Xanthan Gum Process

8/13/2019 Xanthan Gum Process

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2.0 PROCESS SELECTION AND DESCRIPTION

2.1 PROCESS SELECTION

Alcohol or agent

Alcohol or agent

Alcohol or agent

MAINTENANCE

(L strain)

Xanthomonas

Campestris

GROWTH

INOCULUMBIOREACTORPRODUCTION

MEDIUM AND

OPERATIONAL

CONDITIONS

ERMENTATION BROTH

PASTEURI!ATION

SEPARATION

WASHING" DEWATERING an#

DR$ING

%ANTHAN GUM

CELL REMO&AL

PRECIPITATIONDISTILLATION


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The process for making xanthan gum is shown in the flow chart above. irst! the selecte"

microbial strain is preserve" for possible long#term storage b$ proven metho"s to maintain the

"esire" properties. A small amount of the preserve" culture is expan"e" b$ growth on

soli" surfaces or in li%ui" me"ia to obtain the inoculum for large bioreactors. The

growth of the microorganism an" xanthan pro"uction are influence" b$ factors such as

the t$pe of bioreactor use"! the mo"e of operation &batch or continuous'! the me"ium

composition! an" the culture con"itions &temperature! p(! "issolve" ox$gen concentration'.

The ke$ steps of a t$pical xanthan pro"uction process are summari)e" in the table below.

Pr'ss st* Sa+ an# '*rati'n S,**'rts

Culture preservation

ofX. campestris

Long-term: lyophilized; Strain improvement;

test for culture viabilityInoculum build-up Shake flasks; inoculum

fermenters

Growth medium

composition;

controlled operational

conditions;

tests for contaminantsProduction stage Lab scale batch fermentor Euipment design;

production medium

composition;

fermentation conditions;

controlled operational

conditionsHarvest !hermal" chemical" or

enzymatic;

#entrifugation or filtration

$rocess development of

cell

deactivation and removal

Isolation $recipitation; filtration %evelopment of e&traction

and

purification methods


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This illustrates the t$pe an" scale of each step! an" provi"es an in"ication of the associate"

anal$tical an" "evelopmental support necessar$ to achieve the optimum process performance.

There are two t$pes of fermentation s$stem involve" in the pro"uction of xanthan gum an" it is

usuall$ carrie" out accor"ing to the preferences an" after an anal$ses on the s$stems. In our

pro*ect! we have selecte" batch s$stem after anal$)ing the a"vantages of the s$stem against

continuous s$stem. The relevant factors which contribute" towar"s the selection of batch s$stem

is "iscusse" in section +.,., an" the "escription on the process selection will be "iscusse" further

in section +.-.

2.1.1 SELECTION O BATCH AGAINST CONTINUOUS S$STEM

Although batch culture is commerciall$ preferre" having fewer parameters to be

controlle" an" well un"erstoo"! a problem of operation in batch culture is that the environment

for cell growth keeps changing throughout the growth c$cle/ an" coul" give a"verse

con"itions! such as toxic pro"ucts or extreme p( an" exhaustion of nutrients. 0hile in

continuous culture! the growth me"ium is continuousl$ supplie" to the culture vessel! extreme

con"itions will not occur as me"ium is continuousl$ "ilute" an" remove" from the vessel.

1ecker et al. have also pointe" out that continuous process shows reasonabl$ high conversion

rates of substrate to pol$mer of 234536! but also mention problems of maintaining the sterilit$

an" the risks of emergence of fast#growing mutants that "o not pro"uce the "esire pro"uct!

xanthan gum. Nevertheless! the continuous process gives a cost competitive s$stem! an" with

suitable growth con"itions consi"erable $iel"s of pol$sacchari"es can be maintaine" for more

than +333 h! thus the continuous process coul" be the choice rather than the batch mo"e.

Although conventional metho"s can be improve" b$ continuous fermentation! there is still aclassic problem that the pro"uct contain cells an" cell "ebris! which gives lower the filterabilit$

of the xanthan solution an" limits its application. The pro"uction of cell#free xanthan gum is

therefore "esirable. In ,722! 8sso 9ro"uction :esearch ;ompan$ foun" that continuous film

fermentation reactions can be rea"il$ carrie" out b$ a continuousl$ "epositing a suitable

substrate on the surface of a rotating "rum or moving belt or similar "evice! an" appl$ing a


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culture containing selecte" microorganisms to the film! an" then continuousl$ removing the

fermentation pro"uct after sufficient resi"ence time has elapse". Tests have shown that such a

process makes it possible to use the substrate in higher concentrations! permits surprisingl$

effective utilisation of the substrate! re"uces the time re%uire" for carr$ing out the fermentation

reaction! minimises variation in pro"uct %ualit$! an" simplifies recover$ of the fermentation

pro"ucts.

2.2 RAW MATERIAL

Matria+s ,s# in %ant-an Pr'#,ti'n

The main material emplo$e" in xanthan gum pro"uction is the bacteria itself< =anthomonas

campestris &N::L 1#,>?7'. This microorganism can be obtaine" as a l$ophili)e" sample.

,. @ me"ium< ,3 gBL D#glucose purissimoC ? gBL bacteriological peptoneC - gBL $east extractC

- gBL malt extract. 0hen @#agar me"ium is emplo$e"! Agaragar purissimo &+3 gBL' must be

a""e". 1acteriological peptone! $east extract an" malt extract have to be store" at >;.

+. @#T me"ium< ,+ gBL D#glucose purissimo! +.? gBL bacteriological peptone! ,.? gBL $east

extract! ,.? gBL malt extract! ,.? gBL 9O>(&N(>'+! +.? gBL 9O>(E+! an" 3.3? gBL gSO>. The p(

has to be a"*uste" to 5.3 b$ a""ition of (;l.

-. 9ro"uction me"ium< >3 gBL sucrose! +., gBL citric aci"! ,.75 gBL N(>NO-! +.F22 gBL E(+9O>!

3.?35 gBL g;l+! 3.3F7 gBL Na+SO>! 3.332 gBL (-1O-! 3.332 gBL GnO! 3.3+3 gBL e;l-.2(+O!

3.3+3 gBL ;a;O-! an" 3.,- mLBL (;l cc. The p( has to be a"*uste" to 5.3 b$ a""ing NaO(. All

the pro"ucts must be pure.

Matria+s Us# in %ant-an G, Is'+ati'n

ermentation broths obtaine" as previousl$ "escribe" were precipitate" using several agents

such as< ethanol! I9A! an" acetone! all of in"ustrial %ualit$! that is 726 &wBw'! F?6 &wBw'! an"

7F6 &wBw'! respectivel$! an" salts &Na;l! ;a;l+' of pure %ualit$! aroun" 776 &wBw' in purit$.


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;haracteri)ation of xanthan pro"uce" was performe" b$ the measurement of "ifferent

parameters. Acetate an" p$ruvate content were measure" using en)$matic kits! acetate using

1oehringer#annheim no. ,>F+2, an" p$ruvate with 1oehringer#annheim no. ,+>7F+.

:heological behavior was "etermine" using a viscosimeter &1rookfiel" LHT#S$nchrolectric'.

This viscosimeter has a microcapsule for sample thermostation &1rookfiel" S;>#,FB,-:'. A

1rookfiel" no. ,F spin"le was usuall$ emplo$e". Ash an" moisture contents in xanthan were

measure" using a Dupont 7?, thermogravimetric anal$)er.


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2./ PROCESS DESCRIPTION

Several steps must be followe" to carr$ out xanthan gum pro"uction successfull$< strain

maintenance! inoculum buil"up! an" xanthan pro"uction itself. The evolution of the pro"uction

s$stem can be followe" accor"ing to the "ifferent anal$sis techni%ues that are "escribe" at the

en" of this section.

2./.1 PROCEDURE OR %ANTHAN PRODUCTION

Strain Maintnan

The first step in xanthan gum pro"uction is to ensure that the strain to be use" in fermentation is

L#strain &the real gum pro"ucer'. The microorganism has to be plate" on @#agar plates an"

incubate" at +?; for - ". The colonies obtaine" must be bright $ellow an" between > an" ? mm

"iameter. To avoi" "egra"ation of L#strain to strains Sm &+4- mm "iameter' an" Hs &, mm

"iameter'! not bright an" pale $ellow! =. campestris has to be transferre" to fresh @#agar

me"ium ever$ ,> " an" incubate" un"er the same con"itions "escribe" above &>!,>'.

In',+, B,i+#,*

The si)e of the inoculum to be emplo$e" is aroun" ?4,36 of the total fermentation volume. The

microorganism state to be use" for pro"uction has a great influence on overall process evolution

rate. In xanthan fermentation this step is reall$ important! because xanthan is the bacterial

capsule of =. campestris! an" the gum is pro"uce" when the microorganism is growing. The

xanthan gum pro"uce" "uring this stage is not welcome because of its great resistance to nutrient

uptake b$ the cells. The culture nee"e" for inoculation for pro"uction has to be in the

exponential growing phase an" must have an important biomass concentration without xanthan

gum. This inoculum must be built up in "ifferent stages! that is! from "ifferent growth c$cles.

0hen the final fermentation volume is aroun" + L! two stages are enough. &,' The protocol for


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on! being previousl$ polari)e" for at least 2 h. Afterwar"s it has to be calibrate" b$ means of

ox$gen "esorption with nitrogen an" absorption with air emplo$ing the stirrer spee" of the first

stage in the fermentation &for + L of final work vol the initial stirrer spee" is aroun" +,3 rpm'.

5. The operational con"itions "escribe" have been optimi)e" to obtain high xanthan

concentrations.

ii) In',+ati'n

,. The microorganism is intro"uce" into the vessel when all the operational con"itions have been

establishe". The volume of the inoculum that is to be intro"uce" at the beginning of the

fermentation to obtain a pre"etermine" biomass concentration can be calculate" from the value

of the inoculum concentration. To "etermine the biomass content in the inoculum! the cultures in

the "ifferent 8rlenme$er flasks have to be mixe" in a vertical laminar work station in a sterile

wa$. A small sample of the mixe" culture is anal$)e" b$ means of a spectrophotometer at ?>3

nm to know the biomass concentration &see Subhea"ing -.,.>.,.'. The volume of inoculum to be

emplo$e" for fermentation is calculate" from the concentration obtaine".

+. The culture is intro"uce" throughout a membrane emplo$ing a sterile s$ringe.

iii) rntati'n

,. The fermentation takes place when inoculum has been intro"uce" into the vessel. In the first

part of the fermentation! there is a strong "ecrease of "issolve" ox$gen concentration

correspon"ing to the growth of the microorganismC this "ecrease in "issolve" ox$gen

concentration is faster as inoculum biomass concentration is higher.

+. During the process! the fermentation broth becomes bright $ellow with a great increase of its

viscosit$. The viscosit$ obtaine" pro"uces a ver$ important "ecrease of the ox$gen transport

rate! an" as a conse%uence a "ecrease in the "issolve" ox$gen concentration. or successful

pro"uction this concentration has to be maintaine" higher than ,36 of saturation value.

-. This must be "one b$ increasing the stirrer spee"! with a stirrer spee" program "uring the

fermentation time. ermentations carrie" out in a +#L work volume fermentor usuall$ finish at


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stirrer spee" aroun" ,333 rpm gives a t$pical evolution of the s$stem! showing the concentration

evolution of several compoun"s an" variables "uring xanthan pro"uction.

Ana+tia+ Mt-'#s

To check the state of the fermentation! some anal$tical techni%ues must be use". The main

components to be anal$)e" are< biomass! xanthan! carbon source &sucrose'! an" nitrogen source

&ammonium' concentrations. Dissolve" ox$gen is also a ver$ important component to consi"er!

but it is usuall$ monitore" on line b$ means of an electro"e.

Bi'ass Ana+sis

1iomass concentration is obtaine" b$ means of the measurement of the optical "ensit$ at ?>3 nm

of the "ilute" broth. 1iomass concentration can be "etermine" accor"ing to?#m filter. =.

campestris is 3.>43.5 m in wi"th an" 3.54,.F m in lengthC therefore! a cake hin"ering the

correct filtration process can %uickl$ be forme". It is necessar$ to stir the fermentation broth at

+333 rpm to make a suspension an" to avoi" cake formation.

-. Salt is a""e" to the filtere" xanthan pro"uction broth &free of =anthomonas cells' an"

"issolve" b$ agitation. Jsuall$ 3.? gBL of Na;l is a"vise".

>. Then I9A is a""e" in enough %uantit$ to pro"uce total pol$mer precipitation. If an$ cells pass

through first filter! alcohol a""ition can be use" to l$se the cells! so the$ can be eliminate" b$

successive washing.


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?. =anthan precipitate is separate" from the broth b$ a new filtration through a ,33#m filter.

Now broth! l$se" cells! salt! an" isopropanol pass through the filter while xanthan is retaine".

I9A ma$ be recovere" b$ "istillation.

2. =anthan obtaine" is washe" with a mixture of I9A#water &approx -


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2.0 MASS BALANCE

=anthan gum is pro"uce" using =anthomonas campestris in batch culture. In this pro*ect!

for each gram of sucrose utilise" b$ the bacteria! 3.+- g ox$gen an" 3.3, g ammonia are

consume"! while 3.5? g gum! 3.37 g cells! 3.+5 g gaseous ;O + an" 3.,- g (+3 are forme".

Other components of the s$stem such as phosphate! magnesium can be neglecte". e"ium

containing sucrose an" ammonia "issolve" in ? litres water is pumpe" into a batch fermenter

an" inoculate" with =.campestris. Air is sparge" into the fermenterC the total amount of off#

gas recovere" "uring the entire batch culture is ,+?3 kg. 1ecause of the high viscosit$ an"

"ifficult$ in han"ling xanthan#gum solutions! the final gum concentration shoul" not be

allowe" to excee" -.? wt6.

Step ,

:eaction e%uation is as follows+9' kg cells

&3.+5 kg' &3.3,+29' kg ;O +

&3.,- kg' &3.332359' kg (+3.


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&3.+--A' kg 3 +in K 3 kg 3 +generate" # 3 +out K &3.3,359' kg 3 +consume".

.. 3+out &3.+--A # 3.3,359' kg. &+'

N +balance

N +is a tie component.

&3.525A' kg N+in N +out.

.. N +out &3.525A' kg. &-'

;O +balance

3 kg ;O +in K &3.3,+29' kg ;O+generate" # ;O+out K 3 kg ;O+consume".

.. ;O +out &3.3,+29' kg. &>'

The total mass of gas out is 3.-, kg. Therefore! a""ing the amounts of O +! N +an" ;O +

out from &+'! &-' an" &>'25 9' kg. &2'

N(-balance

N( -in K 3 kg N(-generate" # 3 kg N( -out K &3.333>59' kg N(-consume".

.. N(-in &3.333>59' kg. &5'

0e can now calculate the total mass of the fee"! 5,59' kg &F'

0e can now use &F'an" &?'in &,'5,59' K &3.-, # 3.33,79' &3.-, K 9'

? 3.7?>5- 9

.. 9 ?.+> kg.

.. um out 3.,F kg.

Substituting this result in &?'an" &F'


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A 3.-33 kg

?.+? kg

rom Table

3+ in 3.35 kg

N+ in 3.+- kg.

Jsing the results for 9! A an" in &+'! &-'! &>'! &2'an" &5' kg

N +out 3.+- kg

;O+ out 3.322 kg

Sucrose in 3.+ kg

N( -in 3.33+? kg

;ell balance

3 kg cells in K &3.33>+9' kg cells generate" cells out K 3 kg cells consume".

.. ;ells out &3.33>+9' kg

;ells out 3.3++ kg

(+3 balance

? kg (+3 in K &3.332359' kg (+3 generate" (+3 out K 3 kg (+3 consume".

.. (+3 out ? K &3.332359' kg


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(+3 out ?.3- kg

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Xanthan Gum Process

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