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4?Improvements in Fermentation Processes

4?Improvements in Fermentation Processes

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226



M. Kumar and K. Gayen



Bagasse, Barley straw

Wheat straw, Corn stover,

Switchgrass



Lignocellulosic feedstocks



Wet grinding



Acids/Enzymes



Dilute sulfuric acid

Alkaline peroxide



Hydrolysis



Pretreatment of fermentation

inhibitors



Solid/Liquid Separation



Inoculum



Distillation

Gas stripping

Pervaporation

Perstraction

Adsorption



Fermentation



Solid residue



Carbon dioxide,

Hydrogen



Recovery



Butanol



Acetone

Ethanol



Fig. 7.2 Schematic process flow diagram for lignocellulosic materials to butanol. Hydrolysis

methods and inhibitors of broth vary from substrate to substrate. Figures are partly adapted from

Ling Tao and Andy Aden’s study [57]



7.4.1 Batch and Fed-Batch Fermentation Processes

For lignocellulosic materials, the performance of batch and fed-batch fermentation

processes has been examined using different bacteria. In one previous study, five

different combinations of pretreatment of wheat straw and batch fermentation

process have been performed. Results demonstrated that simultaneous hydrolysis

and fermentation with agitation by gas stripping showed maximum productivity



7 Biobutanol: The Future Biofuel



227



Table 7.1 Various feedstocks and their compositions for ABE fermentation

Raw materials

Composition

Bacterial strain



References



Barley straw



C. beijerinckii



[26]



C. beijerinckii



[23, 58, 60]



C. beijerinckii



[25]



C. beijerinckii



[22]



C. beijerinckii



[22]



C. acetobutylocum



[61]



C. saccharobutylicum



[34]



C. acetobutylocum



[29]



C. beijerinckii



[36]



C. acetobutylocum



[44]



C. beijerinckii

Co-culture of B. Subtilis

and C. butylicum

C. acetobutylocum



[53]

[62]



42% Cellulose, 28%

hemicellulose, 7%

lignin, 11% ash

Wheat straw

38% Cellulose, 29%

hemicellulose, 24%

lignin, 6% ash

Corn fiber

20% Starch, 50–60% nonstarch polysaccharides

Corn stover

38% Cellulose, 26%

hemicellulose, 23%

lignin. 6% Ash

Switchgrass

37% Cellulose, 29%

(Panicumvirgatum)

hemicellulose, 19%

lignin

Domestic organic waste 59% Sugars, 13% lignin,

17% ash

Sago

86% Starch, small amounts

of mineral and

nitrogenous matters

Defibrated-sweetStarch

potato-slurry

(DSPS)

Degermed corn

73% Starch, 3% ash, 13%

proteins

Extruded corn

61% starch, 3.8% corn oil,

8.0% protein, 11.2%

fiber

Liquefied corn starch

39% Starch, 45% moisture

Cassava

70% Starch, 2.7% protein,

2.4% fiber, 0.2% ash

Whey permeate

5% Lactose, 0.36% fat,

0.86% protein



[38, 41, 54]



of 0.31 g l-1 h-1 over the other combinations (fermentation of pretreated wheat

straw, separate hydrolysis and fermentation of wheat straw without removing

sediments, simultaneous hydrolysis and fermentation of wheat straw without

agitation, simultaneous hydrolysis and fermentation of wheat straw with addition

of sugar supplements). Also, the successful consumption of hydrolysate sugars

(glucose, xylose, arabinose, galactose, and mannose) of wheat straw has been seen

during the study [33]. For the same feedstock, fed-batch process enabled to produce butanol with more than twofold productivity of batch fermentation

(0.77 g l-1 h-1) [60]. Further, pH-stat fed-batch fermentation synthesized

16 g l-1 of butanol with 72% higher productivity than conventional batch processes. The pH was maintained constant by adding butyric acid. The other

advantage of butyric acid was observed in enhancing the solventogenesis phase

during the metabolic pathways of clostridium bacteria [43]. Solventogenesis phase



(iii) Cells recycling and bleeding



(ii) Immobilized cells continuous

fermentation



Continuous fermentation

(i) Free cell continuous fermentation



Fed-batch fermentation



Batch fermentation



saccharobutylicum DSM 13864

beijerinckii BA101

beijerinckii BA101

acetobutylicum P262

acetobutylicum 824A

acetobutylicum ATCC 55025

acetobutylicum P262

beijerinckii BA101

saccharoperbutylacetonicum N14



C.

C.

C.

C.

C.



beijerinckii P260

beijerinckii P260

beijerinckii BA101

beijerinckii P260

beijerinckii P260

beijerinckii P260

saccharoperbutylacetonicum

N1-4



C.

C.

C.

C.



C.

C.

C.

C.

C.

C.

C.



Table 7.2 Various fermentation processes for butanol production

Fermentation process

Strain used



Lactose and yeast extract

Corn

Defidered-sweet-potato-slurry

Synthetic medium

Synthetic medium



Sago starch

Degermed corn

Starch and glucose

Whey permeate



Barley straw

Wheat straw

Corn fibers

Corn stover and switchgrass (1:1)

Switch grass

Wheat straw

Synthetic medium with butyric acid



Substrate



1.43

12.50 (butanol)

7.73

8.8

8.58



9.1

14.28

9.9

8.6



26.64

21.42

9.3

21.06

14.61

16.59

16.0



[65]

[30]

[29]

[33]

[66]



[34]

[36]

[35]

[38]



[26]

[23]

[25]

[22]

[22]

[24]

[32]



Production of ABE References

(g/l)



228

M. Kumar and K. Gayen



7 Biobutanol: The Future Biofuel



229



is the second phase of metabolic pathway, which includes the production of solvents (acetone, butanol, and ethanol) on the consumption of acids (acetic and

butyric acid), produced in first phase or acidogenesis.

The productivity and performance of fermentation processes also depend on

pretreatment or hydrolysis methods, which convert the complex biomass composition into simple sugars. Two traditional pretreatment methods such as acidic and

enzymatic methods were compared on the basis of yield of batch fermentation. It

was observed that enzymatic method eliminates some inhibitors from the sugar

solution and generates better yield (0.35 g g-1) over the acidic method [25]. Other

studies also suggested that the inhibitors of cellulosic hydrosylates, produced in

hydrolysis processes, can be reduced by treating with Ca(OH)2 [49, 63].

The integrated fermentation process with recovery has provided significant elevation to total solvent concentration in the broth. It was recorded 51.5 g l-1 with the

comparison 24.2 g l-1 in non-integrated batch process using C. beijerinckii BA 101.

In integrated process, sugar consumption was also increased to as high as 150 g l-1

over 60 g l-1 in the case of non-integrated process [64]. Due to the toxicity of high

sugar concentration to the bacterial culture, the fed-batch process also has great

advantages [67]. In fed-batch reactor, a total concentration of 165.1 g l-1of the

solvent was achieved compared to 25.3 g l-1 in batch reactor [68].



7.4.2 Continuous Fermentation Process

From the solvent productivity point of view, batch process produced solvents with

such a low productivity of 0.35–0.40 g l-1 h-1 [64]. For scaling up of such a

process, high volume of broth, high capital cost ,and operational cost became severe

problems.This leads to uneconomical production of solvents. Continuous fermentation reactors show several advantages over batch reactors such as one inoculum

culture is sufficient for the long time, drastically reduced sterilization and inoculation time. Various continuous processes, such as free cells, immobilized cells, and

cell recycling and bleeding, have been investigated. However, immobilized cell

process has shown significant potential with as high as 15.8 g l-1 h-1 of solvent

productivity (about 40 times than batch process) [69]. Also, cell immobilization

allowed long survival time to cells of C. acetobutylicum in solventogenesis phase,

which resulted that 20% higher yield than conventional fermentation [30].



7.5 Recovery Techniques Integrated with Fermentation

Process

Butanol inhibition is one of the most crucial problems for developing industrial

scale production of butanol. Butanol-producing bacteria can rarely tolerate more

than 2% butanol in broth [70]. More precisely, 1% exposure of butanol caused a



230



M. Kumar and K. Gayen



Table 7.3 Integrated systems for enhancing the production of ABE fermentation

Recovery

Type of

Max. titer of ABE

Max. titer of ABE (with References

method

reactor

(without online recovery) online recovery) in g/l

in g/l

Gas stripping Batch

Batch

Fed-batch

Fed-batch

Batch

Batch

Fed-batch

Batch

Pervaporation Fed-batch

Fed-batch

Perstraction

Batch

Fed-batch

Fed-batch

Adsorption

Batch

Fed-batch

Fed-batch

(repeated

cycles)



8.7





17.7

18.4

18.2



17.7

25.3



7.72

7.72

19

13.5

13.5

13.5



70.0

69.7

120

232.8

23.9

26.5

81.3

75.9

165.1

154.97

136.58

57.8

33

23.2

59.8

387.3



[39]

[40]

[40]

[75, 76]

[53]

[53]

[53]

[76]

[63]

[77]

[41]

[41]

[78]

[79]

[79]

[79]



20–30% increment in the fluidity of cell membrane [71, 72]. C. acetobutylicum

was found sensitive to the higher concentration of butanol than 12–13 g l-1 [43,

73]. Various attempts are being made at the organism and process level for

reducing the butanol inhibition. One attractive development in process is as an

integrated system of fermentation and recovery processes, which allows simultaneous production and selective removal of solvents illustrated very significant

results at laboratory scale studies. The common butanol recovery techniques are

adsorption, liquid–liquid extraction, perstraction, reverse osmosis, pervaporation,

and gas stripping, which can be integrated with ABE fermentation for online

removal of products (Table 7.3) [74].

Gas stripping (Fig. 7.3) comprises the most advantageous characteristics such

as simple and economical process (no need of expensive equipments), does not

harm the culture, does not remove the nutrients and reaction intermediates, and

reduces butanol inhibition effectively [39, 40]. At laboratory scale, gas stripping

showed the significant results as integrated with various kinds of fermentation

processes, batch [39, 40, 53], fed-batch [53, 75], and continuous [40]. While

nitrogen [40] and gases produced in fermentation (hydrogen and carbon dioxide)

[39, 53, 75] are used for stripping purpose, whereas utilization of nitrogen gas

reflected more effective recovery than other gases.



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