photobacterium leiognathi are luminous.
The chemistry behind bioluminescence
Microbial bioluminescence rendered visible as shining colonies in agar
plates or as glowing liquid broth culture is truly the most awe-inspiring
phenomenon that microbiology can unravel before human eyes. Imagine
living organisms generating their own cold bright bluish-green light in the
dark. All life processes are possible and sustained by getting energized
electrons in molecules found in foods called carbohydrates. These
energized electrons from carbohydrates are brought inside the cell and once
inside, the energy that they possess is removed causing them to be de-
energized. The energy that removed from the electrons is needed to sustain
and maintain the life of all cells. Living organisms will die if not provided with
these energized electrons into their cells, which come only from the foods
consumed. Carbohydrates are describe as the main “source of energy” for
living organisms precisely because they are the major source of energized
electrons. These substances are also the most abundant type of food found
in the biosphere. However, on a weight-to-weight basis, fats and oils contain
140
far more energized electrons than carbohydrates. In all living systems, the
energy removed from the energized or activated electrons is use to produce
a universally known energy molecule known simply as “ATP”. ATP is a
versatile molecule that can be used to either withdraw energy from when
needed or store energy into when there is an abundance of it. It is like
money that can be withdrawn from banks when needed and can be
deposited in banks if there is a surplus.
The movement of the energized electrons in the process of light
production (bioluminescence) is actually an alternative pathway or a detour
(Shunt) of the electron transport chain or respiratory chain. This light-
generating metabolic process is the responsibility of the enzyme Luciferase,
an enzyme present as well in other bioluminescent organisms like firefly.
Luciferase accepts activated electrons from a special molecule coming from
their food. The energized electrons instead of being passed on to the
“cytochromes” the usual terminal route of the traffic of the energized
electrons in respiration, are instead rerouted to the enzyme Luciferase,
which orchestrates the bioluminescence reactions. Luciferase carrying the
highly energized electrons passes them on to oxygen, where the energized
electrons are stripped of their energy thus ending up as low energy electrons
141
bound tightly in water. The energy difference between the initial energized
electrons and the final de-energized electrons is emitted as the cold bluish-
green light that is visible as bioluminescence, the “gift of light”.
Humans also get energy in practically the same way as described
previously for the luminous microbes. Electrons coming from high calorie
foods like sugars, bread and French fries are in the activated or energized
state. Once these foods are eaten the activated electrons are transported
inside our cells by special molecules where they are then stripped of the
energy they carry. The energy removed from them is utilized to power life
processes like thinking, walking, talking, and breathing. The once energized
electrons, now devoid of energy, go down to a lower energy level and taken
up by oxygen that ends up as water produced in cells. To prevent the gain of
weight through the consumption of large quantities of fats and
carbohydrates, one can burn off these excess calories through rigorous
workouts performed called aerobic exercises. Exertion is a form of burning
off the excess calories or chemical energy through heavy aerobic or oxygen-
requiring workouts. Breathing gets deeper because of the need for more
oxygen from air by our respiring cells. Heat is produce in our body at the
peak of these strenuous exercises resulting in the production of massive
142
amount of perspiration. This oxygen-requiring metabolic process of burning-
off excess calories is called “Respiration”. Water comes out of our skin
dripping profusely and evaporates rapidly into the surrounding air taking with
it the excess heat that the body has produced. This cooling effect is
important for human survival for without perspiration and water evaporation;
heat is trap in our body causing body temperature to rise rapidly to
dangerous levels causing irreparable damage to tissues and organs.
Respiration is similar to bioluminescence; both processes need oxygen to
extract the energy from electrons present in high energy or calorie
molecules. However, in respiration, the energy obtained from the electrons
is released as heat while in bioluminescence, the energy is released in the
form of visible light. In physics, light propagates itself through space in the
form of waves. The length of a wave from crest to crest is its wavelength and
wavelength determines the color of the light that humans can see. The light
produced by luminous microbes has a wavelength of approximately 490 nm
making the radiation appear bluish-green to our sight. The abbreviation “nm”
stands for nanometer, which is a very small unit of measurement. A
nanometer is a unit of measurement for length; a ruler for instance contains
305,000,000 nanometers. A light with a wavelength of 410 nm will appear
143
violet and 700 nm appears red in color. The overall reaction for the
bioluminescence of the marine microbes is shown as follows:
Reduced Flavin Mononucleotide (RFM) + Oxygen + Long Chain Aldehyde (LCA)
Oxidized Flavin Mononucleotide (OFM) + water + Long Chain Fatty Acid (LCFA) The bioluminescence reaction is not complicated at all; it can just so
easily be likened to a game of basketball. The terms above, in the first row,
are the players in a basketball game and the ball is the electron. The
energized electron or the ball initially comes from LCA, a high-energy
biomolecule. LCA then passed on the ball to RFM. RFM accepts the ball
and passes it on further to oxygen. Oxygen accepts the ball and holds on to
it permanently. In the process, oxygen is immediately converted to water
located in the second row. The electron in water’s possession is in the de-
energized form. The energy removed from the initially energized electron is
emitted as the visible cold bluish-green light of bioluminescence. LCA is
converted to LCFA as it gives up the electron to RFM. And RFM is
converted to OFM as it loses the electron when it passes on the ball to
oxygen. Oxygen is the final electron acceptor and it gets transformed
ENERGY (LIGHT)
144
immediately into water. The electron now deprived of its energy ends lockup
in water. The LCFA can be broken down into several two-carbon long
chemical compounds by a special pathway known in biochemistry as beta-
oxidation. The two-carbon long compounds can be fed into a terminal cyclic
oxidative pathway called the Kreb’s Cycle in biochemistry where it is
converted to carbon dioxide and water and the production of more energy in
the form of ATP.
Focus your attention on oxygen and water (bold terms), it can be seen
that bioluminescence, the generation of light, depends on the availability of
oxygen. If the amount of oxygen in the reaction is restricted compared to the
other the upper row chemical reactants, then the bioluminescence reaction
can be made to measure the amount of available oxygen in a liquid medium
where the luminous microbes are found. More oxygen translates into longer
sustained periods of bioluminescence and less oxygen means shorter
periods of bioluminescence. This is the principle of the award-winning
project of the QueSci students.
Vibrio fischeri and the other microbes made their debut on earth billions
of years ago, three and a half to be exact, in a world that is totally different
from what it is now. They were the very first living organism that appeared
145
on earth at the dawn of time; only after around a billion years after earth was
formed as a planet in its fiery cauldron of birth. The microbes were very
much ahead of the emergence of the dinosaurs, the fishes, the plants, the
trilobites and all the other groups of living organisms existing today. The
world in which they originally found themselves is a world totally devoid of
oxygen where processes so seemingly natural as burning and combustion
cannot and do not happen at all. Fire simply does not exist. The absence of
oxygen in this primeval world was created by the massive geological
upheaval that imprisoned gaseous oxygen. Oxygen was bound forcefully in
gaseous compounds in the atmosphere, in liquid water with hydrogen and
in the solid form of hard rocks and minerals that form the earth’s crust. An
atmosphere without gaseous molecular oxygen thus tenaciously acquired
the initial stronghold and blanketed the world with such endurance that it
lasted for something like 80% of earth’s history. Using Carl Sagan’s analogy
of compressing in a one-year period, the history of planet earth, then that
would mean that the atmosphere was devoid of oxygen (anaerobic) from
January to October and attained its present aerobic condition only in
November to December. What caused the atmosphere to change abruptly
on a global scale? Something momentous must have occurred that altered
the course of planetary evolution forever. Some of Vibrio fischeri’s relative
146
microbes were able to develop and acquire a green, light-reactive pigment.
The green pigment enabled them to become the first producers; capturing
energy from sunlight in an instant and utilizing it to convert a gas carbon
dioxide and water into carbohydrates, food for all. That is the main reaction.
However, a side reaction was able to split water into hydrogen and oxygen.
This made the big difference in atmospheric evolution. In short, this special
group of microbes was able to invent an essential biological process called
“photosynthesis”. Photosynthesis enabled the chlorophyllous
microorganisms to virtually conquer the planet and vanquished the other
microbes simply by religiously producing oxygen. Microbes that remained
strict anaerobes cannot tolerate oxygen and were killed by its accumulation
in the atmosphere. The photosynthetic microbes are known as the “Blue-
green Algae”, “Blue-green bacteria” or “Cyanobacteria” and from them
descended more complex type of algae and ultimately all modern plants
existing today. A whole new kingdom was born, the realm of the green
whose members possess the light-sensitive pigment - chlorophyll. The
chlorophyllous organisms performed photosynthesis with fidelity for billions
of years of their existence on this planet changing the world completely and
eventually setting the stage for the emergence of another Kingdom - the
animals. Oxygen accumulated in the atmosphere converting it from being
147
anaerobic to aerobic. Ozone, one of the chemical manifestations of oxygen,
was formed high up in the sky shielding the earth from harmful
extraterrestrial radiation. This important shield paved the way for the
eventual colonization of the land by living organisms. Life, which originated
in the sea finally ventured into land and it started an entirely new way of
living on the continents. Life previously marine has now become terrestrial.
Fire too finally showed up in the world; a sign that the processes of burning
and combustion are now possible. For the realm of the tiny, the microbes,
this is a huge change to contend with. Some of them adapted by becoming
aerobic organisms and those that cannot tolerate oxygen, the strictly
anaerobic microbes were almost wiped out from the face of the planet
surviving today only in habitats totally devoid of oxygen. These anoxic
environments are found deep in the mud and sediments, in the great depths
of the ocean floors and would you believe inside the animal’s intestinal tract.
Vibrio fischeri must have been a survivor; it developed the ability to grow in
the presence of oxygen. In microbiological term, it is “Facultative” meaning it
can grow in the presence or absence of oxygen. In a world that has gone
aerobic Vibrio fischeri was able to produce something wonderful, the “gift of
light”. Indeed some scientists believe that the process of bioluminescence is
Vibrio fischeri’s way of removing oxygen from the atmosphere. If it were true,
148
it is a huge exercise in futility but a beautiful one at that! Bioluminescence is
like burning or combustion in producing light and radiance but unlike burning
or combustion almost no heat is produced. Bioluminescence can be likened
to a special fire giving off light but does not generate heat, a cold fire!
The bluish-green wavelength of bioluminescence carries more energy
than green, yellow, orange, and red wavelengths. It can be concluded that
since the conversion of chemical energy to light energy in bioluminescence
is highly conserved, so much so that very little of the transformed energy is
wasted as heat thereby highly energetic blue-green light is generated. Since
the blue-green light can travel farthest under the sea and can therefore
reach as many eyes as possible compared with other visible colors, then
evolution thus favored a bluish-green color for bioluminescence as a means
of signaling for the marine denizens. Many scientists had surmised that the
marine luminous microbes played a significant role in the evolution of the
vertebrate eyes and therefore of vision. Since most of the lower animal life
forms are from the sea, a molecule that can respond sensitively to
bioluminescence must have first evolved. This light-sensitive molecule was
later on incorporated in special light sensitive organs called “Eye”. Eyes in
higher life forms can then generate visions. Sea creatures, great, and small,
149
in the unbelievable vastness and depths of the world’s oceans used
bioluminescence as the major means of communication. It is the main
reason why the pictures or films of the marine world appear in shades of
blue allowing blue-green light to be absorbed less and to travel far
distances.
Hand grasping a flask containing billions of happily shining cell
suspension of Vibrio fischeri
References: Jones, B.W. and M.K. Nishiguchi. “Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda), Marine Biology 2004
Kaplan, H.B. and E P Greenberg (1985) Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system. J Bacteriol. 163(3): 1210–1214.
150
Nealson, K. and Hastings, J.W. (1991) The luminous bacteria. In: The Prokaryotes 2nd Edition (A. Balows, H.G. Trüper, M. Dworkin, W. Harder and K.H. Schleifer, eds.) pp. 625-639, Volume I, Part 2, Ch. 25. (Springer-Verlag, NY)
“The emergence of complex life”. www.globalchange.umich.edu/globalchange1/current/lectures/complex_life/complex_life.html
151
Chapter III: Of Vision
His eyes were like fireballs, fearfully blazing – Cats
Catty, the praying tabby Cat
The Eye Vision is a complex reaction of life processes triggered by light that
enables us to perceive and interpret the world around us in full living colors.
Science tells us that the stimulus that ultimately creates vision comes to us
in the form of light particles called photons. A photon is a particle of light
that carries a fixed package of energy. Countless numbers of photon come
152
from outside and enters the eye through the cornea containing a special lens
for focusing them. They then continue traveling by traversing the main
internal transparent gel-like portion of the eye known as the vitreous body.
The photons finally end their journey when they hit the back portion of the
eye known as the optic part of the retina. The retina contains hundreds of
millions of photoreceptive cells, the rods and cones, which interact
sensitively with light. Photons hit a special biomolecule called rhodopsin
found inside the eye’s photoreceptor cells and transfers its fixed package of
energy to it resulting in a cascade of chemical reactions that generates a
nerve impulse. The nerve impulse, much like an electric current, travels
along the optic nerve bringing it ultimately to the brain. The optic nerve is like
a copper wire connecting the eye and the brain. Eventually, the swiftly
traveling nerve impulse reaches the brain creating a sensation in the mind
known as vision. During daytime or in well-lit conditions the cones are
activated and function in receiving signals resulting in bright or photopic
vision. However, in the dark, the cones lose their functions to very dim light
and the rods take over. Activating the rods results in another kind of vision, a
vision called scotopic or dark vision. Dark vision is not well known since one
does not normally find himself in a dark environment completely devoid of
light. The use of artificial lights of various kinds: incandescent and
153
fluorescent, powered by electrical energy has brightened up the
surroundings even during the night that caused photopic vision to still be
employed by the eyes. This might eventually cause scotopic or dark vision to
be relegated into permanent disuse and once a biological structure or
function is rendered useless, evolution will eventually remove it completely.
Many times in the past when people discover new, isolated ecosystems
found deep underground where not a single photon of light can ever enter
penetrate, creatures previously with eyes that get trapped in this dark world
loses their sight and vision. Their useless eyes degenerate into non-
functional or vestigial organs eventually making them completely blind.
Evolution directs this course of development since there is no use eyes in a
world without light.
The luminous marine microbes must have played a leading role in the
emergence and evolution of the first eye since they have been producing
light in the realm of the earth’s vast hydrosphere even before the first animal
appeared on earth. Since all life in general and animal life in particular first
emerged in the sea, the first eye must have been biologically developed to
respond to the bluish-green color of microbial bioluminescence. In its most
primitive form or first step in the evolution of vision, a molecule must be
154
developed inside a special cell of an organism that can alter its shape when
exposed to the light of bioluminescence. This special cell must have been
incorporated as part of a system of cells or an organ, which ultimately
acquired the form of an eye. Thus Vibrio fischeri must have been a crucial
participant in the evolution of eyes and vision.
Why do humans possess scotopic or dark vision that responds most
sensitively to the bluish-green color of bioluminescence when we are
terrestrial dwellers not marine? Remember that bioluminescence is the
major means of communication by the denizens of the marine world. The
answer was simply there all along; it wonderfully verifies the very essence,
the very foundation of biology itself – evolution. Scotopic vision in humans is
the most likely remnant of the vision that our great great fore creature had
used in the darker marine world five hundred million years ago. This first
period in the first and longest of three major biogeologic time scales known
as the Paleozoic Era or “ancient life” is called the Cambrian period. It is the
time when the first fish appeared and existed as the dominant and most
intelligent creature of that time. This fishy forefather left the sea and became
a land dweller roughly four hundred million years ago, scotopic vision must
have been relegated to secondary status by evolution with another type of
vision called photopic vision. Photopic vision is suitable in a terrestrial world,
155
out of the sea, where it is much brighter compared to the dimmer marine
world where scotopic vision is preferred.
Scotopic vision is what I used in observing the dimmer manifestations
of microbial bioluminescence specially the light coming from PIBiT. Scotopic
vision requires absolute darkness and so there is a need to stay long in a
dark room. Upon entering a dark room one does not immediately see some
of the dimmer manifestations of microbial bioluminescence because it takes
time for the eye to switch from photopic to scotopic vision. A period of
around fifteen to twenty minutes of immersing oneself in absolute darkness
called dark adaptation.
The standard microbial bioluminescence toxicity test
The cold bluish-green light generated by the various species of marine
luminous microbes like Vibrio fischeri, Vibrio harveyi, Photobacterium
phosphoreum and Photobacterium leiognathi, is a visually observable and
measurable biological property known as bioluminescence. Since
bioluminescence is the product of a stable and complex metabolic activity, it
lends itself well as a highly accurate and sensitive indicator of the well being
and viability of the luminous microbes. If the luminous microbes are happy
156
and are in a state of maximum well being; they can tell us by shining brightly
over time. But if they are sad and their state of well being is low their light
dims rapidly and even fades out to the point of complete inhibition. Any
physical agent like heat and toxic chemicals that can damage the integrity of
the cell will cause partial or complete inhibition of their light output. The rapid
dimming of the luminous cells exposed to inhibitory compounds usually
takes place almost instantly from a few seconds to several minutes to an
hour or more depending on the degree of toxicity and the concentration of
the chemical agent.
The conventional method of the microbial bioluminescence toxicity test
in the evaluation of the toxic nature of a water sample if it is safe or unsafe,
is to add the right amount of luminous microbes in the water sample itself.
Before adding the luminous microbes in the water sample to be tested, it
must first be made three-percent saline by adding the right amount of table
salt. This is required since the luminous microbes are of marine origin;
without the required amount of salt, the luminous microbes will die instantly.
The water sample to be tested is placed inside a small glass test tube, which
glows bluish-green upon the addition of luminous microbes. Another tube
contains the negative control. The negative control simply contains clean
157
and pure water. Luminous microbes are likewise added in the negative
control. The two tubes, one bearing the water sample and one bearing the
negative control, are both inserted into the luminometer side by side. A
luminometer is an instrument that measures quantitatively the luminance of
the two tubes. There are two possible outcomes from this toxicity test: the
luminance of the water sample fades rapidly as compared with the
luminance of the negative control after 30 minutes to an hour. An alternative
result is when the luminance of the water sample does not fade and remains
just as bright as the luminance of the negative control after 30 minutes to an
hour. If there is a big difference between the luminance of the water sample
and the negative control with the water sample much dimmer than the
negative control, then the water sample is dirty and unsafe to drink it
contains something toxic. If there is no difference between the luminance of
the water sample and the negative control with the water sample persisting
just as bright as the negative control then the water sample is as clean and
pure as the negative control. The water sample does not contain any toxic
agent. The presence of toxic substances in the water sample harms the
luminous microbes causing them to decrease and at times even shut down
completely their light production. Any unknown compound or substance,
158
whose toxicity is suspected, can likewise easily be verified and evaluated
using this method.
Outcome of a Clean and Pure Water Sample Contained in Tubes
After half to one hour
Water Negative Water Negative Sample Control Sample Control
Outcome of a Dirty and Unsafe Water Sample Contained in Tubes
After half to one hour
Water Negative Water Negative Sample Control Sample Control
N.B. Yellow means sustained luminance; gray means decreased luminance
The luminance of the negative control should persist brightly over a
period of time since it contains pure water with no substance to harm the
luminous microbes. It is the luminance of the water sample that will either
persist or decrease. That is the simplicity of the bioluminescence toxicity test
159
making it very useful and practical. The only stumbling block for its
widespread use in the developing countries is the very expensive
luminometer. A luminometer costs approximately one and a half million
pesos depending on the brand.
The Bioluminescence oxygen demand (BiOD) for water pollution
Water pollution is the undesirable change in the quality of water due to
human or non-human activities. Polluted water can exert irreparable damage
the ecosystem. The quality of water is measured based on the following
parameters: pH, dissolved oxygen, temperature, and dissolved chemical
substances. The pH of non-polluted water usually ranges from 6 to 9.
Dissolved oxygen (DO) an important parameter that measures the amount
of oxygen in mom-polluted water is usually 6 ppm. DO support aquatic
animal life like arthropods and fishes. Should DO fall much lower than 6
ppm, animal life dies due to insufficient oxygen. An aquarium needs
continuous aeration to increase DO and thereby providing ample amount of
oxygen for the resident fishes. If the aeration is stopped, then not enough
oxygen can be provided to the resident fishes and soon they die.
Temperature is also a factor in determining water pollution. High
160
temperature can cause the amount of dissolved oxygen to decrease.
Dissolved chemicals like magnesium, calcium, sulfate, bicarbonate, chloride
and others determine water hardness. Water hardness if high can affect the
ability of soap to foam. Bacterial bioluminescence is also affected by pH,
oxygen and temperature. So theoretically, bioluminescence be used to
evaluate the quality of the water sample, its ability to sustain aquatic life,
based on the response of the luminous microbes in it. Once again, salt has
to be dissolved in the wastewater samples to a 3 percent solution contained
in test tube before the addition of the luminous microbes. Brightly shining
blue-green colonies can easily be scraped off from the nutrient agar plates in
a small volume of 3 percent saline to make a heavy suspension. These
brightly shining suspensions are then added to wastewater samples
contained in screw capped test tubes. After capping and inverting the tube
twice to homogenize the distribution of the luminous cells, the entire liquid
shines bluish-green in the dark. However, the bioluminescence after a while
breaks apart, fades and blacks out completely except for a thin zone of
bioluminescence that remains on the surface of the liquid. Tube
bioluminescence will fade once dissolved oxygen in the liquid (wastewater
sample) is depleted. The longer the length of time of the bioluminescence in
the tube, the less polluted the water is. The shorter the length of time of the
161
bioluminescence in the tube, the more polluted it is. Bioluminescence like
respiration is a biological process that needs oxygen. So if there is little of
dissolved oxygen in the water, indicative of heavy pollution, the shorter is the
length of time to sustain. The tube bioluminescence fades swiftly to the point
of blacking out completely. Below is a picture of homogeneous tube
bioluminescence except for the second tube from left that is already in the
process of blacking out. Using BiOD, it is hereby proposed that a
wastewater sample blacking out, undergoing tube luminescence extinction,
in 90 minutes and less is classified as heavily polluted. A tube luminescence
extinction of more than 90 to 120 minutes for a water sample tested with
BiOD is classified as moderately polluted and a period of tube luminescence
extinction of greater than 120 minutes is non-polluted.
The tube bioluminescence extinction technology (TuBET)
This innovation making used of bioluminescence in test tubes for
various assays is given the name “Tube Bioluminescence Extinction
Technology” or in short “TuBET”. Tubet can be used to determine the
“freshness” of various food samples like milk, pork products, beef products,
chicken products, sea foods, and many dried foods. Since freshness
connotes cleanliness and something that has not yet undergone degradation
or decomposition then bioluminescence conducted in test tubes can be
162
employed for its evaluation. Meat and milk products that have undergone
significant stages of degradation due to autolysis or microbial action
generate volatile amines resulting from protein breakdown. The presence of
large amounts of these biogenic amines in meat products due to extensive
autolysis and microbial activity put into question the “freshness” of the food
samples and concomitantly its safety. It is primarily the reason why solutions
or suspensions of meat products become alkaline when they undergo
microbial spoilage. Bacteria break down proteins to produce these offensive
smelling volatile amines like putrescine and cadaverine. Meat coming from
double dead animals are specially worrisome since undesirable changes in
its quality set in immediately at the time of death. Since TuBET is very
sensitive to high bacterial load and the biogenic amines, it can readily be
used for the evaluation of “freshness” of the samples. Saline washings of
meat and other food samples that yield short periods of extinction using
TuBET would indicate a condition of high bacterial load and high biogenic
amines, which reflects the unhygienic and dirty state of the tested samples.
Parameters and standards of clean and acceptable food quality can be
based on sound investigations using TuBET. Furthermore, the use of TuBET
should make the analyses of food for “freshness” faster and cheaper but
without sacrificing assay sensitivity.
163
From left, tubes 1, 3, and 4 still exhibit homogeneous tube luminescence while tube 2 has undergone extinction (black out). Take note of the bright thin layer of luminescence at the
surface is due to an abundant supply of oxygen from the air-space.
Aside from a novel parameter for the classification of water pollution
using TuBET, other applications include rapid determination of the bacterial
load of and sterilized quality of pasteurized and UHT treated liquid milk,
chocolate drinks, and other non-acidic beverages. I never thought before
that a brightly shining suspension of the luminous microbes can make
opaque drinks like milk and chocolates shine in the dark. Indeed
bioluminescence carries much energy that it can make plenty of liquids
shine in darkness.
164
Tubes containing UHT-treated and pasteurized milk mixed with a suspension of
luminous microbes. The 3 tubes from left have undergone bioluminescence extinction and the 4th tube from left is starting to “black out”.
Preparation of small paper-discs Marine luminous microbes have been found to adhere readily on
practically any solid surface of various compositions like glass, plastic,
metal, and paper. They have an outer covering that is rich in carbohydrates
and fats making it quite sticky. Once the luminous microbes are rendered
motionless on a solid material many useful and amazing things can be done
with them. Paper among other materials holds the promise of availability and
ease of use for immobilizing the luminous microbes. Whatmann filter papers
165
have been found to be suitable for this purpose. Whatmann is a special type
of paper that is sold in the market used by wet laboratory researchers and
analysts. Whatmann filter paper comes in different grades and each grade
exhibits specific properties for thickness, porosity, etc required for a scientific
investigation. I used Whatmann grade one filter paper, the most commonly
employed grade that comes in large white sheets the size of a gift-wrapping
paper. I then cut it into even smaller sheets the size of a table napkin, which
is subsequently punched out into small discs using an ordinary office paper
puncher. The “punch out” paper discs have a diameter of roughly six
millimeter, which were all collected in a petri dish.
How to make the paper-discs bioluminous
It is very easy to make these paper-discs bioluminous. A species of
Vibrio fischeri USTCMS 1063, is sensitive to toxicants and also shine
brightly over long period. This strain with an accession number 1063 was
isolated from squids (Loligo sp.) was bought at the Trabaho Market near
UST. It was preserved and maintained in the USTCMS (University of Santo
Tomas Collection of Microbial Strains) and was used for the innovative
paper-disc immobilized bioluminescence technology (PIBIT). Vibrio fischeri
166
USTCMS 1063 was streaked in a nutrient agar plates. After the streaking
procedure, the plates were incubated at room temperature in a locker for
about fifteen hours. After the incubation period, the plates were examined for
luxuriant growth as shown by the presence of numerous brightly shining
colonies in the dark.
Three percent saline was prepared by dissolving three grams of table
salt in approximately two cups of distilled water. A little volume of the saline
was poured into the bottom dish containing the nutrient agar. Saline was
poured on the brightly shining colonies of Vibrio fischeri just enough to flood
the surface of the agar. Using a wire loop, the colonies were gently scraped
off from the surface of the agar medium. This was done easily by using the
usual streaking procedure described previously. The heavily turbid saline
suspension was poured into a bigger glass flask. The suspension in the flask
was vigorously shaken by hand for about a minute to homogenize it. It was
then examined in the dark for its bright bioluminescence. In darkness, the
liquid inside the flask should glow brightly bluish-green as though it were
radioactive.
167
A brightly shining suspension of luminous microbes
The turbidity of the cell suspension was then adjusted according to the
level of turbidity of a McFarland Number One Barium Sulfate Turbidity
Standard. There is a need to adjust the turbidity of the cell suspension
because it can vary widely. It’s like being ask how should the degree or
intensity of the turbidity of the cell suspension be, should it be less or more
with respect to what standard of comparison? Without adjusting the degree
of cloudiness or turbidity to a McFarland Number 1 Standard there is no way
on earth by which you and I can arrive at the same level of turbidity and also
of luminance intensity. The level of turbidity used can surely affect the
results of the toxicity test. Microbiologists are well verse in preparing these
168
increasing levels of McFarland Standards numbered one to ten. A
McFarland Number One is just a barely cloudy cell suspension when visually
examined in a test tube but a cell suspension of similar turbidity contains
300,000,000 cells in a milliliter of the cell suspension. The degree of
turbidity of a McFarland Number One can be reproduced in the kitchen by
mixing a cup of water with more or less two to three drops of liquid whole
milk. By adding more saline in the flask containing the heavily turbid cell
suspension it can be diluted to something less turbid, less cloudy and
ultimately approximating that of a McFarland Number One Turbidity
Standard. After the level of the turbidity of the cell suspension has been
adjusted to the turbidity of a McFarland Number One, it can now be used for
the very simple procedure of immobilizing the luminous cells.
Using a stainless steel curved forceps with serrated tips; one paper-
disc was taken out from a collection of it contained in a petri dish. The single
paper-disc held at the tip of the forceps is simply dipped into the previously
standardized McFarland Number One cell suspension. Simply immersing
the paper-disc into the luminous cell suspension is enough to cause large
numbers of luminous cells to be adsorbed and entrapped in the paper-disc
resulting in their immobilization. I estimated that there must be something
like fifteen million luminous cells immobilized in a paper-disc. This large
169
numbers of luminous cells packed and immobilized together like sardines in
the small confines of the paper-disc is important in the cells’ continuous
state of bright bioluminescence. Recall that these cells require a “sense of
quorum” of being part of a very large population for them to continue shining
brightly. Packing millions of luminous cells in the matrices of a small paper-
disc tricks them into thinking exactly just that of the cells’ being a part of a
big population. Dispersing the luminous cells in a big volume of saline will
make them lose their bright luminosity for lack of close contact with one
another. This would make them think that their population is small and so
the individual cells become dim.
The bioluminous paper disc is then directly transferred into and allowed
to sink in a screw-capped test tube containing the water sample or chemical
solution to be tested for toxicity. The bioluminous paper-discs are introduced
individually into four tubes containing water of known purity, which serve as
the negative control. A negative control simply means water free of any toxic
contaminant; known to be clean and pure. Next step would be to introduce
bioluminous paper-discs individually into four test tubes containing a water
sample or a chemical solution whose toxicity is to be determined. The water
sample can be from bottled water, groundwater, river and even seawater or
it can also be a solution of any chemical whose toxicity is suspected. In all
170
cases, the fresh water samples have to be made saline by adding table salt
to make a three-percent solution. Likewise, chemicals to be tested will have
to be dissolved in a three-percent saline. The luminous microbes require the
three-percent salinity because of their marine origin. Another essential factor
to be checked before introducing the bioluminous paper-disc is the pH of the
water sample or chemical solution prepared. The pH is a measure of the
acidity or alkalinity of a water sample or any solution. For this toxicity test to
be valid, the pH must be in the range of about five to nine. A pH of seven will
be perfect for this purpose since it is neutral neither acidic nor alkaline. The
luminous microbes love a neutral pH, which is pH seven. Examples of acids
are vinegar and tomato sauce. Vinegar has a pH of three and tomato sauce
has a pH of four. Examples of alkaline substances are milk of magnesia and
baking powder. Milk of magnesia has a pH of 11 and baking powder has a
pH of 9. Exposure of the luminous microbes to extreme acidic or alkaline pH;
pH values below 5 or above 9 will cause the cells to die resulting in the
fading of their luminance.
171
Tales of light from the bioluminous paper-discs
The four screw-capped test tubes each containing the bioluminous
paper-disc are examined in the dark. All the test tubes will have to be placed
in a metal rack, which can hold a maximum of forty tubes. I would hold and
lift up the rack above my head and observe the tubes from their “butts” so to
speak in a dark room. In this way, the tubes disappear in the dark and I see
only an array of bioluminous paper-discs arranged in rows and columns. The
luminance of the four bioluminous paper-discs contained in the negative
control tubes as well as those in the four water sample tubes was examined.
If the period of luminance of the bioluminous paper-discs in the sample
tubes lasted as long as those in the negative control tubes then the water
samples tested is as clean as the water in the negative control tubes. The
water sample is therefore clean and safe to drink. But if the luminance of the
bioluminous paper-discs in the water samples fades rapidly and at times
black out in comparison to that of the negative control tubes then there is
something toxic in the water samples tested. The water sample is not safe to
drink since they contain toxicants that caused the luminance of the paper-
discs inside them to fade out rapidly. These are the two possible outcomes
of this simple toxicity test; either the luminance in the sample persists as
172
bright as the negative control or the luminance of the sample fades out much
faster than that of the negative control. The longer the luminance of the
bioluminous paper-discs persists, the safer and the less toxic the sample or
the chemical solution is. Degree of toxicity can also be determined as well
by dissolving each chemical to be tested at the same concentration in saline
and monitoring the length of time each will cause the bioluminous paper-disc
to fade out or even to “black out completely”.
Outcome of a Clean and Pure Water Sample Using PIBiT
Negative Control After half to one hour Negative Control
Water Sample Water Sample
Outcome of a Dirty and Unsafe Water Sample Using PIBiT
Negative Control After half to one hour Negative Control
Water Sample Water Sample
N.B. Yellow means sustained luminance; gray means decreased luminance
The shorter the time period to fade completely the more toxic the
chemical is. And the longer the time period for the luminance of the solution
173
to fade out completely, the less toxic the chemical. Using this method,
chemicals can be arranged individually from least toxic to the most toxic.
Background information behind bioluminosity and immobilization
A paper-disc when dipped into a brightly shining blue liquid absorbs the
millions of luminous microbial cells and will retain its bright luminosity even
when transferred and immersed in another saline. The paper disc absorbs a
small amount of liquid and becomes bioluminous because of the millions of
luminous microbial cells rendered bound, intertwined, and trapped in the
network of cellulosic fibrils found in the paper disc itself. The body of the
luminous microbes contains sticky substances on their body’s outer covering
making them adhere to the cellulosic fine and intertwining fibers of paper.
This simple method causes the immobilization of millions of luminous
microbes in the paper-disc making it brightly bluish-green when viewed in
the dark.
The readily visible bioluminous paper discs thereby precludes the use
of expensive and complex light-measuring instruments called luminometers
in testing the toxicity of water samples and unknown solutions. The human
eye can distinguish between varying degrees of brightness and are very
174
sensitive even to low magnitude light sources when it has undergone dark
adaptation. Dark adaptation enlarges the opening of the iris allowing more
photons to get into the eye and activates the photoreceptor rod cells of the
retina. Dark adaptation usually takes fifteen to thirty minutes of switching our
vision from photopic to scotopic, from bright to dark vision. Science has
determined that scotopic or dark vision is so sensitive to light that even a
single photon that reaches the retina will elicit a vision. A bioluminous paper-
disc contains millions of luminous microbes each of which is producing
hundreds of photons per second. Collectively the luminous cells in a single
bioluminous paper-disc can send photons into our eyes by the millions.
Paper-disc Immobilized Bioluminescence Technology (PIBiT)
The method of using bioluminous paper-discs for the bioluminescence
toxicity test using scotopic or dark vision is hereby presented with the
proposed term “Paper-disc Immobilized Bioluminescence Technology” or
simply the acronym “PIBiT”. PIBiT is an innovative, simple and highly
economical, non-instrument and non-culture medium based technique with
wide ranging biotechnological applications. The use of scotopic vision in
PIBiT thereby precludes the use of complex and expensive luminometers.
175
Moreover, since saline, a non-growth medium for microbes is used all
through out the procedure in the preparation and in the testing of samples,
the required microbiological measures to “keep out” unwanted microbial
contaminant called “aseptic techniques” can be abandoned completely.
PIBiT is user-friendly because of its simplicity and environment-friendly due
to the significantly reduced consumption of chemicals, manpower, and
energy.
A luminous suspension contained in an eppendorf tube roughly half the
size of our little finger can produce one hundred bioluminous paper-discs. A
hundred bioluminous paper-discs can test twenty-four water samples with
four replicates per sample and four replicates as well for the negative
control. One brightly shining plate culture can give a luminous suspension
that produce hundreds of thousands of bioluminous paper discs! In addition,
PIBiT can be made resource sustainable, which should make it very useful
and practicable in poor countries where a need to regularly monitor their
drinking water for safety and cleanliness is needed.
176
Punched out filter paper-discs contained in a petri dish, held by a forcep ready to be immersed in a flask containing suspension of luminous microbes and contained in a screw-capped tube whose water content is being tested for toxicity.
Clean and safe drinking water “I was born in the sign of water and it’s there that I feel my best, the
albatross and the whales they are my brothers” is a part of a lyric of a
beautiful song. The song entitled “Cool Change” by the Little River Band,
appealed to very much for Indeed I was also born in the sign of water and
my winning entry in the Young Inventors’ Awards has been designated in the
issue of the Far Eastern Economic Review magazine as “Testing the
Waters”. It really seems that I have such close affinity with everything that
has to do with water though I really never learned how to swim! Water is
defined by science as the universal solvent. Universal means that compared
to other chemical solvents more substances: solid, liquid and even gas;
crystalline and powder are soluble in water. Many of these substances are
associated with or components of living organisms and are appropriately
called biomolecules (Life Molecules). Our cells are bathed in and out with
177
water. Seventy to eighty percent of the composition of many living
organisms is water; for jellyfishes the water content probably approaches a
hundred percent. Water is the best medium in which our life processes can
take place. Chemical reactions and cellular processes occurring at the
molecular level are happening in a solution of water and salts. Since today’s
terrestrial organisms have move out of the sea a long, long time ago; there
is a need to continuously replenish the water within them from an external
source. Animals lose water from urination, perspiration, and respiration.
Plants lose theirs by transpiration. That is why animals have mouths and
plants have roots; for food assimilation and to replace the water that
escapes constantly to the environment. Thus, water becomes an essential
resource for the sustenance and maintenance of life. Water’s primal
significance means that access to clean and safe water must be ensured at
all cost. Simply putting it arithmetically, safe and clean water equal healthy
and long life for all living organisms. Dirty and polluted water is unhealthy
because it poisons living organisms resulting in diseases and therefore short
life.
The old adage that water is life has never meant so much to us now
than ever before. Cyanide contaminated seawater in Sorsogon, Philippines;
178
benzene in a river in China; arsenic in the groundwater in Bangladesh, and
mercury, chromium, lead, and cadmium in a lake in Massachusetts are just
some of the news that grabs the headlines in the media and internet these
days. Contamination of our ecosystem in general and drinking water in
particular by toxic chemicals has caused serious health problems like
poisoning, cancer and birth defects. Indeed, our drinking water’s cleanliness
and safeness is threatened as never before by a plethora of chemicals
artificially created in large quantities spewed out by a rapidly industrializing
world. Metallic and non-metallic pollutants abound within our immediate
vicinity that their presence though at minute amounts have already been
detected in the water that we drink and in the fresh produce that we eat.
Many of these chemicals have the ability to react with and change the
chemical structure of DNA, the blue-print of life. Changing DNA can lead to
birth defects if what has been affected is the DNA of sperms, egg cells or the
developing fetus itself. However, if what is affected is the DNA of one of the
billions of cells of our bodies then this can lead to cancer and malignancy.
On a grand scale, chemicals can also destroy swathes of ecosystems and
put many organisms at the risk of extinction. Chemical toxicants like heavy
metals, chlorinated hydrocarbons, aromatic compounds, formalin, cyanides,
detergents, phenolics, pesticides, mycotoxins and even some disinfectants
179
and antiseptic agents have already found their way into our bodies. Like a
book, no one can judge water by its appearance; water can look crystal
clear, smell right but deadly! Very small concentrations of various toxicants
at the parts per million (ppm) or even parts per billion (ppb) level will hardly
affect the physical qualities of water but can cause chronic life threatening
diseases when taken over long periods of time. The detection of toxicants in
water can be expensive and complicated using the instrumental analysis
employed today in modern laboratory. PIBiT is definitely the solution to this
seemingly helpless predicament of safeguarding the cleanliness of our
water. PIBiT is first and foremost to be used for the simple, rapid and cheap
toxicity testing of all forms of water samples like groundwater, surface water
as well as bottled mineral and distilled water. The beauty of using PIBiT is
that it can sensitively detect a wide variety of toxicants: metallic and
nonmetallic. Mercury, lead, cadmium, barium, nickel, cobalt, chromium,
arsenic, copper, cyanides, formaldehyde, dyes, detergents, and pesticides
are all detectable at their maximum allowable concentration in water as
specified by the United States Environmental Protection Agency (EPA) in as
short as half to an hour. PIBiT tells us if the water is toxic or not; whether it is
safe or unsafe to drink. However, PIBiT cannot tell us the identity of the
toxicant or toxicants that are present in the water.
180
In detail, metals like Lead is detectable by PIBiT in mineral water at its
U.S. EPA maximum allowable concentration of 0.015 ppm in just one hour;
cadmium at 0.005 ppm also in one hour; barium at 2 ppm in one hour and
the transition metals nickel at 0.1 ppm in one and half an hour and copper
at 1.3 ppm in one and half an hour. The organic toxicants like the detergent
sodium dodecylsulfate detectable at 0.05 ppm in two hours; the stain
methylene blue at 0.1 ppm in two hours; the chlorinated hydrocarbon
Chloroform at 0.05 ppm in an hour; Phenol at 0.5 ppm in one and half an
hour and the preservative formalin at 0.05 ppm in one hour.
PIBiT’s ability to detect mercury in mineral water is at 10 ppb (parts per
billion) in thirty minutes. When the bioluminous paper-discs are exposed to 1
ppb of mercury even om hard water, the luminance fades in an hour. One
ppb is mercury’s maximum allowable concentration in drinking water as
specified by U.S. E.P.A. Moreover, when exposed to 0.1 ppb the luminance
of the bioluminous paper-discs fades in two hours and finally when exposed
to a mercury concentration of 0.01 ppb or 10 ppt (parts per trillion) the
bioluminous paper-discs fades in three hours. One part per trillion is the limit
of detection for mercury in mineral water or hard water using PIBiT. While
the bioluminous paper-discs in the negative control blacks out completely in
six hours. These results show how ultra-sensitive PIBiT can be in detecting
181
a wide array of inorganic toxicants like heavy metals. PIBiT rivals the
detection ability of a sophisticated instrument called the Atomic Absorption
Spectrophotometer (AAS). An AAS can cost three hundred thousand US
dollars but PIBiT cost only a mere one dollar to analyze one thousand or
more water samples for toxicity.
In addition to heavy metals and other organic toxicants, PIBiT can also
detect the presence of volatile organic compounds (VOCs) in bottled distilled
water. Distillation process units can produce toxic distilled water that can
cause the bioluminous paper-discs to black out in as short as fifteen minutes
due to the presence of VOCs. But if the distilled water is boiled for ten
minutes in an open container, the resulting water can sustain the brightness
of the bioluminous paper-discs for as long as five hours. How come there is
a big difference in the length of time of luminance of the bioluminous paper-
discs between distilled water and boiled distilled water? Why did boiling
reduce the toxicity of the distilled water? The answer is very simple. Boiling
expels the highly volatile VOCs from the distilled water. These VOCs are
chlorinated hydrocarbons and therefore have the potential to be
carcinogenic. VOCs are produced when the surface water undergoes
chlorination in the water treatment plant converting it into tap water. When
tap water is distilled, the VOCs being more volatile than water evaporates
182
faster and so accumulates largely in the condensing water as it cools down
and collected. The distillation of tap water has an undesirable side effect, the
VOCs present in the tap at low concentrations get heavily concentrated in
the distilled water. Is it possible to remove VOCs from distilled water? The
answer is yes. A special steam vent should be present in the distillation
apparatus that will result in the removal of VOCs from distilled water. Can
the VOCs be removed completely from distilled water during the distillation
process most probably not. The distilled water will have to be passed
through special filtration columns containing activated carbon that should
remove all of the VOCs. A distilled water sample in which the luminance of
the boiled is significantly longer than that of the unboiled should be
suspected of containing prohibitive concentrations of VOCs. Ozone
treatment of water can also produce toxic byproducts in water, which can be
detected by PIBiT.
Brands of shining mineral water containing luminous microbes
183
Between bottled distilled and mineral water, PIBiT favors the bottled
mineral water. This indicates that the bioluminous paper-discs remain bright
much longer in mineral or hard water than in distilled water. Mineral water
contains dissolved substances like magnesium, calcium, bicarbonate, etc
that have beneficial effects on the integrity of the luminous cells. Besides,
humans have been drinking mineral water since the dawn of time for almost
three million years now and distilled water has only been around for about
twenty years. Among the commercially available distilled water in the
Philippines, PIBiT stays brighter longer in Brand B than in Brand C and
among the mineral water, PIBiT favors Brand A over Brand D and Brand D
is better than brand E an ozone treated mineral water. It would be better if
we take a second look in the use of ozone to clean up mineral water for
drinking purposes because doing so may bring in more harm than good.
Many nasty chemicals can be produce in water by an ozone treatment and
most of these are carcinogenic. Ozonolysis or the degradation of minute
amount of organic compounds in water using ozone is one of the treatments
utilized by German industrial plants for their wastewater. The higher
incidence of cancer in the population can be traced to prolonged exposure to
a plethora of artificial chemicals found in our modern way of life. In
summary, a water sample tested for toxicity using PIBiT that resulted in the
184
rapid inhibition of luminance in comparison with a negative control in as
short as an hour or less should be questionable as to its cleanliness and
safety.
PIBiT’s applications and its resource sustainability
PIBiT can be an alternative and novel method of antimicrobial assay
without making use of the conventional nutrient broth and nutrient agar for
the antimicrobial testing. Complex and expensive nutrient agar media used
initially for the cultivation of the marine luminous microbes can readily be
replaced by local substitutes. I found out that skimmed milk powder, which is
available in all local market, could be used as a good substitute for the
cultivation of marine luminous microbes. The skimmed milk powder once
suspended in water can first be treated with the protein-degrading enzyme
papain. Papain can be obtained from the milky latex of the papaya tree
when its stem or the immature fruit is cut. The papaya tree is abundantly
found in all the countries of the tropics. Casein, milk protein, is a large
molecule that needs to be digested by proteolytic enzymes to shorter
peptides and even amino acids so that it becomes soluble and more
absorbable to microbial cells. Another possible substitute for papain is the
185
pancreatic juice or juice from the small intestine of slaughtered pigs or cows.
The juice contains the enzyme trypsin that can breakdown the large protein
molecule in milk called casein into smaller more soluble portions that can be
absorbed easily by the luminous microbes. Agar is also a substance that is
readily available in the tropical coastal regions particularly from seaweeds.
The glass petri dishes can be re-used.
Luminous microbes growing and glowing brightly on self-made locally available
seawater salt, skimmed milk and agar-agar bars
Coarse salt can always be taken easily from the sea by sunlight-
powered evaporation and this is the kind of salt that the marine luminous
microbes are really accustomed to. Turbidity standardization using a
McFarland Standard # 1 contained in screw-capped test tube can be
prepared and reused repeatedly. The whatmann paper can possibly be
replaced by commercially available rough paper towel or some dried leaves
186
that can be punched out into small discs. PIBiT can find applications in the
following:
Using this method, fifteen different plants prepared as one percent
extracts in saline caused the bioluminous paper-discs in garlic, ginger,
onion and bitter gourd to black out much faster than the negative
control. Garlic causes the bioluminous paper-discs to black out in as
short a period as five minutes. PIBiT can therefore be used to screen
plant extracts rapidly for antimicrobial activity. Three plant extracts
studied in UST showed potent antimicrobial activity with complete light
inhibition being achieved in less than five minutes, three extracts are
moderately active with complete light inhibition being achieved in three
to four hours and eight showed no antimicrobial activity indicating
much longer period of sustained luminance.
PIBiT also responds sensitively to common disinfectants containing
chlorox and phenolics and health-care products like soaps and tooth
pastes with triclosan, rubbing alcohol, contact lens solution, eye drops,
mouthwashes etc. making their antimicrobial assays easier, cheaper
and simpler. Results showed that chlorox and chlorox-based
disinfectants are most potent disinfectants since they can achieve
187
inhibition of light output of the bioluminous paper-discs at a much
lower dilution levels than the phenolic based disinfectants. Soaps and
toothpastes containing triclosan likewise inhibit light output of the
bioluminous paper-discs faster and at much lower dilutions than
identical commercial products without it.
Acute toxicity testing of grains like rice and corn, cereal-based snacks,
food colorings and chemical additives, copra, root crops like cassava,
vegetables and other foods for the presence of mycotoxins,
Benzo(a)pyrenes, pesticides, cyanides, formalin etc. can be easily and
cheaply monitored to ensure safe and clean foods for the people.
Toxicity testing of fishes and seafoods for the presence of malachite
green and other poisonous compounds.
Fishermen can probably use PIBiT as natural, cheap, luminous and
attractive baits for various saltwater fishes. Fishes seemed to be
attracted to the cold bluish-green light of marine bioluminescence.
Thousands of luminous paper discs can easily be prepared in the
laboratory and released into the sea at night to attract various fishes
thereby facilitating their capture. Since the luminous bacteria used
have not been genetically modified and indeed are of marine origin,
their use would be a very natural process. In addition, the movement
188
and luminosity of these bioluminous discs in the sea can easily be
monitored for probable oceanographic studies.
Epidemiological studies and diagnosis of chemical poisoning of the
populace can be achieved by collecting and analyzing urine samples
and testing it for heavy metal poisoning.
Marine plant extracts can be assayed using PIBiT to screen for
antivibriosis compounds. Luminous vibriosis is responsible for the
infestations of prawn farms. Extracts from sea plants and seaweeds
can be screened and subsequently utilized for antivibriosis activity.
This should be more environmentally appropriate than the use of
antibiotics and other synthetic antimicrobial agent.
It is also highly probable that since luminous bacteria are normal
microflora of the intestine of marine fishes, they may play a probiotic
role (beneficial) enhancing the well being of these marine animals.
PIBiT can also be used to introduce these bacteria in the gut of
saltwater fishes raised in captivity by mouth so as to increase their
resistance against microbial pathogens.
Since bacterial bioluminescence is highly dependent on dissolved
oxygen, PIBiT can most likely be utilized to measure dissolved oxygen
(DO) in water as well as well as evaluate the BOD at a much cheaper
189
and faster rate. The amount of dissolved oxygen in water is used to
determine the pollution level in the water. The more oxygen is
dissolved in a water sample the less polluted it is. The less oxygen, the
more polluted the water is.
PIBiT can also be made to monitor cheaply the amount of residual
chlorine in drinking water. Chlorine in drinking water protects us from
waterborne diseases.
PIBiT can also be used to easily determine the amount of salt in
perspiration, which can be utilized for the initial diagnosis of cystic
fibrosis.
PIBiT is also sensitive to ultraviolet radiation and can determine their
effectiveness for disinfecting purposes in the operating rooms of
hospitals and clean benches of research institutions.
TuBET can rapidly determine the “Freshness” of fishes, seafoods,
pork, beef, and chicken.
Lastly, PIBiT can be used to check any unknown liquid or solution
whose toxicity is suspected. Paints and organic materials suspected to
contain heavy metals like lead, mercury, and cadmium can first
undergo incineration. The resulting residue is dissolved in saline and
tested using PIBiT. Here in UST, I intend to check unknown liquids
190
dripping from any of the tubes hanging up in our ceilings or even water
coming as condensate from an autoclave. Any unknown solid that is
readily soluble in water can likewise be tested for toxicity.
The use of PIBiT can be programmed in such a way that it can be
prepared during daytime and observation of the glowing discs can favorably
and easily be done during nighttime. This should facilitate the testing
process and the evaluation of the results. PIBiT is very natural,
environmentally friendly, non-harmful and almost zero energy consuming
analytical process. The paper-discs are highly biodegradable material. It
should significantly reduced consumption of chemicals and culture media,
glasswares, manpower, and energy than is conventionally required in a
typical chemical analysis. Significantly, less consumption of commercially
available chemicals and other materials would mean significantly less
industrial output of the greenhouse gases. These characteristics will surely
endear PIBiT to people concerned with environmental protection and
delaying global warming problems. PIBiT can address the “Philippine Clean
Water Act” with certainty. PIBiT can safeguard the protection of the La Mesa
Watershed through its highly sensitive capability to detect the presence of
minute amounts of toxic chemicals that can come from without. Mies once
191
said. “less is more”. PIBiT is just exactly that; its simplicity and minimalistic
attributes hold the promise of affordability to the vast number of poor people
throughout the world. Its simplicity and minimalistic attributes also hold the
promise of being an alternative to existing technologies that even contribute
to the strength of climate change. PIBiT’s presents itself as an
environmentally friendly technique now that the apocalyptic effects of global
warming threaten the world.
EQ phone NASA
It was very touching for me to hear from Pres. George Bush, the
planned construction of the International Space Station by the National
Aeronautics and Space Administration or NASA. What a great achievement
that will be for mankind, imagine a permanent abode in the firmaments
which will eventually serve as the launching pad for modern space crafts
that will carry humans to the outer planets of the solar system and beyond.
People and animals will someday dwell in the ISS. Ever since man’s great
voyages from the Old World to the New, a substantial supply of clean and
safe drinking water is needed. Water in the ISS, will be a very precious
commodity that requiring it to be rationed and reused. Water present in all
192
human secretions like urine, sweats, mouth water vapor and hand washings
will have to be recycled by special machines. These special machines
installed in the ISS will be fed with the collected bodily fluids of the
occupants. These fluids will then converted into ultra-pure drinkable water!
Between the urine and the drinkable water are series of continuous
purification operations and processes. More often than not, contaminants
can gain entry into the water as it undergoes various purification treatments.
If I may recommend, NASA can make use of PIBiT to check on a regular
basis the quality of their recycled drinking water. PIBiT’s ultra-sensitivity to
minute amounts of toxicants in water can safeguard the health and well
being of the space crews for many years. Bringing the marine luminous
microbes in space will be the least of all problems. They can be stored in
small plastic cryotubes and preserved easily in small canisters of liquid
nitrogen or in small ultra-low temperature freezers. The luminous microbes
kept under suspended animation can be revived easily into viable cells and
subsequently utilized in PIBiT. Microbial bioluminescence can also be
employed to directly measure the life support system of other planets that
we humans might consider to reside on. Physiological conditions like
temperature, pH, oxygen availability and ionic concentration needed to
sustain life are also compatible with those of Vibrio fischeri. For indeed,
193
bioluminescence is oxygen and oxygen is bioluminescence. Man’s inherent
exploratory nature will bring us to extra-terrestrial worlds someday. Surely, in
those days bioluminescence and PIBiT will play an important role in
ensuring safe and clean drinking water to the space travelers. In addition,
since Vibrio fischeri and Photobacterium leiognathi are honest-to-goodness
denizens of the earth’s marine hydrosphere, then we can used them to
monitor bodies of water in other planets like the oceans of Europa or the ice-
cold geysers of Enceladus for their similarity to the physic-chemical
properties of earth’s seawater.
Future plans for PIBiT
Scientists share the results of their findings by publishing it in technical
journal devoted to a particular discipline. The other alternative is to submit
research studies or findings in the form of an abstract for oral and poster
presentation in a special annual gathering of colleagues and fellow
scientists. In both cases, peers in the scientific community review the
soundness of the hypotheses, experimental results and validity of the study.
After reviewing the submitted abstract based on established protocols, a
group of scientists working together as a committee makes a decision as to
194
its acceptance or rejection for presentation. This special gathering is called a
congress, convention, or symposium and is organized by a small highly
specialized group or a large international organization of scientists. It is with
much excitement that I dream of someday presenting my findings in a
scientific organization held in the US or Europe.
I was so thrilled last February 2006 when my submitted abstract on
PIBiT’s ultra-sensitive ability to detect mercury in hard water got accepted
for a poster presentation in the prestigious 106th annual convention of the
“American Society for Microbiology” (ASM). The ASM will be held in
Orlando, Florida in May of 2006. My abstract was peer reviewed by ASM
and was accepted for presentation. It is indeed an honor and a privilege for
a research work to be recognized by an international body of scientists like
the prestigious American Society for Microbiology. Although excited, as I
was to participate in the ASM, my planned participation fell through for lack
of support from the UST’s College of Science administration. Presumably,
for the administration, financial assistance for travel is only awarded for an
oral and not for a poster presentation. It was truly disappointing for I have
looked forward with much enthusiasm to visit Florida and to participate in the
convention. Hopefully, in 2008 in Boston, I can be allowed to participate in
195
the prestigious annual convention of the American Society for Microbiology.
Before ASM, my abstract on PIBiT was also accepted for a poster
presentation in the gathering of the International Union of Microbiological
Societies (IUMS) late in 2005. IUMS was held at the Moscone Center in San
Francisco. Again for lack of funds, I was not able to attend and participate. In
both IUMS and ASM, I am pleased that my abstract on PIBiT underwent
rigorous screening and was considered worthy of presentation. In fact, ASM
screens submitted abstracts thoroughly based on high standards and
accepted protocols before an acceptance is granted to the author. In June
2005, another aspect of PIBiT, submitted as an abstract, was accepted for
poster presentation in the 24th International Congress of Chemotherapy. The
International Society of Chemotherapy, based in London, sponsors the
Congress for Infection and Cancer. However, hope indeed springs eternal.
One day while searching the internet, I came across two other special
gatherings in the US that still receives submitted papers for presentation that
deals on my research area. One of these is a smaller and more specialized
meeting of the “International Society of Bioluminescence and
Chemiluminescence” (ISBC). The 14th annual convention of the ISBC will
take place in San Diego, California in October of 2006. This convention is
probably the one suited for me since it specializes in bioluminescence and
196
chemiluminescence and that is what PIBiT is all about. The other gathering,
much bigger, is the annual convention of the “American Association for the
Advancement of Science” AAAS. It will be held in San Francisco in February
of 2007. Its theme on sustainable practices to protect the environment and
provide safe drinking water makes PIBiT suitable for a poster presentation.
Besides, PIBiT presented in the AAAS can be made known to a wider and
bigger community of scientists coming from various disciplines. The “World
Water Forum” held biannually in different countries the last one was in
Mexico will also be one of the best venues, where I can present PIBiT. The
World Water Forum deals with how water is utilized, managed, protected,
distributed and studied in third world countries. Lastly, I am still searching for
annual conventions and symposia in the U.S. concerned with water, the
environment or microbiology. PIBiT will surely be useful in these conventions
for its ability to detect minute amounts of contaminants in water that pose
health risk to the populace. Very recently, my years of work on PIBiT gained
recognition from my colleagues in the Philippine Society for Microbiology
(PSM) when I was awarded the Best Poster Paper Award for 2007. It is
indeed an honor to be included in the roster of the PSM’s Best Poster Paper
Awardees with the likes of Prof. Sanchez, Raymundo, Dogma, Matias,
197
Natividad, Barraquio, Kobayashi, Rivera, Rivero, Monsalud, Tapay, de
Ungria, Dalmacio and many others.
Recognitions through the years on my work with Bioluminescence
Lately, while surfing the web I came across a site (http://tiee.ecoed.net)
the Ecology Education Publications dated April 2005 maintained by the
Ecological Society of America and supported by the National Science
Foundation. Listed in its website was my very first ISI (Institute for Scientific
Information) publication in 2001 on bioluminescence that appeared in the
journal of Biological Education. What an honor indeed it was to receive such
recognition from a prestigious body of teachers and scientists in the U.S.
198
From the www.tiee.ecoed.net
PIBiT was also submitted for poster presentation and accepted in the
15th International Congress of Photobiology (ICP), which will be held on June
2009 in Düsseldorf, Germany. Another prestigious congress where it was
screened and accepted for poster and group discussions presentation was
in the 2009 FEMS (Federation of European Microbiological Societies) that
will be held in July of 2009 in Gothenburg, Sweden.
We dream of a much better place, a distant and perfect world of our
deepest yearning. A world we try so hard to realize in this present life
through our own little ways of achieving it. A world free of pollution, wars,
diseases, defects, intolerance, sadness, senseless killings, and where no
one is poor and deprived of dignity and self-worth. This generation of ours
may never see that distant world come into fruition. It definitely will not come
TIEE TEACHING ISSUES AND EXPERIMENTS IN ECOLOGY a peer reviewed publication of ecological
educational materials by the Ecological
Society of America
CURRENT VOLUME
ALL VOLUMES
ABOUT TIEE
SUBMIT WORK
SEARCH
199
in my lifetime. But to dream and strive for it, little by little, each day of our
lives is like going through a dark tunnel and seeing a light, the distant world,
from afar getting ever so closer.
All of us take pleasure, guidance and strength from a song that comes
along in our life that is full of meaning and purpose; a song that embodies
our dreams and aspirations. For me this song is from Disney’s animated
movie “Hercules” entitled “Go the Distance”. For from where I am now, the
US could be that far off place in space and time where I dream of presenting
PIBiT to an association of the learned men and women of science. To hear
with much anticipation what they think of PIBiT and how it can be used,
applied and even improved for the betterment of life. Here are the inspiring
lyrics to that wonderful song:
I have often dreamed, of a far off place
Where a hero’s welcome, would be waiting for me
Where the crowds will cheer, when they see my face
And a voice keeps saying, this is where I’m meant to be
I’ll be there someday, if I can go the distance
I will find my way, if I can be strong
200
I know every mile, will be worth my while
When I go the distance, I’ll be right where I belong
Down an unknown road, to embrace my fate
Though that road may wander, it will lead me to you
And a thousand years, would be worth the wait
It might take a lifetime, but somehow I’ll see it through
And I won’t look back, cause I can go the distance
And I’ll stay on track, no, I won’t accept defeat
It’s an uphill slope, but I won’t lose hope
Till I go the distance, and my journey is complete
But to look beyond the glory is the hardest part
For a hero’s strength is measured by his heart
Like a shooting star, I will go the distance
I will search this world, I will face its’ harms
I don’t care how far, cause I can go the distance
Till I find my hero’s welcome, waiting in your arms
I will search this world, I will face it’s harms
Till I find my hero’s welcome, waiting in your arms
201
The healing effects of the good blue light
Plate cultures of bioluminescent microbes can generate bright and
stable blue light for the entire nighttime of 12 hours or more. Several shining
plates can be enclosed in special glass receptacles and placed inside hotel
rooms or any dark exhibition lobby or also in science discovery or
exploratory hall. These bioluminescent lamps placed above or near beds
can function as novel fixtures striking a chord with the occupants of our
grand fight against global warming and a new green way of living. This is
particularly effective since microbial bioluminescence creates bright light
without consuming electricity, generating heat or producing green house
gases. In addition, the good blue light of 490 nm wavelength has been found
to repair serious cellular damages due to our exposure to sunlight that can
carry ultraviolet radiation. Ultraviolet radiation causes numerous lesions in
our DNA, the repository of our genetic information, which if left unrepaired
can lead to cancer or programmed cell death. During daytime, we are
exposed to the damaging part of sunlight, its energetic ultraviolet portion. So
why not expose ourselves to bioluminescence coming from special lamps at
nighttime, when we sleep, this should help our cells repair and heal
themselves. Indeed, recent scientific studies showed that it is blue light,
202
which effectively and efficiently repair the damage in our DNA thus allowing
sick cells to heal themselves. Healing using blue light allow them to recover
their original health and vitality. In fact, many scientists believe that one of
the reasons for the existence of bioluminescence, the production of cold
light, is specifically to activate enzymes that repair DNA damaged by harmful
chemicals and physical agents called mutagens. I can see the day when
bioluminescence will become a part of our everyday life. They can indeed
become aesthetic displays in our homes, in hotel rooms, in lobbies and also
science exploration centers like aquaria, plants and flowers.
Stacked luminous plate cultures brightly shining that allows one to read
prints in utter darkness
203
It is rare, even if it ever happens, for Vibrio fischeri to unfurl its brightest
luminous wonders in nature, as it would do so in the laboratory. For
instance, in seawater Vibrio fischeri’s population density is so low probably
occurring in only ten cells in a cup of seawater. This extremely low number
precludes their ability to attain the magic number or the quorum of
10,000,000 cells/mL to initiate a brilliant bioluminescence. Nevertheless,
packing them close together in large numbers to attain the magic number
needed to switch on their lux genes to initiate bioluminescence has been
achieved in the light organs of fishes and squids. But still the kind of
sustained maximum brilliance several folds brighter than the light organs of
fishes and squids and lasting long for several hours has only been achieved
by Vibrio fischeri in the artificial world of the laboratory. Modern microbiology
has provided the right sustenance and the optimum conditions to Vibrio
fischeri for it to shine in full splendor. Perhaps Vibrio fischeri has been
waiting for billions years of its existence on this planet; pre-empting the
emergence of the first sponge, the first crustacean, the first plant, the first
fish, the first amphibian, the first reptile, the first bird, the first mammal and
most of all the first intelligent being. A thinking species finally found the
means to unlock the vast bioluminescent potentials of the marine luminous
204
microbes. Vibrio fischeri, maker of light, originating from the earliest periods
of earth’s history must have been instrumental in the development of vision
in primitive animals that gradually evolved into the most complex organ of
higher life forms - the eye. Luminous microbes were most likely the first
bioluminescent organisms producing blue-green radiant energy that
interacted specifically with light-sensitive chemical pigment known as
carotenoids. The interaction converts light energy into electrical energy,
which travels to the brain of animals where the unique sense of vision is
created. At the peak of creation is man endowed with an intelligent mind for
whom dominion over all creatures, grand and humble, is given and with
whom luminous microbes alone in the dark has made a close encounter of
the “third kind”. The contact can give man a deep sense of appreciation and
an inspiration of awe for this microbe’s wonderful gift of light. The photons
that Vibrio fischeri creates, leave its source from the cell, travel through the
long inter-organismic space to reach human eyes, trigger visual reactions
and bring about a new vision in man. A vision that can inspire us to learn
and value the greatness of this humble speck of life and indeed all other
living organisms, great and small, with which we share this planet with. It is
indeed a new vision of oneness with all living organisms for molecular
biology has proven that we all share a common lineage, a single ancestry.
205
Microbes and plants are our cousins and animals are our brothers and
sisters. A pet is defined as a plant or animal kept for amusement and
entertainment or something loved and cherished. The luminous microbe to
me is more than just a pet. It is the cause for a much greener world and a
better life.
A photo of the first Christmas tree in history illuminated by bioluminescence
206
A photo of the World’s First Bioluminescence Illuminated Christmas Tree serving as a
screensaver for computer desktops, notebooks and tablets
207
Having fun with the bioluminescence – A bioluminescent Santa Claus
They say that the grand symphonies of Beethoven have to await the
evolution of a twentieth century full modern orchestra to unravel their
ultimate beauty and grandeur. The same can be said of Vibrio fischeri, a
twentieth century modern microbiology has finally unlock the full potentials of
the wonders of this microbe and the beauty of its celestial gift:
“Living Light”“Cold Light”
“Milky Sea”
“Light of the Abyss”
“The good blue light of a
Marine Luminous Microbe:
Vibrio fischeri isolated from
a local squid shining in water
without heat and using no
electricity”
Wishing all the grand and humble denizens of our Mother Earth
“A Merry Bright Bioluminescent Christmas for 2008”Edward Quinto, UST, Manila, The Philippines
BIOLUMINESCENCE:
STOP GLOBAL WARMING!
“Light of Life
208
bioluminescence. Observing the awe-inspiring beauty of its blue-green light
has made the study of the intricacies of life processes and the means to
protect it more meaningful, more practical, and less daunting. This smartest
and heavenliest of microbes must have stolen light from the gods like the
mythical Prometheus and gave it to us to see a new world in a different bluer
light. Health as many say is wealth, for truly what is life if it is not lived and
enjoyed in the best of health. Protecting health is what this microbe does; it
gives off its own “Light of Life” to teach us new ways on how to keep our
water and our environment clean. A clean water and environment should
bring us all a healthy and beautiful life. Indeed the “microbes of light” are
giving us lessons in green. Lessons that can make us learn how to protect
effectively the health of the environment, the whole planet, and all of us.
Suddenly, a world tarnished by pollution due to rapid industrialization and a
booming population is showing signs of rehabilitation. I believe that we can
still avert the predicted dawning of the apocalyptic effects of Global Warming
due to our wanton and reckless neglect of the environment. As an amateur
astronomer and a microbiologist, my eyes have seen the light from the
farthest – the Andromeda Galaxy to the deepest – bioluminescence; the light
from outer space to the light of inner space; the light from the hottest – stars
to the coldest – bioluminescence; the light of the nonliving to the living light!
209
Let us see the world in a different light, the light of bioluminescence. What a
beautiful world it will be! It will surely be a greener world, a world where the
water is safe to drink and the environment is clean and free of harmful
pollutants. After having shared my personal and fulfilling experience with the
“Microbes of Light” with you, I implore and enjoin all to begin the exploration
of new ways of living through bioluminescence, a clean form of life energy.
To bring to light the public’s awareness on the usefulness of
bioluminescence, the good blue light, let us strive to request UNESCO to
declare 26 January as “World Bioluminescence Day”. This day was not
chosen because it’s my birthday or it’s Australia Day or it’s the Day of the
Republic of India but on this day the brightest recorded apparition of a “Milky
Sea” that appeared in the Arabian Sea was recorded by a U.S. satellite.
Therefore, with utmost urgency I plead to all our “Biophiles”, the lovers
of life of this world to help me launch this bioluminescence revolution, a new
way of green living for all of us. It will surely be a new way of healthy and
aesthetic life for all of us, a beautiful life illuminated by the good blue light
shining a pathway to the future, to a clean and safe new world.
210
A beautiful and clean view of the West Philippine Sea from the top of the world
References: Aruldoss, J.A., Viraraghavan, T., (1998) Toxicity Testing of Refinery Wastewater Using Microtox. Bull. Environ. Contam. Toxicol. 60 456 - 463
Bulich, A.A., Tung, K.K. and G. Scheibner (1990) The Luminescent Bacteria Toxicity Test. It’s Potential as an In Vitro Alternative. J. Biolumin. Chemilumin. 5(2) 71 – 77.
Peter, S., Siersdorfer, C., Kaltwasser, H. and M. Geiger (1995) Toxicity Estimation of Treated Coke Plant Wastewater Using the Luminescent Bacteria Assay and the Algal Growth Inhibition Test. Environ. Tox. And Water Quality. 10: 179 – 184.
211
Quinto, E. A Simple Water Toxicity Test Using Photobacterium leiognathi. Journal of Biological Education. 35(2) pp. 89 – 92
Schnapf, J., "How Photoreceptors Respond to Light", Scientific American, April 1987
Tchounwou, P.B. and L. Reed (1999) Assessment of Lead Toxicity to the marine bacterium, Vibrio fischeri, and to a Heterogeneous Population of Microorganisms Derived from the Pearl River in Jackson, Mississippi, USA. Reviews on Environmental Health 14(2) 51 – 61.
Yates, I.E. and J.K. Porter (1982) Bacterial Bioluminescence as a Bioassay for Mycotoxins. Appl. Environ. Microbiol., 44(5):1072-1075.
Water on the Space Station. http://science.nasa.gov/headlines/y2000/ast02nov_1.htm
212
About the Author
The author is a tenured faculty member teaching microbiology and molecular biology at
the Department of Biological Sciences, College of Science; University of Santo Tomas.
He completed his B.S. Chemistry in 1982 and M.S. in Microbiology in 1998 in UST and
earned a postgraduate certificate course in Biogeography and Environmental
Assessment (Aufbaustudium) from the University of Saarland, Germany in 1993. He is a
registered chemist and an elected regular member of the National Research Council of
the Philippines (NRCP) in the division of Biological Sciences. Recently, he was elected
Diplomate of the esteemed Philippine Academy of Microbiology (PAM). A contributor of
the section in microbiology of the book: Guidebook to Plant Screening: Chemical and
Biological that won the National Academy of Science and Technology (NAST)
Outstanding Book Award in 2006. His national awards include winning three times (1997,
1999 and 2000) in the professional category of the Best Poster Competition sponsored
by the Philippine Council for Health Research and Development (PCHRD) of the
Department of Science and Technology (DOST). Recently, he won the celebrated 2007
Best Poster Paper Award, which was followed by the 2008 Best Oral Paper Award of the
Philippine Society for Microbiology (PSM). UST awarded him three consecutive Silver
Series Awards (2000, 2002 and 2004), the International Publication Award, Dangal ng
UST Santo Domingo Award for Best in Innovation, and the Parangal ng Science Award
(2006 and 2007). His international awards include the Gold in the 2001 Young Inventors’
Awards sponsored by the prestigious Far Eastern Economic Review (FEER) and
Hewlett-Packard (HP) Invent and in 2005, the Tom Bergan Memorial Award on the
occasion of the 24th International Congress of Chemotherapy in Manila given by the
213
International Society of Chemotherapy (ISC - London). He is listed in the 2006 - 2007
Marquis Who’s Who in Science and Engineering. In 2003, he founded and launched the
University of Santo Tomas Collection of Microbial Strains (USTCMS) whose maiden
catalogue and brochure were printed through a generous grant provided him by the
Philippine Council for Advanced Science and Technology Research Development
(PCASTRD). His collected strains of marine luminous bacteria served as the initial
microbial holdings that evolved into the present USTCMS. He is an active full member of
the American Society for Microbiology (ASM) and an active life member of the Philippine
Society for Microbiology (PSM), the Biology Teachers Association of the Philippines
(BIOTA) and the Philippine Association of German Academic Exchange Scholars
(PAGAES). He is a recipient of scholarship grants from the German Academic Exchange
Service (DAAD) in 1992-1993 and in 2000; the Carl Duisberg Gesellschaft (CDG) in 1990
and the Gesellschaft für Biotechnologische Forschung (GBF) in 1988. He has been
deeply involved in microbiology and chemistry education in the Philippines with 23 years
of experience through his numerous publications in local and international peer-reviewed
journals, seminar-workshops, training courses, lectures, patents, paper and poster
presentations, and the writing of laboratory manuals. His research interests are in the
fields of microbial bioluminescence, microbial taxonomy, bioactive microbial natural
products, and lactic acid fermentations. For years now, he has supervised many high
school students in their science projects that went on to win national and international
awards especially in the Intel Science and Engineering Fair (ISEF). His hobby includes
amateur astronomy, traveling, reading, and writing.