Abstract This chapter details the recent advances made on bioconversion of lignocellulosic biomass to butanol, a superior biofuel that can be used in internal combustion engines or transportation industry. It should be noted that butanol producing cultures cannot tolerate or produce more than 20–30 g/L of acetone-butanol-ethanol (ABE) in batch reactors of which butanol is of the order of 13–18 g/L. This is due to toxicity of butanol to the culture. In order to overcome this challenge, two approaches have been applied: (1) developing more butanol tolerant strains using genetic engineering techniques and (2) employing process engineering approaches to simultaneously recover butanol from the fermentation broth thus not allowing butanol concentrations in the reactor to accumulate beyond culture’s tolerance. By the application of the fi rst approach, a number of butanol producing strains have been developed; however, none of these accumulated greater than 1,200 mg/L (1.2 g/L) butanol, while using the second approach total ABE up to 461 g/L has been produced. Attempts to improve the newly developed strains are continuing.
Chapter 15 Cellulosic Butanol Production from Agricultural Biomass and Residues: Recent Advances in Technology *
N. Qureshi , S. Liu , and T. C. Ezeji
N. Qureshi (*) United States Department of Agriculture (USDA), Agricultural Research Service (ARS) , National Center for Agricultural Utilization Research (NCAUR), Bioenergy Research Unit, Renewable Products Technology , 1815 N University Street , Peoria , IL 61604 , USA e-mail: [email protected]
S. Liu United States Department of Agriculture (USDA), Agricultural Research Service (ARS) , National Center for Agricultural Utilization Research (NCAUR), Renewable Products Technology , 1815 N University Street , Peoria , IL 61604 , USA
T. C. Ezeji Department of Animal Sciences and Ohio State Agricultural Research and Development Center (OARDC) , The Ohio State University , 305 Gerlaugh Hall, 1680 Madison Avenue , Wooster , OH 44691 , USA
* Mention of trade names or commercial products in this article is solely for the purpose of providing scienti fi c information and does not imply recommendation or endorsement by the United States Department of Agriculture. USDA is an equal opportunity provider and employer.
248 N. Qureshi et al.
Lignocellulosic substrates have been used to produce butanol due to their abundant availability and economical prices usually in the range of $24–60/ton as opposed to corn prices which have been in the range of $153–218/ton during recent months. It should be noted that lignocellulosic substrates require separate hydrolysis prior to fermentation. In a more recent approach, hydrolysis and fermentation (and simulta-neous recovery) have been integrated or combined to reduce the cost of butanol production from cellulosic substrates. Using such an approach, up to 192 g/L ABE was produced from 430 g/L cellulosic biomass/sugars. Additionally, this chapter provides details of process integration and simultaneous product recovery technologies for butanol production.
1 Introduction
Throughout the world, countries are promoting biofuel development with mandates and directives. The US, by using corn as the primary feedstock, produced 10.6 billion gallons of ethanol in 2009, and more than 12 billion gallons was produced in 2010 (Renewable fuels association 2010). Concomitantly there was a marked increase in the cost of corn, an important livestock feed component. The extent to which these two trends are associated is unknown and is subject to considerable debate. In any case, there is considerable interest in the production of biofuels using alternative substrates, such as lignocellulosic biomass. Butanol (also known as n -butanol), a superior biofuel than ethanol, can be produced by fermentation from lignocellulosic biomass and contains more energy on per gallon (or per lb) basis. In this fermentation, all three components (acetone, butanol, ethanol; ABE) are produced simultaneously with butanol being the major product.
Butanol, currently manufactured with petroleum feedstocks, is an important chemical with many applications in the production of solvents, plasticizers, butylamines, amino resins, butyl acetates, etc. [ 14 ] . In 2008, global consumption of butanol was estimated as 350 million gallons, according to the chemical giant BASF. Butanol has several advantages over ethanol as a fuel extender or fuel substitute. It has an energy content that is similar to gasoline, which means fewer gallons are required than ethanol to achieve the same energy output [ 42 ] . It has a lower vapor pressure than ethanol, which makes it safer to transport and use in combustion engines [ 14 ] . A car can also use butanol as a fuel with little or no modi fi cation to the engine [ 3 ] . In addition, the existing gasoline station and transport infrastructures can continue to be used for butanol transport without modi fi cation because butanol is less hygro-scopic and less corrosive to the pipelines than ethanol. Currently, butanol is not used as a biofuel due to several challenges. These challenges include substrate cost, butanol toxicity/inhibition to the fermenting microorganisms, and the low butanol titer in the fermentation broth which results in high energy requirements for the recovery of butanol from this dilute stream. To solve the problem of butanol toxicity to the culture, a signi fi cant amount of research has been performed on the use of alternative fermentation and product recovery technologies for biobutanol production.
24915 Cellulosic Butanol Production from Agricultural Biomass and Residues…
Technologies involving the use of immobilized and cell recycle continuous bioreactors, adsorption, gas stripping, separation using ionic liquids, liquid-liquid extraction, pervaporation, aqueous two phase separation, supercritical extraction, and perstraction have allowed the use of concentrated sugar solutions (up to 500 g/L) for butanol fermentation and the production of a highly concentrated butanol product stream [ 16, 20 ] . Typically, ABE fermentation by Clostridium beijerinckii proceeds in two phases. The fi rst phase is called the acidogenic phase (acetic and butyric acid are produced) and is growth associated and the second is a solventogenic phase characterized by the uptake of acids and ABE production and which is relatively nongrowth associated [ 16, 20 ] .
Scienti fi c research including that published by the authors and others, demonstrates the ability of solventogenic Clostridium species to use pentose and hexose sugars, the major sugar components of lignocellulosic biomass, for growth and ABE pro-duction [ 15 ] . Lignocellulosic biomass represents the most abundant renewable energy resource on the planet. The “Billion Ton Study” published by the US Department of Energy (DOE) in 2005 indicated that there could be 1.3 billion dry tons of biomass available per year, enough to produce biofuels to meet more than one-third of the current demand for transportation fuels. Recent breakthroughs in the development of hybrid and electric cars could further reduce petroleum needs and increase this estimate to two-thirds. Collectively, while there is compelling evidence that solventogenic Clostridium species are the best natural butanol producing microorganisms with the capacity to use pentose and hexose sugars for butanol production, these microorganisms, including other fermenting microorganisms, use lignocellulosic biomass hydrolyzates poorly due to the presence of inhibitory compounds [ 15, 16, 18, 20 ] .
Because of the recalcitrance of biomass, pretreatment is commonly used for the hydrolysis of the hemicellulose fraction and the disruption of the lignin sheath of bio-mass so that enzymatic hydrolysis of the cellulose fraction to glucose can be achieved with greater yield. Unfortunately, during pretreatment and hydrolysis, a complex mix-ture of microbial inhibitors is generated. Even biomass hydrolyzates produced from the most benign pretreatment and hydrolysis processes can contain some microbial inhibitors because some of the inhibitors are components of hemicellulose and lignin structures of biomass [ 19 ] . Consequently, removal of inhibitory compounds from hydrolyzates is typically necessary to facilitate ef fi cient microbial growth and biofuel production. Considering the need of keeping low process costs, the removal of inhibi-tors from hydrolyzates prior to fermentation is not economically viable given the costs associated with additional processing steps and potential loss of fermentable sugars.
Development of inhibitor tolerant and hyperbutanol producing microbial strains that ef fi ciently metabolize mixed sugars, and compatible advanced fermentation and recovery technologies will accelerate the development of a sustainable lignocel-lulosic biomass-to-biofuels industry. Many laboratories, including those of the authors, are currently involved in research directed toward strain development for ef fi cient conversion of biomass to butanol and advanced fermentation and recovery techniques for butanol production. This chapter details the recent developments that have been made in this direction.
250 N. Qureshi et al.
2 Butanol Producing Cultures
2.1 Traditional Strains
The naturally occurring butanol producing microbes are Gram-positive endospore-forming obligate anaerobes. They belong exclusively to the genus Clostridium which includes C. acetobutylicum , C. aurantibutyricum, C. beijerinckii , C. cadav-eris, C. pasteurianum, C. saccharoperbutylacetonicum, C. saccharobutylicum (P262) , C. sporogenes, and C. tetanomorphum [ 30 ] . Among these producing strains, the highest butanol production trait was found in C. acetobutylicum and C. beijer-inckii species. Extensive studies related to understanding the molecular mecha-nisms, developing butanol tolerant Clostridial strains were performed with C. acetobutylicum ATCC 824 , C . beijerinckii NCIMB 8052, C. beijerinckii P260, and C. beijerinckii BA101 [ 6, 8, 26, 34, 68, 70, 73 ] .
The physiological state of the cells is directly associated with ABE fermentation. Butyric and acetic acids are produced by C. acetobutylicum strains during the rapid anaerobic growth phase (acidogenesis phase), resulting in a decrease of the medium pH. Then the butyric and acetic acids are partially re-assimilated and converted into ABE (solventogenic phase) when the culture progresses into the stationary phase, con-sequently, the pH of the culture is stabilized or increased slightly [ 31 ] .
The biochemical and molecular events that trigger the switch from acidogenesis to solventogenesis have begun to unfold with time. The decreases of acidogenic enzymes and increases of solventogenic enzymes were reported earlier [ 22, 29, 31 ] . The intracellular concentrations of coenzyme A (CoA) and derivatives were found playing regulatory roles [ 10, 46 ] . A recent study demonstrated increases of butyryl-phosphate (BuP) during the switch of solvent production and suggested a role of BuP in regulating the transition from acidogenesis to solventogenesis [ 75 ] .
The completion of genome sequencing of both C. acetobutylicum ATCC 824 [ 44 ] and C. beijerinckii NCIMB 8052 [ 67 ] have facilitated microarray analyses to elucidate the molecular mechanism of the shift from acidogenesis to solventogene-sis [ 2, 67 ] . A response regulator gene spo0A positively controls sporulation and promotes the expression of the solvent formation genes ( aad, ctfA, ctfB, and adc ) during stationary phase, thus enhancing solvent formation. Inactivation of spo0A in C. acetobutylicum led to the asporogenic strain SK01 with much reduced butanol production [ 25 ] . The overexpression of the spo0A in C. acetobutylicum ATCC 824 (pMSPOA) resulted in accelerated endospore formation but again decreased butanol production. The spo0A plays a regulatory role on sporulation vs. solvent gene expres-sion. The overexpression apparently tips the balance in favor of accelerated sporula-tion at the expense of overall solvent production [ 25 ] . The expression of sporulation genes spo0A and sigF operon in C. beijerinckii NCIMB 8052 was induced during the acidogenic phase and increased signi fi cantly during the onset of solvent formation [ 67 ] . Interestingly, these genes were induced but the level of induction is two to eight-fold lower in the hyperbutanol-producing C. beijerinckii BA101 strain [ 67 ] . Based on the above-mentioned fi ndings, the fi ne tuning of spo0A and sigF at transcriptional
25115 Cellulosic Butanol Production from Agricultural Biomass and Residues…
and translational levels via pathway engineering will help to delay sporulation and to improve solvent production in Clostridia . However, it is challenging to perform genetic manipulations using the solvent producing Clostridium species. Major barri-ers include: (a) the strict anaerobic growth requirement, (b) the slow growth, and (c) the small number of genetic tools available to modify the Clostridium strains. Therefore, metabolic engineering of other fast-growing, nonspore-forming microbes should be explored for cost-effective butanol production.
2.2 Genetically Engineered Strains
The current fermentative production of butanol is not cost effective because of (1) a spore-forming life cycle, (2) butanol toxicity, (3) slow growth and instability of the producing strains and (4) production of other unwanted byproducts including butyrate, acetate, acetone, and ethanol [ 31 ] . In addition, no commercial microbes are available to ferment various lignocellulosic hydrolyzate mixtures into butanol. Thus, new microbes are needed for fermentative conversion of these hydrolyzates to butanol biofuel.
In the recent years, the fermentative production of butanol has been demonstrated in engineered strains of Escherichia coli [ 4, 30, 43 ] and Saccharomyces cerevisiae [ 71 ] . The entire butanol production pathway from Clostridium has been recon-structed and introduced into these model hosts. More recently, the pathway recon-struction strategy was applied to more robust and butanol tolerant species including Pseudomonas putida , Bacillus subtilis [ 43 ] , Lactobacillus brevis [ 7 ] , Lactobacillus buchneri [ 35 ] , and Corynebacterium glutamicum [ 69 ] . Although the polycistronic expression of the butanol production pathway genes are achieved in these robust host cells, the butanol titers of these recombinant organisms are relatively low (Table 1 ) and has yet to exceed 19.50 g/L, a production level that can be achieved by Clostridium species [ 57 ] .
Three of the highest butanol producing strains are the engineered E. coli strains JCL187, EB4.F, and BUT2, which can produce 552, 580, and 1,200 mg/L of butanol, respectively (Table 1 ). Although the E. coli BUT2 was reported as producing more butanol, the cells were fi rst grown aerobically and later resuspended for anaerobic fermentation. Furthermore, these strains suffer from butanol toxicity (very sensitive to butanol) and can be killed by the accumulation of no more than 15 g/L butanol [ 32 ] . So far, no breakthrough improvement of butanol production strains from ligno-cellulosic biomass hydrolyzates has been reported and yet, more research is needed for strain development.
2.3 Potential of Gram-Positive Bacteria for Butanol Production
Gram-positive bacteria possess several desirable traits, including the ability to fer-ment multiple sugars simultaneously, to grow at lower pH values, and for some strains, to grow at a temperature range from 30 to 50°C [ 9 ] . The Gram-positive Lactic acid bacteria (LAB) are considered attractive biocatalysts for biomass to
252 N. Qureshi et al.
Table 1 Genetically modi fi ed butanol producing strains
Culture
Max. tolerance (g/L)
Max. production (mg/L) Substrate
Fermentation conditions and reference
Saccharomyces cerevisiae ESY7
Unknown 2.5 Glucose Semianaerobic; Steen et al. [ 71 ]
Escherichia coli JCL 187 15 552 TB glucose or glycerol
Aerobically; Atsumi et al. [ 4 ]
E. coli JCL16, Kivd + ADH2 ilva + leuABCD
Unknown 44–237 Glucose and l -threonine
Nonfermentative pathway; Atsumi et al. [ 5 ]
E. coli BUT1 Unknown 320 Glucose Resuspended cells for anaerobic fermenta-tion; Inui et al. [ 30 ]
E. coli BUT2 Unknown 1,200 Glucose
E. coli BL21 EB4.G 10 580 Glucose Aerobically; Nielsen et al. [ 43 ]
Pseudomonas putida PS1.0
7.5 120 Glycerol Aerobic, TB medium + 5 g/L glycerol; Nielsen et al. [ 43 ]
PS2.0 7.5 112 Glycerol
Bacillus subtilis BK1.0 12.5 24 Glucose or glycerol
Anaerobic, TB medium + 5 g/L glucose or glycerol; Nielsen et al. [ 43 ]
Corynebacterium glutamicum pKS167
20 140 Glucose Aerobic; Smith et al. [ 69 ]
Lactobacillus brevis pHYc-bcs
20–30 300 Glucose Semiaerobically; Berezina et al. [ 7 ]
Lactobacillus brevis pHYc-thl-bcs
20–30 250 Glucose Semiaerobically; Berezina et al. [ 7 ]
Lactobacillus buchneri pTRKH2692Thl
25–30 66 Glucose Anaerobic; Liu et al. [ 35 ]
Lactococcus lactis pTRKH2692Thl
25–30 28 Glucose Anaerobic; Liu et al. [ 35 ]
biofuels for several reasons. LAB have GRAS (Generally Recognized As Safe) status, lack cytochromes, and possess aero-tolerant or anaerobic and obligatory fer-mentative pathways. They ferment a variety of carbohydrates (both hexoses and pentoses) naturally for growth and fermentation [ 9 ] . LAB have relative small genomes [ 39 ] that range from 1.7 to 3.3 Mb. Genetic engineering tools are available in several model strains, and in fact, recombinant strains have been developed for production of B-vitamins, mannitol and sorbitol, lactic acid, bacteriocins, exopoly-saccharides and oligosaccharides, and other chemicals [ 28 ] . In addition, LAB were isolated as source of spoilage in ABE production processes [ 76 ] , and most LAB species are butanol tolerant ( [ 32 ] and our unpublished data). Certain species, such as L. brevis and L. buchneri, are known to grow in the presence of inhibitors derived from plant materials such as wine polyphenolics or hop acids present in beer [ 36, 66 ] , thus LAB and other nonspore-forming Gram-positive species should be further explored for biomass to butanol production by metabolic pathway reconstructions.
25315 Cellulosic Butanol Production from Agricultural Biomass and Residues…
3 Production of Butanol from Agricultural Residues
Production of butanol is adversely affected by the high costs of traditional substrates such as glucose, corn, sugarcane molasses, and whey permeate. To reduce the cost of production, this biofuel could be produced from economically available renewable feedstocks such as corn stover, wheat, barley, and rice straws, corn fi ber, switchgrass, alfalfa, reed canary grass, sugarcane bagasse, miscanthus, waste paper, distillers dry grains and solubles (DDGS), and soy molasses. Currently, costs of corn stover, grasses, and straws are in the range of $24–60/ton as opposed to corn which has ranged from $153–218/ton during recent months. It should be noted that while prices of these residue feedstocks are low, they are associated with additional process steps such as pretreatment, and hydrolysis prior to fermentation. Additionally, fermentation inhibitors are generated during the pretreatment process which either halt or slow down reaction rates or fermentation. This section describes production of butanol from wheat straw, barley straw, corn stover, switchgrass, corn fi ber, and DDGS and challenges that are faced when handling these feedstocks for the production of this biofuel.
3.1 ABE Production from Wheat and Barley Straws, Corn Stover, Switchgrass, and Dried Distillers’ Grains and Solubles
Wheat straw was found to be a novel substrate for the production of ABE in batch fermentation with total ABE production of 25 g/L. In this system, an ABE produc-tivity of 0.60 g/L h was observed which is over 200% that obtained in a control glucose fermentation run ( [ 57 ] ; Table 2 ). It was speculated that dilute sulfuric acid pretreated and enzymatically hydrolyzed wheat straw (wheat straw hydrolyzate, WSH) contained fermentation stimulating components that enhanced both ABE production levels and productivity. A detailed discussion of fermentation stimulat-ing components present in WSH has been given in Sect. 3.3 .
Barley straw is another substrate that is economically available and appears to be similar to wheat straw. Initially, it was thought that the rate of fermentation of barley straw hydrolyzate (BSH) would be similar to that of WSH. However, it was observed that BSH is toxic to C. beijerinckii P260. In order to overcome this toxicity prob-lem, a number of treatments were applied including diluting the hydrolyzate with water, mixing with WSH, and overliming. Although all three techniques were suc-cessful, overliming resulted in the highest production of ABE (26.64 g/L) suggest-ing that fermentation inhibitors were removed by overliming [ 61 ] . The control fermentation containing equivalent amount of glucose resulted in the production of 21.06 g/L ABE.
Corn stover, which is available in large quantities in the Midwestern region of the United States, can be converted to butanol after pretreatment with dilute sulfuric
254 N. Qureshi et al.
acid and hydrolysis with enzymes. The reader is informed that untreated corn stover hydrolyzate (CSH) did not support cell growth and fermentation, suggesting that it was toxic to the culture. In order to relieve toxic effect, treatments similar to those used for BSH were applied followed by fermentation using C. beijerinckii P260. The lime treated hydrolyzate resulted in the production of 26.27 g/L ABE (Table 2 ) compared to 21.06 g/L using glucose as control [ 62 ] . Parekh et al. [ 45 ] also pro-duced ABE from CSH employing C. acetobutylicum (renamed as C. saccharobu-tylicum P262). These investigators pretreated corn stover employing SO
2 followed
by hydrolysis using enzymes. In their studies total ABE concentration of 25.70 g/L was achieved with a yield of 0.34 and a productivity of 1.07 g/L h. It is noteworthy to mention that their hydrolyzate was not toxic to the culture and required no addi-tional treatment such as overliming prior to fermentation. It is likely that SO
2 pre-
treatment does not generate inhibitors that inhibit culture’s viability and fermentative capacity. It is also possible that Clostridium sacchrobutylicum P262 is a more toler-ant strain than C. beijerinckii P260. The reader is informed that productivity obtained in their system cannot be compared with that achieved in our CSH fermentations as these authors employed cell recycle fermentations that result in signi fi cantly improved productivity due to increased cell concentration in the reactor. Other authors that reported butanol production from CSH and corncob hydrolyzate (CCH) include Marchal et al. [ 41 ] . In these investigations corncobs were pretreated with steam expansion followed by enzymatic hydrolysis and fermentation in large reac-tor (48,000 L) which resulted in an ABE concentration of 20.50 g/L (Table 2 ).
Attempts have been made to produce butanol from switchgrass hydrolyzate (SGH). The switchgrass was pretreated and hydrolyzed in a similar manner as barley straw and corn stover and the hydrolyzate was subjected to butanol fermentation.
Table 2 Production of butanol from various agricultural residues
Biomass ABE before lime treatment (g/L)
ABE after lime treatment
Reference ABE (g/L) Yield (−) Productivity
(g/L h)
Control 21.06 a a a Qureshi et al. [ 61 ] WSH 25.00 a a a Qureshi et al. [ 57 ] BSH 7.09 26.64 0.43 0.39 Qureshi et al. [ 61 ] CSH b 26.27 0.44 0.31 Qureshi et al. [ 62 ] CSH 25.70 c a a a Parekh et al. [ 45 ] CCH 20.50 d d d d Marchal et al. [ 41 ] SGH 1.48 e f f Qureshi et al. [ 62 ] DDGS b 12.8 0.31 0.18 Ezeji and Blaschek [ 15 ]
a No treatment required as fermentation was good without treatment b Resulted in no growth and no fermentation c Cell recycle experiment (calculated values: yield 0.34, and productivity 1.07 g/L h). Culture used Clostridium acetobutylicum P262 d Detoxi fi cation not reported (perhaps it did not require) e Poor cell growth and fermentation f Not calculated due to poor growth and fermentation
25515 Cellulosic Butanol Production from Agricultural Biomass and Residues…
The untreated SGH did not result in the production of more than 1.48 g/L ABE [ 62 ] . When the SGH was diluted twofold with water, the culture produced 14.61 g/L ABE. In order to improve it further the SGH was mixed with WSH and fermented using C. beijerinckii P260. In this fermentation the culture produced 8.91 g/L ABE, while lime treated SGH resulted in poor cell growth (0.20 g/L) and no fermentation. It is likely that SGH still contained cell growth and fermentation inhibitors.
Studies were also performed to produce ABE from corn fi ber hydrolyzate (CFH; [ 49 ] ). It was found that untreated CFH was also toxic to the culture and it resulted in the production of 1.7 g/L ABE, while XAD-4 resin (trade name) treated CFH resulted in the production of 9.3 g/L ABE. This suggested that fermentation inhibi-tors were removed by the resin. Further investigations were performed on the pro-duction of ABE from corn fi ber arabinoxylan and 9.60 g/L ABE was produced from this substrate in a batch system [ 51 ] .
In an attempt to produce butanol from DDGS, Ezeji and Blaschek [ 15 ] pretreated this lignocellulosic substrate with dilute sulfuric acid, hot water, and ammonia fi ber expansion (AFEX) followed by enzymatic hydrolysis. Upon hydrolysis 52.6, 48.8, and 41.4 g/L total sugars were obtained, respectively, from 150 g/L DDGS total solids. Fermentation of these hydrolyzates was then performed using a number of solventogenic cultures including C. beijerinckii P260. These cultures were not able to grow in the hydrolyzate due to the presence of toxic chemicals generated during dilute sulfuric acid pretreatment process, indicating that removal of toxic chemicals was essential prior to fermentation. Toxic chemicals were removed by overliming the hydrolyzate and the detoxi fi ed hydrolyzate supported cell growth and fermenta-tion thus producing 12.8 g/L total ABE using C. beijerinckii P260. This system resulted in a productivity of 0.18 g/L h and an ABE yield of 0.31 [ 15 ] .
As indicated above, conversion of cellulosic biomass to butanol requires pretreatment employing dilute sulfuric acid or dilute sodium hydroxide, or alkaline peroxide. During the pretreatment and neutralization process, inhibitors such as salts (sodium acetate, sodium chloride, and sodium sulfate), and chemicals including furfural, hydroxymethyl furfural (HMF), syringealdehyde, and acids (acetic, glucuronic, ferulic, and r -coumaric) are produced. Some of these chemicals are toxic to the culture. In a recent study it was observed that sodium sulfate [ 18 ] , sodium chloride [ 59 ] , glucuronic, ferulic, and r -coumaric acids, phenol, and syringealdehyde [ 18 ] were toxic to a butanol producing culture. Ferulic acid, at a concentration as low as 0.3 g/L, was a strong inhibitor to both cell growth and fermentation. On the contrary, syringealdehyde (0.3–1.0 g/L) was not so toxic to the cell growth; how-ever, it resulted in complete arrest of ABE production [ 18 ] . As expected, phenol was found to be inhibitory to both cell growth and ABE production.
In order to produce ABE from agricultural residues successfully, inhibitors present in the hydrolyzates must be removed prior to fermentation. In recent studies, Qureshi
256 N. Qureshi et al.
et al. [ 61, 62 ] attempted to produce butanol from untreated and treated BSH and CSH. ABE was successfully produced from the treated hydrolyzates, however, strong inhibition of cell growth was still observed. In case of BSH and CSH, 0.80 and 0.77 g/L cell mass was obtained as compared to 2.66 g/L in the control experiment in which glucose was used. It is likely that chemical inhibitors were removed from the hydrolyzates by overliming leaving behind salts that were generated during neutralization. In order to reduce or eliminate cell growth inhibition, it is recom-mended that salts also should be removed from the medium by electrodialysis [ 59 ] followed by fermentation. Possibly, removal of salts could improve both cell growth and productivity.
3.3 Hydrolyzate Fermentation Stimulators
In an interesting investigation, it was observed that some of the cellulosic hydrolyzate chemicals that are generated during the pretreatment or the neutralization process stimulate both cell growth and ABE production. These chemicals include sodium acetate, furfural, and HMF. In the presence of 8.9 g/L sodium acetate cell growth was slightly improved. In the control experiment 17.8 g/L ABE was produced while in the presence of 8.9 g/L acetate 20.3 g/L ABE was produced, showing an increase of 14%. An improvement in fermentation performance on supplementation of acetate has previously been documented [ 11, 24 ] . Inclusion of furfural and HMF (0.3–2.0 g/L) in the fermentation medium improved both cell concentration and ABE production [ 18 ] . It is suggested that acetate, furfural, and HMF are bene fi cial to this fermentation within a certain concentration range.
4 Product Separation Techniques
Butanol fermentation results in low butanol concentration in the fermentation broth due to toxicity of this product to the culture. A maximum concentration of butanol or ABE that can be produced in a batch process is limited to 20–30 g/L. This low con-centration of ABE possesses the following problems: (1) low butanol/ABE produc-tivity usually of the order of 0.30–0.50 g/L h; (2) use of dilute sugar solution as feed usually in the range of 50–60 g/L; and (3) energy inef fi cient recovery of the fi nal product. Recovery of such a low amount of product (20–30 g/L) in combination with butanol’s high boiling point (118°C; higher than water) requires a very high amount of energy for distillation thus making the process of butanol production uneconomic. Use of dilute sugar solution as feed for this fermentation requires more energy to prepare and sterilize it (feed) thus resulting in elevated capital and process costs.
In order to make butanol production economic from renewable biomass/residues, one or both of the following approaches should be considered: (1) developing a cul-ture that can produce and tolerate high concentration of this product by application of
25715 Cellulosic Butanol Production from Agricultural Biomass and Residues…
microbial genetics and/or (2) simultaneous removal of the toxic product from the fermentation broth using energy ef fi cient alternative product recovery technique, an engineering approach. During the last 3 decades, progress in the fi rst direction has been limited and no culture as yet has been developed that can tolerate or produce butanol ( n -butanol) concentration in excess of 20 g/L, while approach number two has made signi fi cant strides in this direction. Simultaneous recovery of butanol from the fermentation broth has been bene fi cial for this process and following advance-ments have been made: (1) productivity in free cell fermentations has been improved by a factor of 2–3 (0.98 g/L h, [ 17 ] ; and 15.8–16.2 g/L h in immobilized cell reactors, [ 33, 63 ] ); (2) concentrated sugar solution up to 500 g/L have been used; and fermen-tations have been prolonged thus eliminating down time. Table 3 shows ABE pro-ductivity, type of reactor employed, reactor life, and concentration of sugar solution that has been used. Some of the techniques that have been employed for simultane-ous product removal include adsorption [ 50, 74 ] , N
2 gas stripping [ 13, 52 ] , CO
2 & H
2
(fermentation gases) gas stripping [ 38, 55 ] , pervaporation [ 23, 37, 56, 72 ] , liquid-liquid extraction [ 53, 55, 64, 65 ] , perstraction [ 54, 55 ] , and reverse osmosis [ 21 ] . Details of these product separation techniques are published elsewhere [ 12, 37, 48 ] .
5 Process Integration
The purpose of process integration is to combine more than one unit operations into a single unit to reduce both capital and operational cost. In butanol fermentation, early reports on process integration were published in the late 1980s and the early
Table 3 Selected bioreactor systems employed for the production of butanol
a Integrated system with simultaneous product removal b Some of the parameters are calculated values;—not reported
258 N. Qureshi et al.
1990s [ 13, 52 ] where butanol fermentation was integrated with product separation. In the case of butanol production by fermentation, following process integrations can occur depending upon the feedstock used:
1. Fermentation and recovery of butanol. 2. Hydrolysis of feedstock and fermentation to butanol. 3. Hydrolysis of feedstock, fermentation, and separation of butanol.
In the case of fermentation and recovery, feedstock does not require hydrolysis such as glucose or if it requires hydrolysis, the later can be performed by the butanol producing culture while fermentation occurs. An example of this is the production of butanol from whey permeate (a byproduct of cheese making industry) and simul-taneous recovery by gas stripping. Whey permeate contains lactose (a disaccharide of glucose and galactose) which can be hydrolyzed by the culture into monomeric sugar units. Then both of these sugars can be converted to butanol. Butanol that is produced by the culture can be recovered simultaneously by one of the product recovery techniques mentioned in Sect. 4 .
The second group is where a feedstock requires hydrolysis using exogenous enzymes. In this case, the butanol producing culture is not capable of hydrolyzing the substrate and hence either enzymes are added to the reactor or hydrolytic enzyme producing culture is propagated with the butanol producing culture. The conditions under which enzymes perform hydrolysis need to be close to the cultivation condi-tions of butanol producing culture. In this system, hydrolysis and fermentation are all combined. This group is called SSF (Simultaneous Sacchari fi cation and Fermentation). The third group is where hydrolysis, fermentation, and recovery are combined. In this case, enzymes or a hydrolytic enzyme producing culture hydroly-ses the feedstock such as cellulosic biomass, the butanol producing culture performs fermentation, and application of product (butanol) recovery technique recovers the product simultaneously from the reactor. The third process can be abbreviated as SSFR (simultaneous sacchari fi cation, fermentation, and recovery).
5.1 Separate Hydrolysis, Fermentation, and Recovery
Although the cost of agricultural residues is much lower than the cost of other conventional substrates such as corn, the process of butanol production from residues requires additional process steps. One such step is the hydrolysis of residues to simple sugars prior to their conversion to butanol. Unless these residues are converted to simple sugars, they cannot be used by butanol producing cultures. The hydrolysis fi rst requires pretreatment using dilute sulfuric acid or dilute alkali at 121°C or higher. This is done to make cellulosic fi bers accessible to enzymes. For our studies dilute sulfuric acid was used in order to make the process simple [ 57 ] . Following pretreatment, the biomass was hydrolyzed with enzymes which were then fermented to butanol. Butanol or ABE was then recovered using the gas stripping technique [ 57 ] . It is suggested that any of the product recovery techniques described by
25915 Cellulosic Butanol Production from Agricultural Biomass and Residues…
Maddox [ 37 ] or Qureshi [ 48 ] can be used for ABE removal. The overall process of production of butanol from cellulosic biomass by this process requires three sepa-rate steps (hydrolysis, fermentation, and recovery) and is called “Separate Hydrolysis, Fermentation, and Recovery (SHFR).” Figure 1a shows a schematic diagram of butanol production by this process.
5.2 Simultaneous Hydrolysis, Fermentation, and Recovery
The process described under this category aims at reducing the number of process steps (SSFR) as opposed to SHFR. In the SSFR process, after pretreatment of the cellulosic biomass, enzymes are added to the reactor and at the same time the reactor is inoculated with a butanol producing culture. Since optimum pH for hydrolytic enzymes and culture that produce butanol is the similar (5.0), these two unit opera-tions can be performed simultaneously in the same reactor. It should be noted that enzymes perform more ef fi ciently at 45°C while the optimum temperature for butanol producing culture is only 35°C. In spite of the different optimum temperatures for the enzymes and the culture, this process performs well. In order to make this
Hydrolysis(Step I)
+
Fermentation(Step II)
Recovery(Step III)
Feed Product
An integrated/combined process of hydrolysis (Step I), fermentation(Step II), and product recovery (Step III)
+Feed Product
a
b
Fig. 1 A schematic diagram of production of butanol/ABE from lignocellulosic biomass employ-ing Clostridium beijerinckii P260. ( a ) SHFR (separate hydrolysis fermentation and recovery pro-cess); ( b ) SSFR (simultaneous sacchari fi cation, fermentation, and recovery process; also known as an integrated process)
260 N. Qureshi et al.
process more ef fi cient, it is recommended that new hydrolytic enzymes be developed with 35°C as their optimum temperature. Since butanol is toxic to the microbial cells, the product should be removed simultaneously. Continuous removal of butanol from the fermentation broth would also prolong the reaction thus improving ef fi ciency of the process further. A schematic diagram of the SSFR process is shown in Fig. 1b . The overall process bene fi ts from this system as all three unit operations are performed in a single reactor. This system has been applied to butanol produc-tion from wheat straw [ 49, 58 ] .
In a study, Marchal et al. [ 40 ] produced ABE in an integrated system where hydrolysis and fermentation were combined. These authors did not apply simulta-neous product removal technique to remove ABE from the system/fermentation broth. In this system wheat straw was pretreated with alkali followed by washing the straw several times with tap water. Cellulase enzyme was prepared from Trichoderma reesei Cl-847 and added to the fermentation medium which was inoc-ulated by C. acetobutylicum IFP 921. These investigations were performed in a 6 L bioreactor with 2 L medium containing 194 g (dry weight) of pretreated wheat straw, 12 g of dried corn steep liquor and 360 mL of undiluted enzyme preparation. Approximately 17.3 g/L ABE was produced from the wheat straw.
5.2.1 Batch Fermentation and Recovery
The studies on butanol production employing a SSFR process were performed using wheat straw as a substrate [ 60 ] . In this process wheat straw was pretreated with dilute sulfuric acid at 121°C for 1 h followed by cooling the mixture to 45°C and adjusting pH to 5.0 with concentrated NaOH solution. During these studies a num-ber of experiments were performed with the following conclusions: (1) presence of sediments in the reactor does not inhibit fermentation; (2) agitation by gas stripping was necessary to improve mass transfer which helped wheat straw to hydrolyze to near completion and remove butanol simultaneously; and (3) hydrolysis of wheat straw to sugars using enzymes was slower than sugar utilization by the culture to produce butanol and often the culture was found de fi cient in sugar. This may have been due to different optimum temperatures for enzymes (45°C) and butanol fer-mentation (35°C). Although, SSFR in a batch reactor was successful, it had some problems that are listed below: (1) aseptic transfer of pretreated wheat straw to the bioreactor was dif fi cult, introducing the possibility of contamination; (2) liquid sampling from the reactor was problematic due to the presence of signi fi cant amount of solids in the reactor; and (3) axial agitation of biomass and cell broth affects the culture negatively and hence agitation by gas stripping was considered as an option. While use of gas stripping was helpful, rate of butanol removal from the broth was low thus requiring a large amount of gas recycle. In a batch reactor where SSFR was applied a productivity of 0.31 g/L h was observed (when wheat straw was used) as compared to 0.30 g/L h when glucose was used as a substrate. In this integrated batch process 21.42 g/L total ABE was produced from 86 g/L wheat straw. This system resulted in an ABE yield of 0.37 (g ABE/g sugar released) based on 95% hydrolysis of wheat straw (Table 4 ) .
26115 Cellulosic Butanol Production from Agricultural Biomass and Residues…
Table 4 Production of butanol/ABE from wheat straw in simultaneous sacchari fi cation, fermenta-tion, and recovery (SSFR) process
Production of butanol in a fed-batch reactor is another system where hydrolysis, fermentation, and recovery can be combined (SSFR). Using such a system, butanol was produced from wheat straw [ 58 ] . The reactor was loaded with 86 g/L pretreated wheat straw, enzymes, and the butanol producing culture. The system was operated for 533 h at a pH 5.0 and temperature 35°C. During the initial period of 120 h, wheat straw was hydrolyzed completely by the added hydrolytic enzymes. In order to ascertain that the culture was not de fi cient in sugar, a mixture containing glucose, xylose, arabinose, galactose, and mannose was fed to the reactor to mimic their proportion in wheat straw. In a 1 L reaction mixture a total (including sugar present in WS) of 430 g sugar was used thus producing 192 g total ABE with a yield of 0.44 (Table 4 ). In this reactor, a productivity of 0.36 g/L h was achieved which is 20% higher than achieved (0.30 g/L h) in a control reactor. One of the major problems associated with this fermentation was that the culture had dif fi culty utilizing xylose in the later part of fermentation. In this system, gas stripping was used to agitate the treated biomass and recover ABE.
6 Economic Evaluation of Agricultural Residues to Butanol
In a recent economic study on production of butanol from WSH, it was identi fi ed that utility costs are one of the most signi fi cant factors that impact price of butanol. This was largely due to distillative recovery of butanol from fermentation broth. Wheat straw was treated using dilute sulfuric acid at 121°C and hydrolyzed using enzymes prior to fermentation to butanol. Fermentation was performed in batch reactors employing C. beijerinckii P260 followed by recovery by traditional distil-lation. It was estimated that distillative recovery of butanol would result in the production price of $1.37/kg ($4.26/gal) for a grass rooted/or green fi eld plant while for an annexed plant this price would reduce to $1.07/kg ($3.33/gal). Recovery of butanol using a pervaporation membrane would further reduce this price to
262 N. Qureshi et al.
$0.82/kg ($2.55/gal). This price is based on 2010 equipment purchase cost. In an interesting report, commercial production of acetone-butanol was achieved in Russia (then Soviet Union) from hemp waste, corncobs, and sun fl ower shells [ 76 ] . In addition to acetone-butanol, equal emphasis was placed on recovery of gases, vitamin B12, and methane production by digesting the ef fl uent waste thus bene fi ting from all these coproducts which added to the pro fi tability of the AB plant.
Acknowledgments N. Qureshi would like to thank Michael A. Cotta (United States Department of Agriculture, National Center for Agricultural Utilization Research, Bioenergy Research Unit, Peoria, IL) for reading this manuscript and providing valuable and constructive comments. Part of this work was supported by the hatch grant (Project No: OHO01222; Department of Animal Sciences, The Ohio State University) to T.C. Ezeji.
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