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Orange peel inhibited hup and enhanced hydrogenevolution in some purple non-sulfur bacteria

Amal W. Danial, Refat Abdel-Basset*

Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt

a r t i c l e i n f o

Article history:

Received 8 July 2014

Received in revised form

24 October 2014

Accepted 7 November 2014

Available online 4 December 2014

Keywords:

Hydrogen gas

Hup

Orange peel

Photosynthetic (purple nonsulphur)

bacteria

* Corresponding author. Current address: BioE-mail address: rbasset@aun.edu.eg (R. A

http://dx.doi.org/10.1016/j.ijhydene.2014.11.00360-3199/Copyright © 2014, Hydrogen Ener

a b s t r a c t

The studied bacterial strains grew and evolved hydrogen utilizing orange peel as the sole

carbon and nitrogen source, as much as their respective controls grown in R€AHmedia only.

Noticeably, orange peel inhibited the uptake hydrogenase (Hup) activity and simulta-

neously enhanced the cumulative hydrogen evolution levels. The cumulative hydrogen

was enhanced as hydrogen oxidation/recycling (through the electron transport chain), that

is catalyzed by Hup has been inhibited. Therefore, the role played by orange peel surpassed

its intended role as a source of nutrients. Rather, it seems to act as a specific inhibitor of

Hup activity. Orange peel, in the literature, is famous for its oils and pharmaceutical

components rather than nutritional value. Conversion efficiency of orange peel to

hydrogen was not proportional with its reducing sugars content; it has been inhibited by

high concentrations most probably due to pharmaceutical compounds in orange peel.

The studied strains are purple non-sulfur bacteria (PNS), which have been newly iso-

lated from local sewage water samples at Assiut and Sohag cities (Upper Egypt). Orange

peel together with PNS, in this respect, is unique. PNS generally cannot use biowastes for

growth, as they do not release exozymes to hydrolyze large molecules (e.g. cellulose, starch

or proteins) into absorbable molecules (sugars, organic acids, amino acids, etc.).

Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

Introduction

Biomass to biofuel is anticipated to cover part of the world'senergy needs and may represent an environmentally safe

removal of biowastes. However, hydrolysis of agricultural

lignocelluloses (e.g. massive amounts of rice straw, molasses

and other plant residues) are yet inefficient [1,2]. Sewage

sludge usage in energy regeneration and other uses is

expanding [3,4]. Poultry feather is being studied as well [5,6].

Orange peel is one of the common biowastes, which is

produced in millions of tons worldwide; their carbon and

logy Department, Facultbdel-Basset).44gy Publications, LLC. Publ

nitrogen content can be used as a nutrient medium for mi-

croorganisms to grow and evolve hydrogen. However, unlike

other wastes, orange peel is not famous for its nutritional

values and its use in this concert is scarce. Its content of

pharmaceutical compounds is prior to its nutritional values.

However, only recently, Martin et al. [7] raised the question if

orange peel is a waste or energetic resource, as they could use

it in methane production.

Hydrogen gas is an ideal energy carrier although technol-

ogies and biotechnologies are not developing by the magni-

tude we need. In this work we were concerned with: (1)

Producing hydrogen gas biologically as a clean biofuel using

y of Science, Taif University, Saudi Arabia.

ished by Elsevier Ltd. All rights reserved.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 9 4 1e9 4 7942

local and newly studied isolates of bacterial (purple non-

sulfur) strains. (2) Getting rid of biowastes in a beneficial and

environmentally safe approach. The waste concerned with, in

this work, was orange peel. Recently, research on utilizing

orange peel in microbial growth and biofuel production such

as bioethanol [8] and biogas [9] production is attracting

attention of scientists. However, we could not approach any

published work dealt with using orange peel in microbial

nutrition and hydrogen evolution. Several bacteria of varied

capacities of transforming biomass to biofuel are isolated and

studied [10]. The herein presented work includes growth of

purple non-sulfur bacteria (newly isolated from local sewage

water samples (Assiut and Sohag cities, Egypt) as well as their

potential for hydrogen evolution utilizing this type of waste.

Uptake hydrogenase activity (Hup), as one of the limiting

factors to net hydrogen yield, has also been followed.

Material and methods

Isolation, medium and characterization

Photosynthetic purple non-sulfur bacterial isolates (PNSB)

were isolated fromAssiut and Sohag cities (Upper Egypt). They

were grown on R€AH medium [11] and they were incubated

anaerobically at 30 �C at a light intensity of about 5,000 Lux.

After 1e2 weeks of incubation, a purplish red color developed

in the medium. The PNSB were analyzed macroscopically

considering colony pigmentation, length and width. The col-

ony size and shape were determined using light microscopy

and the phenotypic characterization of the different isolates is

assessed.

Minimal medium (MM) was prepared by dissolving 1.0 g of

disodium hydrogen phosphate (Na2HPO4), 0.2 g of potassium

chloride (KCl) and 0.2 g of magnesium sulfate (MgSO4) in 1 L of

distilled water. The media were then supplemented with or-

ange peel (0, 1, 2, 3 g/700 ml culture media) which are corre-

spondent to 0, 1.42, 2.85 and 4.28 gm per liter culture media.

Control cultures containing no orange peel were included.

Physiological analysis

Determination of reducing sugars content in OPDinitrosalicylic acid (DNSA) reagent method [12] used to assay

the amount of reducing sugars released into the growth me-

dium as described by Boboye and Alao [13]. One gram orange

peel (OP) was suspended in 10 ml distilled water and shaken

for 10 min. The supernatant (0.5 ml) was mixed with 0.5 ml

DNSA reagent, left for 15min at 28 �C and boiled for 5min. The

tubes were rapidly cooled under tap water and the optical

density of the reaction mixture was measured at 540 nm. The

OD values were referred to a glucose standard curve to esti-

mate the amount of reducing sugars/g OP.

Assessment of bacterial growthBacterial growth was followed by measuring the optical den-

sity of the cell suspension at 660 nmm at different time in-

tervals. Protein contents were estimated using Lowry method

[14].

Hydrogenase activityThe sum uptake activity of Hup (uptake hydrogenase) and of

the bidirectional hydrogenase assay mixture contained 1 ml

bacteria, 2.75 ml phosphate buffer (50 mM), 0.25 ml methyl

viologen (50 mM), 1 ml sodium dithionite (100 mM), flushed

with nitrogen to remove oxygen followed by hydrogen, as

conducted by Yu et al., [15] and Colbeau et al., [16]. The

reduction of methyl viologen by Hup and hydrogen was

monitored at 540 nm (spectrophotometer thermoscientific,

double beam spectrophotometer, Evolution 160, UV-VIS,

Germany).

Hydrogen collection and detectionBottles containing cultures cocktail (70 ml phosphate buffer,

70 ml early log phase bacteria, 1, 2, or 3 gm OP and completed

with H2O to 700 ml). Then, bottles were stoppered and stirred

by magnetic stirrer as long as hydrogen is evolving. The gas

produced was captured in a cylinder inverted in water, and

connected with NaOH solution to absorb carbon dioxide.

Molecular hydrogen has being detected by gas chromatog-

raphywith a thermal conductivity detector [17], the carrier gas

was nitrogen.

Results and discussion

Three strains of the purple non-sulphur bacterium Rhosop-

seudomonas (Rh1, Rh2 and Rh3) in addition to one Rhodobacter

(Rd) have been isolated from local sewage water samples at

Assiut and Sohag cities (Egypt). The data concerning their

phenotypic characterization are presented in Table 1. After-

ward, these strains have been studied for their ability to grow

and evolve hydrogen utilizing orange peel as the sole carbon

and nitrogen resource. Purple non-sulphur bacteria of Egyp-

tian origin are scarcely studied and they are virgin in hydrogen

biotechnology. Recently, Danial [10] isolated, characterized

and introduced several some PNSB in her Ph.D. into hydrogen

evolution. Highest growth as protein contents of Rhosop-

seudomonas sp TUT 36422 strains (Rh1 and Rh2) was recorded

at 3 g peel/700 ml culture, while lowest growth was recorded

at control cultures (R€AH medium) as shown in Fig. 1a and b,

respectively).

Growth as optical density displayed similar responses

(data not shown). However, Rh3 exhibited its highest growth

level at 1 and 2 g orange peel/culture (700 ml); depending on

age (Fig. 1d) while Rd at 2 g orange peel/700ml culture (Fig. 1e).

Co-cultures of Rh1 with Rh2 as well as that of Rh3 with Rd

exhibited growth trends similar to their uni-bacterial -cul-

tures (Fig. 1c and f). Rh1 and Rh2 were combined together as

they have been isolated from the same environment (Assiut)

and Rh3 and Rd from another locality (Sohag) i.e. adapted to

the same habitat.

Orange peel has not been extensively studied as a micro-

bial nutrient or as a bioenergy source, although it is a global

waste produced in massive amounts worldwide. The major

component of the primary cell walls (peels) of many higher

plants is pectin [18,19]. The backbone of this pectic poly-

saccharide (de-esterified pectin) is built up with blocks of

ae1,4 linked polygalactosyluronic acid residues inter-spersed

Fig. 1 e Protein contents of PNS bacteria grown on different concentrations (1, 2 or 3 g) of orange peel; control cultures were

growing on R€AH medium; Rh1 (a), Rh2 (b), Rh1 þ Rh2 (c), Rh3 (d), Rd (e), Rh3 þ Rd (f). Other legends are same as in Fig. 1 and

series at all figures are as follows.

Table 1 e Mophological, biochemical and physiological characteristics of isolates Rh1, Rh2, Rh3 and Rd.

Strains Rh1 Rh2 Rh3 Rd

Color of cell suspension Red to pink Red Red Red

Shape Ovoid to rods Oval-rod Rod Rod

Length (mm) 0.6e3.2 0.7e2.7 0.6e2.9 0.5e1.6

Width (mm) 0.3e0.5 0.4e0.6 0.2e0.55 0.2e0.4

Gram stain � � � �Motility þ þ þ þCellular absorption peaks (nm) 371, 800, 851 371, 800, 851 371, 800, 851 371, 800, 851

Bacteriochlorophyll A A A A

Facultative aerobes þ þ þ þCasein hydrolysis � � � �Tween 80 þ þ � �H2S production � � � �Catalase test þ þ þ þpH 6.5e7 6.5e7.5 6.5e7.5 6.5e7.5

Temperature 25e30 25e30 25e30 25e30

Nitrogenase þ þ þ þHydrogenase þ þ þ þH2 production þ þ þ þGlucose þ þ þ þFructose � þ þ þLactose þ þ þ �Maltose þ þ � �Acetic acid � þ � �Succinic acid þ � � �

i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 9 4 1e9 4 7 943

Table

2ePercentagech

anges(increase

dordecrease

drelativeto

theco

ntrol)in

cum

ulativehydro

gen(%

control)andHupactivityofth

edifferentPNSbacteriaasin

fluence

dbydifferentora

ngepeel(O

P)am

ounts.

Control

Rh1

Rh2

Rh1þ

Rh2

Rh3

Rd

Rh3þ

Rd

Cumulative

hydro

gen

H2ase

%Inhibition

Cum

ulative

hydro

gen

H2ase

%Inhibition

Cumulative

hydro

gen

H2ase

%Inhibition

Cumulative

hydro

gen

H2ase

%Inhibition

Cumulative

hydro

gen

H2ase

%Inhibition

Cumulative

hydro

gen

H2ase

%Inhibition

100

0100

0100

0100

0100

0100

0

1gOP

133.9

±01

58.39±0.04

2109±0.13

569±0.05

1359±0.92

46.99±0.20

217.39±0.20

114.39±1.30

92.9

±0.084

42.9

±0.25

64.7

±0.09

300±2.85

2gOP

98.29±0.12

47.29±0.02

173.39±0.11

369±0.06

83.39±0.5

31.39±0.80

304.3

±0.6

57.19±0.1

102.9

±0.45

71.4

±1.02

94.1

±0.05

566.7±1.23

3gOP

160.79±0.8

30.69±0.001

233.39±0.12

169±0.002

183.39±0.36

0259±0.08

173.99±1.2

157.19±1.2

71.49±0.85

74.3

±0.96

170.6

±0.92

66.7±0.084

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 9 4 1e9 4 7944

with regions of alternating galactosyluronic acid and rham-

nosyl residues [20]. Pectate lyase (PL), which is, otherwise,

known as pectate transeliminase catalyzes the eliminative

cleavage of de-esterified pectin [18,19]. Various microbes such

as Xanthomonas campestris, Erwinia and Streptomyces have been

reported to synthesize pectate lyases [21e23]. Therefore, or-

ange peel other than being an environmental pollutant, the

studied strains utilized it for growth and hydrogen

production.

In Rh1, Rh2 as well as their co-cultures, highest inhibition

of Hupwas recorded at 1 g orange peel in 700ml culture (Table

2). This has been also exactly applied to Rh3. However, Rd

exhibited another response i.e. the most inhibitory effect was

exerted by 2 g orange peel.

All the studied bacterial strains exhibited ability to evolve

hydrogen at the expense of orange peel. Rh1 produced maxi-

mally 900 ml/700 ml culture at 3 g orange peel/700 ml culture

that is almost 2 time that of the control culture (Fig. 2a)

whereas Rh2 produced 700 ml/700 ml culture at 3 g orange

peel culture that is 3 times that of the control (Fig. 2b). Co-

cultures of Rh1 and Rh2 produced 1100 ml at 3 g orange

peel/700 ml culture that is 2 time that of the control culture

(Fig. 2c). Rhodopseudomonas sp. (Rh3) produced 700 ml at 2 g

orange peel culture (700 ml), which is 2.5 time that of the

control culture (Fig. 2d). Rhodobacter sp (Rd) produced 700 ml/

700 ml culture at 2 g orange peel that is more or less similar to

its control culture (Fig. 2e). Co-culture of Rh1 and Rh2

enhanced the amount of hydrogen to 1200 ml/700 ml culture

(Fig. 2c) and co-culture of Rh3 þ Rd enhanced hydrogen to

1500 ml/700 ml culture (Fig. 2f). Wilkins [24,25] used orange

peel to produce ethanol using Saccharomyses cervice. Martin

et al. [7] used orange peel to evolvemethane, recommending it

as one of the energy resources rather than a biowaste. The

above two referenceswere the only reportswe could approach

concerning OP and bioenergy. In this work, the studied strain

consumed OP not only for their growth but also for H2

evolution.

Conversion efficiency as hydrogen value per gram reducing

sugar (RS) of orange peel) showed that the amount of

hydrogen does not depend only on the reducing sugars con-

tent in the culture medium (Table 3), since the amount of

hydrogen was not proportional with sugars content. The

highest conversion efficiency was recorded at 1 g OP/700 ml

culture (0.2 g sugar), high concentrations (2 and 3 g/700 ml

culture) were inhibitory to H2 levels in Rh1 and Rh2. Rh3 and

Rd exhibited more or less the same attitude but highest con-

version efficiency was exhibited at 2 g (0.5 or 0.6 g RS). Based

on the results, we hypothesize that other concentrations may

be inhibitory due to pharmaceutical compounds in orange

peel, which are thus prior in effect to the nutrition value.

Hydrogen gas is evolved from microorganisms as an ac-

tivity of hydrogenases and nitrogenases. Hydrogenases are

divided, into uptake (membrane bound) and bidirectional

hydrogenases. Bidirectional (reversible) hydrogenase cata-

lyzes hydrogen metabolism in both directions i.e. reduction

of protons to molecular hydrogen (independent on ATP) or

oxidation of hydrogen molecules (the latter activity is

identical to that of uptake hydrogenase). Hydrogen oxida-

tion catalyzed by uptake hydrogenase (Hup) acts to regain

energy and reducing equivalents lost as H2 e.g. during

Fig. 2 e Cumulative hydrogen of PNS bacteria grown on different concentrations (1, 2 or 3 g) of orange peel; control cultures

were growing on R€AH medium. Other legends are same as in Fig. 1 and series at all figures are as follows.

i n t e r n a t i o n a l j o u rn a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 9 4 1e9 4 7 945

nitrogen fixation [26]. Hup accompanies all nitrogenases in

order to reoxidize molecular hydrogen produced during ni-

trogen fixation. The electrons are thus funneled into the

electron transport chain most probably via the quinine pool

and finally reduce O2 into water in the dark. This reaction is

subsequently, by lowering oxygen levels, helps in installing

anaerobiosis, a prerequisite for H2 evolution. Hydrogen

oxidation (by uptake hydrogenase (Hup) or by bidirectional

hydrogenase activity) occurs at the expense of hydrogen

evolution. Therefore, is one of the factors limiting the

hydrogen evolution levels, which is actually a fragile phys-

iological process, not least due to particular sensitivity to

Table 3 e Conversion efficiency of orange peel to hydrogen gacultures contain 40 mM malate without orange peel (OP).

Rh1

OP/700 ml medium

0 1 g 2 g 3 g 0 1 g

560 750 550 900 300 630

Rh3

OP/700 ml medium

0 1 g 2 g 3 g 0 1 g

230 500 700 400 700 650

oxygen of hydrogenases and nitrogenases. Oxygenic

photosynthetic cyanobacteria like Oscillatoria chalybea are

inhibited from evolving H2 by their own evolved oxygen

[27,28]. There are of course numerous attempts to avoid Hup

activity in order to increase hydrogen yield. For instance,

Masukawa et al. [29] developed a mutant of Nostoc sp. PCC

7422 that its Hup genes were knocked out. The portion of

hydrogen consumed by hup was thus ruled out and

hydrogen yield accordingly was maximized [30]. In addition,

a number of Hup inhibitors have been described in the

literature such as nitrite, nitric oxide, carbon monoxide, and

acetylene [31].

s (ml H2/700 ml culture) by different PNS isolates, control

Rh2 Rh1 þ Rh2

2 g 3 g 0 1 g 2 g 3 g

520 700 600 810 500 1100

Rd Rh3 þ Rd

2 g 3 g 0 1 g 2 g 3 g

720 500 850 550 800 1450

Fig. 3 e Uptake hydrogenase activity (Hup) of PNS bacteria grown on different concentrations (1, 2 or 3 g) of orange peel;

control cultures were growing on R€AH medium. Other legends are the same as in Fig. 1 and series at all figures are as

follows.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 9 4 1e9 4 7946

In this work, orange peel supplementation inhibited Hup

activity. The concentration of 1 g/700 ml culture is a common

dose to inhibit Hup in Rh1 (Fig. 3a) as well as in Rh2 (Fig. 3b),

their combination (Fig. 3c), but not in Rh3 (Fig. 3d) and Rd

which have been inhibited by 2 g (Fig. 3e). Orange peel by

acting as an inhibitor to Hup activity is thus surpassing its

nutritional value, as it is thought or anticipated. The inhibitory

effect on Hup was a different attitude from other cellular ac-

tivities (growth, protein contents and hydrogen yield), which

have been enhanced by orange peel as a nutrient propor-

tionally with the amount provided. The chemical composition

of orange peel contains a considerable amount of volatile oils.

Orange peel and its volatile oils contain several and diverse

compounds which are of significant pharmaceutical impact,

leading to use it in folk medicine and kitchen. The main

components in orange peel are dlimonene (monoterpene),

polymethoxylated flavones (PMFs), vitamins, carotenoid pig-

ments, alkaloids, pectins, etc. Furthermore, orange peel oil

exhibited antimicrobial and anticancer effects [32]. The pros-

pect of purple non-sulfur (PNS) photosynthetic bacteria for

hydrogen production has been analyzed [33].

The impact of orange peel as an inhibitor of Hup at these

purple non-sulphur bacteria should be taken into

consideration in further studies to maximize hydrogen yield.

Furthermore, another wok to separate and specify which

active components of orange peel are inhibitory to Hupwill be

conducted.

Acknowledgments

The authors sincerely thank the STDF (Science and Technol-

ogy Development Fund, Egypt) for financing the project of

“Biological hydrogen Production for fuel and Environment #

972” and its administration for excellent follow up and

guidance.

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