Chapter 6 Figure 6.3.1
2.6 Fermentation of enzymatic hydrolysate to ethanol
In the process of bioethanol production, mainly the hydrolysate obtained from enzymatic hydrolysis of lignocellulosic biomass has been used as substrate for fermentation. However, in some cases hydrolysate obtained after dilute acid pretreatment can also be utilized for ethanol production by employing pentose fermenting microorganisms (Kuhad et al. 2010b). The selection of the type of fermentation process depends on characteristics of biomass, enzyme and microorganisms involved. On the basis of these factors the modes of fermentation and type of microorganisms has been described subsequently. Table 2.3 represents the summary of various reports in literature for ethanol production from lignocellulosic biomass employing various microorganisms and strategies.
Table 2.3 Ethanol production by enzymatic hydrolysis and fermentation using various lignocellulosic materials.
Lignocellulosic
biomass Microorganism Enzyme Mode of
fermentation
Ethanol yield (g/g
sugar)
Ethanol yield (g/g raw biomass)
Reference Paper sludge Kluyveromyces marxianus
Y01070
Commercial cellulase +
β-glucosidase SSF N. A. 0.33 Kadar et al.
2004 Paper sludge Saccharomyces cerevisiae Commercial cellulase +
β-glucosidase
SSF N. A. 0.33 Kadar et al.
2004 Wheat starch pre-
fermentation effluent
Recombinant Saccharomyces cerevisiae strain REF
Commercial amyloglucosidase
SHF 0.47 N. D. Zaldivar et al.
2005 Wheat starch post-
fermentation effluent
Recombinant Saccharomyces cerevisiae strain REF
Commercial amyloglucosidase
SHF 0.46 N. D. Zaldivar et al.
2005
Rice straw Saccharomyces cerevisiae D4A Commercial cellulase SSF N. A. 0.12 Ko et al. 2009 Red sage
(Lantana camara)
Saccharomyces cerevisiae Commercial cellulase + β-glucosidase
SHF 0.48 0.148 Kuhad et al.
2010b Wheat straw Saccharomyces cerevisiae
TMB3400
Commercial cellulase
+ xylanse + β-glucosidase SSCF 0.35 N. D. Olofsson et al.
2010 Kans grass
(Saccharum spontaneum)
Pichia stipitis Cellulase produced by Aspergillus oryzae MTCC 1846
SHF 0.38 N. D. Chandel et al.
2011 Kans grass
(Saccharum sponteneum)
Saccharomyces cerevisiae MTCC170
Cellulase produced by Trichoderma .reesei NCIM 1052
SHF 0.46 N. D. Kataria and
Ghosh 2011 Water hyacinth
(Eichhornia crassipes)
Saccharomyces cerevisiae Commercial cellulase SHF 0.04* N. D. Satyanagalaks-
hmi et al. 2011 N. A., Not applicable; N. D., not determined; *as calculated from data reported in paper, SHF, Separate hydrolysis and fermentation; SSF, Simultaneous saccharification and fermentation; SSCF, Simultaneous saccharification and co-fermentation, CBP, Consolidated bioprocessing
Table 2.3 (…….Continued)
Lignocellulosic
biomass Microorganism Enzyme Mode of
fermentation
Ethanol yield (g/g sugar)
Ethanol yield (g/g raw biomass)
Reference
Prosopis juliflora Saccharomyces cerevisiae HAU
Commercial cellulase +
β-glucosidase SHF 0.45 N. D. Gupta et al.
2012 Corn cob slurry and
molasses
Recombinant strains of Saccharomyces cerevisiae
Enzyme not required SSCF 0.46* N. D. Koppram et al.
2013 Rice straw Saccharomyces cerevisiae
AYH306
Commercial cellulase + β-glucosidase
SSF N. A. 0.13 Sun and Tao
2013 Phosphoric acid swollen
cellulose
Recombinant Saccharomyces cerevisiae
Enzyme not added CBP 0.19* N. D. Yamada et al.
2013 Commercial cellulose Clostridium phytofermentans
and Candida molischiana or S. cerevisiae cdt-1
Endoglucanase CBP N. A. 0.22 Zuroff et al.
2013 Wheat straw Saccharomyces cerevisiae
ATCC 96581
Cellulase +
β-glucosidase + xylanase
SHF N. D. 0.36 Nanda et al.
2014 Pinewood
(Pinus banksiana)
Saccharomyces cerevisiae ATCC 96581
Cellulase + β-glucosidase + xylanase
SHF N. D. 0.35 Nanda et al.
2014 Timothy grass
(Phleum pretense)
Saccharomyces cerevisiae ATCC 96581
Cellulase + β-glucosidase + xylanase
SHF N. D. 0.39 Nanda et al.
2014 Sugarcane bagasse Recombinant Trichoderma
reesei
Enzyme not added CBP N. A. 0.10 Huang et al.
2014 N. A., Not applicable; N. D., not determined; *as calculated from data reported in paper; SHF, Separate hydrolysis and fermentation; SSF, Simultaneous saccharification and fermentation; SSCF, Simultaneous saccharification and co-fermentation, CBP, Consolidated bioprocessing
The characteristics of cellulases and fermenting microorganisms are the deciding factors for the mode of fermentation. Based on these aspects the fermentation processes may be classified as separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and co-fermentation (SSCF) and consolidated bioprocessing (CBP).
In SHF, the enzymatic hydrolysis and fermentation processes being carried out separately, since, the optimum conditions for cellulase activities and growth of fermenting microorganism may not be similar. The main attraction of SHF process is that both hydrolysis and fermentation can be carried out under optimum conditions (Sanchez and Cardona 2008). Kuhad et al. (2010b) carried out ethanol production by SHF of Lantana camara, which resulted in maximum ethanol yield of 148 g/1000 g of pretreated L. camara biomass. The bioethanol production by SHF can be made more manageable by coupling the separate processes as SSF. The SSF is more advantageous over SHF since it eliminates the glucose inhibition to cellulases by reducing the sugar levels in the fermentation broth which also results in reduced the risk of contamination. Also it is more energy efficient process, in view of the utilization of a single vessel for two processes and thus lesser energy consumption for maintenance of physical conditions (Sanchez and Cardona 2008). Stenberg et al.
(2000) used the resulting slurry of the steam pretreatment of SO2-impregnated spruce for SSF and observed that the ethanol productivity was doubled as compared to SHF.
The major limitation of SSF process is the compromise with the optimum conditions of two separate processes viz., enzymatic hydrolysis and fermentation. Most of the cellulases are optimally active at 40-50oC and pH of 4.0-5.0 whereas the optimal
culture conditions of fermenting microorganisms are 30oC and pH of 4.0-5.0. On the other hand, fermentation of pentose sugars is optimally performed at 30-70oC and pH of 5.0-7.0 (Olsson and Hahn-Hagerdal 1996).
Another mode of ethanol fermentation is the inclusion of the pentose fermentation in the SSF, process called simultaneous saccharification and co- fermentation (SSCF). However, the major prerequisite of this mode is the compatibility of both microorganisms in terms of optimal temperature and pH.
Olsson and Hahn-Hagerdal (1996) carried out SSCF of aspen by co-culturing Pichia stipitis and Brettanomyces clausennii at 38oC. The use of recombinant microorganism for utilization of both pentose and hexose sugars has been preferred more currently. Ethanol production by SSCF process was carried out by Olofsson et al. (2010), where acid and enzymatic hydrolyzate of wheat straw were fermented by employing a recombinant Saccharomyces cerevisiae TMB3400 strain. Koppram et al. (2013) have used recombinant strains of Saccharomyces cerevisiae for ethanol production from corn cob slurry and molasses by SSCF process.
The other mode of ethanol production from lignocellulosic biomass is consolidated bioprocessing (CBP), also known as direct microbial conversion (DMC). CBP involves single microbial community to carry out four successive bioconversions, viz., production of cellulases and hemicellulases, hydrolysis of delignified biomass to sugars and fermentation of sugars to ethanol (Cardona and Sanchez 2007). Therefore, the capital and operation costs of cellulase production can be reduced by opting for this strategy (Lynd 1996). However, the optimal conditions for enzyme production, hydrolysis of biomass and fermentation are required to be
entirely compatible. Since, a single microorganism capable of performing all these processes has not been found in nature, only microbial consortium or genetically modified microorganisms can be employed in CBP (Wyman 1994, McMillan 1997, Claassen et al. 1999, Lynd et al. 2002). Zuroff et al. (2013) have used co-cultures of Clostridium phytofermentans with Candida molischiana or S. cerevisiae cdt-1 for ethanol production from cellulose. Recombinant strains of Clostridia (Maki et al.
2013), S. cerevisiae (Sun et al. 2012, Yamada et al. 2013) and Trichoderma sp.
(Huang et al. 2014) has been used in recent studies.