Production of butanol (a biofuel) from agricultural residues: Part II – Use of corn stover and switchgrass hydrolysates

Acetone butanol ethanol (ABE) was produced from hydrolysed corn stover and switchgrass using Clostridium beijerinckii P260. A control experiment using glucose resulted in the production of 21.06 g L^?1 total ABE. In this experiment an ABE yield and productivity of 0.41 and 0.31 g L^?1 h^?1 was achieved, respectively. Fermentation of untreated corn stover hydrolysate (CSH) exhibited no growth and no ABE production; however, upon dilution with water (two fold) and wheat straw hydrolysate (WSH, ratio 1:1), 16.00 and 18.04 g L^?1 ABE was produced, respectively. These experiments resulted in ABE productivity of 0.17–0.21 g L^?1 h^?1. Inhibitors present in CSH were removed by treating the hydrolysate with Ca(OH)₂ (overliming). The culture was able to produce 26.27 g L^?1 ABE after inhibitor removal. Untreated switchgrass hydrolysate (SGH) was poorly fermented and the culture did not produce more than 1.48 g L^?1 ABE which was improved to 14.61 g L^?1. It is suggested that biomass pretreatment methods that do not generate inhibitors be investigated. Alternately, cultures resistant to inhibitors and able to produce butanol at high concentrations may be another approach to improve the current process.     

Simultaneous enzymatic saccharification and ABE fermentation using pretreated oil palm empty fruit bunch as substrate to produce butanol and hydrogen as biofuel

Simultaneous saccharification and acetone–ethanol–butanol (ABE) fermentation was conducted in order to reduce the number of steps involved in the conversion of lignocellulosic biomass into butanol. Enzymatic saccharification of pretreated oil palm empty fruit bunch (OPEFB) by cellulase produced 31.58 g/l of fermentable sugar. This saccharification was conducted at conditions similar to the conditions required for ABE fermentation. The simultaneous process by Clostridium acetobutylicum ATCC 824 produced 4.45 g/l of ABE with butanol concentration of 2.75 g/l. The butanol yield of 0.11 g/g and ABE yield of 0.18 g/g were obtained from this simultaneous process as compared to the two-step process (0.10 g/g of butanol yield and 0.14 g/g of ABE yield). In addition, the simultaneous process also produced higher cumulative hydrogen (282.42 ml) than to the two-step process (222.02 ml) after 96 h of fermentation time. This study suggested that the simultaneous process has the potential to be implemented for the integrated production of butanol and hydrogen from lignocellulosic biomass.     

ABE fermentation from enzymatic hydrolysate of NaOH-pretreated corncobs

Acetone butanol ethanol (ABE) was produced from enzymatic-hydrolyzed corncobs by Clostridium saccharobutylicum DSM 13864. Pretreatment of corncobs was carried out with 0.5 mol L⁻¹ NaOH followed by enzymatic hydrolysis. The yield of total reducing sugars was 917 g kg⁻¹ pretreated (de-lignified) and washed corncobs. The hydrolysate was used without sediments removal for ABE fermentation. A solvent production of 19.44 g L⁻¹ with 12.27 g L⁻¹ butanol was obtained from 55.22 g L⁻¹ sugars, resulting in an ABE yield of 350 g kg⁻¹ and a production rate of 0.54 g L⁻¹ h⁻¹. A control experiment using 55.3 g L⁻¹ mixed sugars resulted in an ABE production of 16.81 g L⁻¹ with 10.26 g L⁻¹ butanol, corresponding to an ABE yield of 300 g kg⁻¹ and a production rate of 0.47 g L⁻¹ h⁻¹, indicating that the enzymatic hydrolysates may contain stimulating compounds that can improve the ABE fermentation.     

Recovery of butanol from fermentation broth by gas stripping

This is an overview of the butanol (usually called acetone butanol ethanol, ABE) fermentation in various types of reactor systems and recovery by gas stripping. Gas stripping is a simple technique which does not require expensive apparatus, does not harm the culture, does not remove nutrients and reaction intermediates and reduces butanol toxicity (inhibition). As a result of butanol removal by gas stripping, concentrated sugar solutions can be used to produce butanol/ABE. Compared to sugar utilization of 30 gl⁻¹ in a control batch reactor, sugar utilization of 199 gl⁻¹ has been reported with 69.7 gl⁻¹ solvent production. In fed-batch reactors concentrated sugar solutions (350 gl⁻¹) have been used. Additionally, the process of ABE production results in concentrated product streams containing 9.1–120 gl⁻¹ butanol/ABE. In the integrated ABE production and recovery systems, selectivities of 4–30.5 have been reported.     

Property change of bagasse as cell-immobilizing carrier and coproduction of hydrogen-butanol in fixed-bed reactor by repeated cycle fermentation

Property change of bagasse as cell-immobilizing carrier and coproduction of hydrogen-butanol in fixed-bed reactor by repeated cycle fermentation was studied. Bagasse would have potential being used as animal feed after four repeated cycles fermentation lasting for 290 h, because cellulose crystallinity was decreased by 33% and protein content was increased by 187%. Specific surface area and total pore volume of bagasse were also decreased. Maximum accumulated hydrogen production, yield and productivity were 11.7 L/L, 1.89 mol/mol and 537 mL/L/h, respectively. Maximum total solvent (acetone, butanol and ethanol) concentration, solvent yield and solvent productivity were 18.2 g/L 0.31 g/g and 0.78 g/L/h, respectively. Modified Gompertz model was used to describe the hydrogen and butanol production and fitting results showed good agreement with the experimental data. The maximum total product (including hydrogen, acetone, butanol and ethanol) energy and energy conversion efficiency during the 4 cycles fermentation were 758.2 kJ/L and 86.3%, respectively.     

Biobutanol production from fiber-enhanced DDGS pretreated with electrolyzed water

DDGS (distiller's dried grains with solubles) is a major co-product in dry-grind ethanol production from corn. A recently developed physical process separates DDGS into two value-added components: a fiber-enriched DDGS and a portion that is rich in oil and protein. Electrolyzed water, a new pretreatment catalyst was employed to pretreat fiber-enriched DDGS. Four temperatures (130, 145, 160, and 175 °C) and three treatment times (10, 20, and 30 min) were examined in the pretreatment with a solid loading of 20% w/w. Other pretreatment methods, such as diluted sulfuric acid, alkaline solution, and hot water, were also tested for comparison purposes. Fifteen FPU cellulase/g cellulose, 40 units β-glucosidase/g cellulose, and 50 units xylanase/g dry biomass were used in the enzymatic hydrolysis at 50 °C and 10% solid loading. The hydrolyzates were fermented by Clostridium beijerinckii BA 101 at 35 °C in an auto-controlled Six-fors fermentor with continuous mixing. The highest sugar yield was achieved when using the acidic electrolyzed water treatment at 175 °C for 10 min, with 23.25 g glucose, xylose and arabinose released from 100 g fiber-enriched DDGS. The C. beijerinckii fermentation produced 5.35 g ABE (acetone, butanol, and ethanol) from 100 g dry fiber-enhanced DDGS. This study demonstrated that DDGS pretreated with electrolyzed water and hydrolyzed with commercial enzymes could be used to produce biobutanol without detoxification.     

Production of butanol (a biofuel) from agricultural residues: Part I – Use of barley straw hydrolysate

Fermentation of dilute sulfuric acid barley straw hydrolysate (BSH; undiluted/untreated) by Clostridium beijerinckii P260 resulted in the production of 7.09 gL^?1 ABE (acetone butanol ethanol), an ABE yield of 0.33, and productivity of 0.10 gL^?1 h^?1. This level of ABE is much less than that observed in a control experiment (21.06 gL^?1) where glucose (initial concentration 60 gL^?1) was used as a substrate. In the control experiment, an ABE yield of 0.41 and productivity of 0.31 gL^?1 h^?1 were observed. This comparison suggested that BSH is toxic to the culture. To reduce this potential toxicity effect, BSH was treated with lime [Ca(OH)₂] followed by fermentation. The treated BSH resulted in a successful fermentation and ABE concentration of 26.64 gL^?1 was achieved. This was superior to both glucose and untreated BSH (initial sugar 60 gL^?1) fermentations. In this fermentation, an ABE yield of 0.43 and productivity of 0.39 gL^?1 h^?1 (390% of untreated/undiluted BSH) was obtained. It should be noted that using lime treated BSH, a specific productivity of 0.55 h^?1 was obtained as compared to 0.12 h^?1 in the control fermentation suggesting that more carbon was directed to product formation.     

Biohydrogen generation from anaerobic digestion of food waste

Biohydrogen generated from the anaerobic digestion of a synthetic food waste with constant composition and a real food waste collected in Hong Kong were studied. This study aims at using a monoculture to increase biohydrogen production and determining optimum conditions for maximum biohydrogen production. Among the nine bacteria screened for biohydrogen production, Escherichia cloacae and Enterobacter aerogenes produced the largest amount of biohydrogen from the anaerobic digestion of synthetic food waste. The optimum anaerobic digestion conditions were determined: initial pH of 7, a water to solids ratio of 5 (w/w), a mesophilic temperature (37 ± 1 °C), and in the presence of 40 mg/L FeSO₄·7H₂O. Anaerobic digestion at the optimum operating conditions using collected food waste with E. cloacae as the bacterial source was also performed. By adjusting the pH in the range of 5–6, a specific biohydrogen production of 155.2 mL/g of volatile solids (VS) in food waste was obtained.     

Isolation of a Clostridium acetobutylicum strain and characterization of its fermentation performance on agricultural wastes

A new solvent-producing Clostridium has been isolated from soil used in intensive rice cultivation. The 16S rRNA analysis of the isolate indicates that it is closely related to Clostridium acetobutylicum, with a sequence identity of 96%. The new isolate, named C. acetobutylicum YM1, produces biobutanol from multiple carbon sources, including glucose, fructose, xylose, arabinose, glycerol, lactose, cellobiose, mannitol, maltose, galactose, sucrose and mannose. This isolate can also utilize polysaccharides such as starch and carboxylmethyl cellulose (CMC) for the production of biobutanol. The ability of isolate YM1 to produce biobutanol from agro-industrial wastes was also evaluated for rice bran, de-oiled rice bran, palm oil mill effluent and palm kernel cake. The highest concentration of biobutanol (7.27 g/L) was obtained from the fermentation medium containing 2% (w/v) fructose, with a total acetone–butanol–ethanol (ABE) concentration of 10.23 g/L. The ability of isolate YM1 to produce biobutanol from various carbon sources and agro-wastes indicates the promise of the use of this isolate for the production of biobutanol, a renewable energy resource, from readily available renewable feedstocks.     

Technological advances in hydrogen production by Enterobacter bacteria upon substrate,luminosity and anaerobic conditions

The enhancement of hydrogen production by Enterobacter aerogenes and Enterobacter cloacae from fermentation of carbon sources such as glucose and lactose (from cheese whey permeate) was investigated. Also, the influence of the luminosity (2200 lux) and anaerobic condition (nitrogen and argon gases) were evaluated. The assays were carried out in 50 mL reactors during 108 h. To E. aerogenes/nitrogen/luminosity condition and using glucose as substrate, H₂ production (73.8 mmol/L.d) was higher than using lactose (15.5 mmol/L.d). In the dark fermentation, hydrogen yields were 1.60 mol H₂/mol glucose and 1.36 molH₂/mol lactose. When using E. cloacae, the light fermentation using nitrogen gas resulted in 77 mmol H₂/L.d and 1.62 mol H₂/mol glucose. In addition, for E. cloacae, hydrogen yields using argon gas and luminosity provided 2.39 mol/mol glucose and 2.53 mol/mol lactose. In general, butyric and acetic acid fermentation were observed and favored the target-product (H₂).     

诱变选育拜氏梭菌利用碱处理木糖渣水解液生产丁醇

试验以拜氏梭菌(Clostridium beijerinckii)NCIMB 8052为出发菌株,采用EMS诱变,并利用含淀粉、2-脱氧-D-葡萄糖的固体培养基对诱变菌株进行多次筛选,最终获得一株诱变菌株Y-3,其在葡萄糖培养基中总溶剂产量为15.8 g/L,丁醇产量为9.4 g/L,较出发菌株分别提高了30.6%和40.3%。利用以木糖渣为底物生产的纤维素酶对经2%氢氧化钠预处理后的木糖渣进行水解,所得的水解液作为Y-3丁醇发酵培养基中的碳源,总溶剂和丁醇产量分别达到16.3 g/L和8.8 g/L。结果表明,诱变菌株Y-3可利用碱处理木糖渣水解液作为发酵底物生产丁醇,且丁醇产量较高,适合于生物质发酵,大大降低了生产成本。     

Simultaneous production of hydrogen and ethanol from waste glycerol by Enterobacter aerogenes KKU-S1

Factors affecting simultaneous hydrogen and ethanol production from waste glycerol by a newly isolated bacterium Enterobacter aerogenes KKU-S1 were investigated employing response surface methodology (RSM) with central composite design (CCD). The Plackett-Burman design was first used to screen the factors influencing simultaneous hydrogen and ethanol production, i.e., initial pH, temperature, amount of vitamin solution, yeast extract (YE) concentration and glycerol concentration. Results indicated that initial pH, temperature, YE concentration, and glycerol concentration had a statistically significant effect (p ≤ 0.05) on hydrogen production rate (HPR) and ethanol production. The significant factors were further optimized using CCD. Optimum conditions for simultaneously maximizing HPR and ethanol production were YE concentration of 1.00 g/L, glycerol concentration of 37 g/L, initial pH of 8.14, and temperature of 37 °C in which a maximum HPR and ethanol production of 0.24 mmol H₂/L h and 120 mmol/L were achieved.     

Feasibility of reed for biobutanol production hydrolyzed by crude cellulase

The aim of this study was to efficiently utilize reed for both cellulase and biobutanol production. The unprocessed cellulase blend produced under solid-state fermentation using reed as the substrate showed a similar reducing sugar yield using Whatman filter paper to the commercial enzyme blend (38.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (29.3%–14.6%) and lignin (17.2%–14.1%) content and increase cellulose content (42.5%–62.3%) from reed. Enzymatic hydrolysis of organosolv-pretreated reed using the crude cellulase with enzyme loading of 25 FPU/g reed, 20% solid content at 50 °C and pH 5.5 resulted in a reed hydrolysate containing 40.01 g/L glucose and 3.55 g/L xylose after 72 h. Fermentation of the hydrolysate medium by Clostridium acetobutylicum produced 9.07 and 14.24 g/L of biobutanol and ABE with yield of 0.21 g/g and 0.33 g/g, respectively. This study proved that crude cellulase complex produced under solid state fermentation and organsolv pretreatment can efficiently provide reed hydrolysate that can be converted to biobutanol without any commercial cellulase usage.     

Hydrogen production from the monomeric sugars hydrolyzed from hemicellulose by Enterobacter aerogenes

Relatively large percentages of xylose with glucose, arabinose, mannose, galactose and rhamnose constitute the hydrolysis products of hemicellulose. In this paper, hydrogen production performance of facultative anaerobe (Enterobacter aerogenes) has been investigated from these different monomeric sugars except glucose. It was shown that the stereoisomers of mannose and galactose were more effective for hydrogen production than those of xylose and arabinose. The substrate of 5 g/l xylose resulted in a relative high level of hydrogen yield (73.8 mmol/l), hydrogen production efficiency (2.2 mol/mol) and a maximum hydrogen production rate (249 ml/l/h). The hydrogen yield, hydrogen production efficiency and the maximum hydrogen production rate reached 104 mmol/l, 2.35 mol/mol and 290 ml/l/h, respectively, on a substrate of 10 g/l galactose. The hydrogen yields and the maximum hydrogen production rates increased with an increase of mannose concentrations and reached 119 mmol/l and 518 ml/l/h on the culture of 25 g/l mannose. However, rhamnose was a relative poor carbon resource for E. aerogenes to produce hydrogen, from which the hydrogen yield and hydrogen production efficiency were about one half of that from the mannose substrate. E. aerogenes was found to be a promising strain for hydrogen production from hydrolysis products of hemicellulose.     

Production of butanol and polyhydroxyalkanoate from industrial waste by Clostridium beijerinckii ASU10

This study demonstrated a biotechnological approach for simultaneous production of low‐cost H₂, liquid biofuels, and polyhydroxyalkanoates (PHAs) by solventogenic bacterium (Clostridium beijerinckii) from renewable industrial wastes such as molasses and crude glycerol. C beijerinckii ASU10 (KF372577) exhibited considerable performance for hydrogen production of 5.1 ± 0.84 and 11 ± 0.44 mL H₂ h^?1 on glycerol and sugarcane molasses, respectively. The total acetone‐butanol‐ethanol (ABE) generation from glycerol and molasses was 9.334 ± 2.98 and 10.831 ± 4.1 g L^?1, respectively. ABE productivity (g L^?1 h^?1) was 0.0486 and 0.0564 with a yield rate (g g^?1) up to 0.508 and 0.493 from glycerol and molasses fermentation, respectively. The PHA yields from glycerol and sugarcane molasses were 84.37% and 37.97% of the dried bacterial biomass, respectively. Additionally, the ultrathin section of C beijerinckii ASU10 showed that PHA granules were accumulated more densely on glycerol than molasses. Gas chromatography–mass spectrometry (GC‐MS) analysis confirmed that the PHAs obtained from molasses fermentation included 3‐hydroxybutyrate (47.3%) and 3‐hydroxyoctanoate (52.7%) as the main constituents. Meanwhile, 3‐hydroxybutyrate represented the sole monomer of PHA produced from glycerol fermentation. This study demonstrated that C beijerinckii ASU10 (KF372577) is a potent strain for low‐cost PHA production depending on its high potential to produce high‐energy biofuel and other valuable compounds from utilization of organic waste materials.     

Optimization of selected salts concentration for improved biohydrogen production from biodiesel-based glycerol using Enterobacter aerogenes

Enterobacter aerogenes have a known ability to convert glycerol (GL) in a fermentative process to yield hydrogen and ethanol as the main by-products. The concentration of some media constituents was optimized to maximize biohydrogen yield and rate of production. E. aerogenes were cultured in aerobic conditions, and then transferred into anaerobic conditions before being cultured in a minimum mineral synthetic media (MMSM) containing 15 g/L GL. The concentration of selected salts were optimized in the following ranges: 0–300 mg/L MgSO₄, 0–14 g/L Na₂EDTA, 0–10 mg/L CaCL₂, 0–10 g/L Na₂HPO₄, and 0–9.7 g/L KH₂PO₄. The results of the full factorial design indicated that the production of biohydrogen required a minimal concentration of 3.5 mg/L EDTA, 200 mg/L MgSO₄.7H₂O and no CaCl₂.2H₂O. A significant interaction between EDTA and MgSO₄ was also observed. Results from the phosphate salts optimization showed that Na₂HPO₄ gave better results than KH₂PO₄. The optimal conditions determined using pure glycerol (commercial grade glycerol), were successfully applied to the fermentation of crude glycerol from biodiesel production. The results indicated promising yields of 0.79 and 0.84 mol/mol of glycerol for bioethanol and biohydrogen, respectively, and this at a faster rate than reported previously for E. aerogenes.     

Photo-fermentative hydrogen production by Rhodopseudomonas palustris CGA009 in the presence of inhibitory compounds

This study investigated Rhodopseudomonas palustris CGA009 biohydrogen production from compounds commonly found in lignocellulosic steam explosion hydrolysate, by examining the effect of individual inhibitory phenolic and furan compounds found in hydrolysates, under photo-fermentative anaerobic conditions. Since lignocellulose is often converted into ethanol via yeast-mediated fermentation, the tolerance of R. palustris CGA009 towards ethanol inhibition was also tested at a concentration range of 0.25–14% (v/v) under anaerobic photo-fermentative conditions. Hydrogen production was enhanced by compounds such as syringaldehyde (0.03 g/L), which accumulated total hydrogen of 960 mL over the cultivation period. In contrast, a reduction in hydrogen production of 1.4 fold was observed in vanillin-containing solutions (0.43 g/L), which obtained accumulated total hydrogen of 576 mL. Increasing ethanol concentrations reduced hydrogen production, but cell growth was not affected up to 1% (v/v), a fairly low concentration. R. palustris CGA009 can tolerate comparatively high concentrations of phenolic compounds, suggesting its use for lignocellulose hydrolysate detoxification and hydrogen production.     

Fermentative hydrogen production by two novel strains of Enterobacter aerogenes HGN-2 and HT 34 isolated from sea buried crude oil pipelines

Present study investigated fermentative hydrogen production of two novel isolates of Enterobacter aerogenes HGN-2 and HT 34 isolated from oil water mixtures. The two isolates were identified as novel strains of E. aerogenes based on 16S rRNA gene. The batch fermentations of two strains from glucose and xylose were carried out using economical culture medium under various conditions such as temperature, initial pH, NaCl, Ni⁺/Fe⁺⁺, substrate concentrations for enhanced fermentation process. Both the strains favoured wide range of pH (6.5–8.0) at 37 °C for optimum production (2.20–2.23 mol H₂/mol-glucose), which occurred through acetate/butyrate pathway. At 55 °C, both strains favoured 6.0–6.5 and acetate type fermentation was predominant in HT 34. Hydrogen production by HT 34 from xylose was highly pH dependant and optimum production was at pH 6.5 (circa 1.98 mol-H₂/mol-xylose) through acetate pathway. The efficiency of the strain HGN-2 at pH 6.5 was 1.92–1.94 mol-H₂/mol-xylose, and displayed both acetate and butyrate pathways. At 55 °C, very low hydrogen production was detected (less than 0.5 m mol/mol-xylose).     

Developing a thermophilic hydrogen-producing microbial consortia from geothermal spring for efficient utilization of xylose and glucose mixed substrates and oil palm trunk hydrolysate

Xylose and glucose are the major sugar components of lignocellulosic hydrolysate. This study aims to develop thermophilic hydrogen-producing consortia from eight sediments-rich samples of geothermal springs in Southern Thailand by repeated batch cultivation at 60 °C with glucose, xylose and xylose-glucose mixed substrates. Significant hydrogen production potentials were obtained from thermophilic enriched cultures encoded as PGR and YLT with the maximum hydrogen yields of 241.4 and 231.6 mL H₂/g sugar_consumed, respectively. After repeated batch cultivation the hydrogen yield from xylose-glucose mixed substrate of PGR increased to 375 mL H₂/g sugar_consumed which was 30% higher than that of YLT (287 mL H₂/g sugar_consumed). Soluble metabolites from xylose-glucose mixed substrates were composed mostly of butyric acid (20.6-21.8 mM), acetic acid (7.2-13.5 mM), lactic acid (8.2-11.7 mM) and butanol (4.4-13.0 mM). Denaturing gradient gel electrophoresis (DGGE) profiles illustrated small difference in microbial patterns of PGR enriched with glucose, xylose-glucose mixed substrate and xylose. The dominant populations were affiliated with low G + C content Gram-positive bacteria, Thermoanaerobacterium sp., Thermoanaerobacter sp., Caloramater sp. and Anoxybacillus sp. based on the 16S rRNA gene. Cultivation of the enriched culture PGR in oil palm trunk hydrolysate, the maximum hydrogen yield of 301 mL H₂/g sugar_consumed was achieved at hydrolysate concentration of 40% (v/v). At higher concentration to 80% (v/v), the hydrogen fermentation process was inhibited. Therefore, the efficient thermophilic hydrogen-producing consortia PGR has successfully developed and has great potential for production of biohydrogen from mixed sugars hydrolysate.     

Comparative study on the production of biohydrogen from rice mill wastewater

This study presents the production of biohydrogen from rice mill wastewater. The acid hydrolysis and enzymatic hydrolysis operating conditions were optimized, for better reducing sugar production. The effect of pH and fermentation time on biohydrogen production from acid and enzymatic hydrolyzed rice mill wastewater was investigated, using Enterobacter aerogenes and Citrobacter ferundii. The enzymatic hydrolysis produced the maximum reducing sugar (15.8 g/L) compared to acid hydrolysis (14.2 g/L). The growth data obtained for E. aerogenes and C. ferundii, fitted well with the Logistic equation. The hydrogen yields of 1.74 mol H₂/mol reducing sugar, and 1.40 mol H₂/mol reducing sugar, were obtained from the hydrolyzate obtained from enzymatic and acid hydrolysis, respectively. The maximum hydrogen yield was obtained from E. aerogenes compared to C. ferundii, and the optimum pH for better hydrogen production was found to be in the range from 6.5 to 7.0. The chemical oxygen demand (COD) reduction obtained was around 71.8% after 60 h of fermentation.