Bio-ethanol from water hyacinth biomass: An evaluation of enzymatic saccharification strategy

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and β-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.     

Ethanolic fermentation of hydrolysates from ammonia fiber expansion (AFEX) treated corn stover and distillers grain without detoxification and external nutrient supplementation

External nutrient supplementation and detoxification of hydrolysate significantly increase the production cost of cellulosic ethanol. In this study, we investigated the feasibility of fermenting cellulosic hydrolysates without washing, detoxification or external nutrient supplementation using ethanologens Escherichia coli KO11 and the adapted strain ML01 at low initial cell density (16 mg dry weight/L). The cellulosic hydrolysates were derived from enzymatically digested ammonia fiber expansion (AFEX)-treated corn stover and dry distiller's grain and solubles (DDGS) at high solids loading (18% by weight). The adaptation was achieved through selective evolution of KO11 on hydrolysate from AFEX-treated corn stover. All cellulosic hydrolysates tested (36-52 g/L glucose) were fermentable. Regardless of strains, metabolic ethanol yields were near the theoretical limit (0.51 g ethanol/g consumed sugar). Volumetric ethanol productivity of 1.2 g/h/L was achieved in fermentation on DDGS hydrolysate and DDGS improved the fermentability of hydrolysate from corn stover. However, enzymatic hydrolysis and xylose utilization during fermentation were the bottlenecks for ethanol production from corn stover at these experimental conditions. In conclusion, fermentation under the baseline conditions was feasible. Utilization of nutrient-rich feedstocks such as DDGS in fermentation can replace expensive media supplementation.     

Effect of pretreatment severity on the conversion of barley straw to fermentable substrates and the release of inhibitory compounds

The production of fermentable substrates from barley straw under various process conditions was studied. Pretreatment included chemical pretreatment with dilute-acid followed by enzymatic hydrolysis; the pretreatment conditions were expressed in a combined severity factor, CS, which ranged in the present study from −1.6 to 1.1. Considering the production of fermentable sugars and the release of inhibitory compounds, the optimal pretreatment conditions were 170 °C, 0% sulfuric acid and 60 min, corresponding to CS −0.4. Under these conditions, 21.4 g glucose/L, 8.5 g xylose/L, and 0.5 g arabinose/L were produced, while 0.1 g HMF/L, 0.4 g furfural/L, 0.0 g levulinic acid/L, 0.0 g formic acid/L, and 2.1 g acetic acid/L were released. The ratio of Σsugars/Σinhibitors proved to be a good tool for evaluating the suitability of a hydrolysate for fermentation purposes.     

Ethanol production from eucalyptus wood hemicellulose hydrolysate by Pichia stipitis

Ethanol production was evaluated from eucalyptus wood hemicellulose acid hydrolysate using Pichia stipitis NRRL Y-7124. An initial lag phase characterized by flocculation and viability loss of the yeast inoculated was observed. Subsequently, cell regrowth occurred with sequential consumption of sugars and production of ethanol. Polyol formation was detected. Acetic acid present in the hydrolysate was an important inhibitor of the fermentation, reducing the rate and the yield. Its toxic effect was due essentially to its undissociated form. The fermentation was more effective at an oxygen transfer rate between 1.2 and 2.4 mmol/L h and an initial pH of 6.5. The hydrolysate used in the experiences had the following composition (expressed in grams per liter): xylose 30, arabinose 2.8, glucose 1.5, galactose 3.7, mannose 1.0, cellobiose 0.5, acetic acid 10, glucuronic acid 1.5, and galacturonic acid 1.0. The best values obtained were maximum ethanol concentration 12.6 g/L, fermentation time 75 h, fermentable sugar consumption 99% ethanol yield 0.35 g/g sugars consumed, and volumetric ethanol productivity 4 g/L day. (c) 1992 John Wiley & Sons, Inc.     

Degradation of starchy substrates by a crude enzyme preparation and utilization of the hydrolysates for lactic fermentation

An enzyme preparation obtained from Aspergillus ustus, possessing cellulase, α-amylase, amyloglucosidase, proteinase and d-xylanase activities, was used along with commercial bacterial α-amylase and amyloglucosidase for the degradation of ragi (Eleusine coracana) flour and wheat (Triticum vulgare) bran. Lactic acid yield from ragi hydrolysate, adjusted to 5% reducing sugars (w/v), was 25% when fermented with Lactobacillus plantarum. The yields increased to 78% and 94% when the ragi hydrolysate was fortified with 20% and 60% (v/v) wheat bran hydrolysate, respectively. When commercial α-amylase and amyloglucosidase were used for the hydrolysis of ragi and wheat bran and L. plantarum was employed to ferment the hydrolysates containing 5% reducing sugars (w/v), lactic acid yields were 10% in ragi hydrolysate and 57% and 90% when the ragi hydrolysate was fortified with 20% and 60% (v/v) of wheat bran hydrolysate, respectively. α-Amylase and amyloglucosidase hydrolysed wheat bran added at 20% (v/v) as the sole source of nutrient to soluble starch hydrolysate (5% reducing sugars) gave 22% yield of lactic acid. The yield increased to 55% by the utilization of A. ustus enzyme preparation in addition to α-amylase and amyloglucosidase for wheat bran hydrolysis.     

Efficient conversion of sugarcane stalks into ethanol employing low temperature alkali pretreatment method

An alternative route for bio-ethanol production from sugarcane stalks (juice and bagasse) featuring a previously reported low temperature alkali pretreatment method was evaluated. Test-tube scale pretreatment-saccharification experiments were carried out to determine optimal LTA pretreatment conditions for sugarcane bagasse with regard to the efficiency of enzymatic hydrolysis of the cellulose. Free fermentable sugars and bagasse recovered from 2 kg of sugarcane stalks were jointly converted into ethanol via separate enzymatic hydrolysis and fermentation (SHF). Results showed that 98% of the cellulose present in the optimally pretreated bagasse was hydrolyzed into glucose after 72-h enzymatic saccharification using commercially available cellulase and β-glucosidase preparations at relatively low enzyme loading. The fermentable sugars in the mixture of the sugar juice and the bagasse hydrolysate were readily converted into 193.5 mL of ethanol by Saccharomyces cerevisiae within 12h, achieving 88% of the theoretical yield from the sugars and cellulose.     

Hydrolysate detoxification with activated charcoal for xylitol production by Candida guilliermondii

A detoxification method using activated charcoal with concentrated rice straw hemicellulosic hydrolysate improved the conversion of xylose to xylitol by the yeast Candida guilliermondii by 22%. This was achieved when the hydrolysate:charcoal ratio was 40 g g^–1, resulting in removal of 27% of phenolic compounds. Under this condition, the xylitol yield factor (0.72 g g^–1) and volumetric productivity (0.61 g l^–1 h^–1) were close to those attained in a semi-defined medium simulating hydrolysate sugars.     

Evaluation of cell recycling in continuous fermentation of enzymatic hydrolysates of spruce with Saccharomyces cerevisiae and on-line monitoring of glucose and ethanol

The maximum growth rate of Saccharomyces cerevisiae ATCC 96581, adapted to fermentation of spent sulphite liquor (SSL), was 7 times higher in SSL of hardwood than the maximum growth rate of bakers' yeast. ATCC 96581 was studied in the continuous fermentation of spruce hydrolysate without and with cell recycling. Ethanol productivity by ATCC 96581 in continuous fermentation of an enzymatic hydrolysate of spruce was increased 4.6 times by employing cell recycling. On-line analysis of CO₂, glucose and ethanol (using a microdialysis probe) was used to investigate the effect of fermentation pH on cell growth and ethanol production, and to set the dilution rate. Cell growth in the spruce hydrolysates was strongly influenced by fermentation pH. The fermentation was operated in continuous mode for 210 h and a theoretical ethanol yield on fermentable sugars was obtained. Received: 25 May 1998 / Received revision: 11 August 1998 / Accepted: 12 August 1998     

Fermentation of sugar cane bagasse hemicellulosic hydrolysate and sugar mixtures to ethanol by recombinant Escherichia coli KO11

Escherichia coli KO11, carrying the ethanol pathway genes pdc (pyruvate decarboxylase) and adh (alcohol dehydrogenase) from Zymomonas mobilis integrated into its chromosome, has the ability to metabolize pentoses and hexoses to ethanol, both in synthetic medium and in hemicellulosic hydrolysates. In the fermentation of sugar mixtures simulating hemicellulose hydrolysate sugar composition (10.0 g of glucose/l and 40.0 g of xylose/l) and supplemented with tryptone and yeast extract, recombinant bacteria produced 24.58 g of ethanol/l, equivalent to 96.4% of the maximum theoretical yield. Corn steep powder (CSP), a byproduct of the corn starch-processing industry, was used to replace tryptone and yeast extract. At a concentration of 12.5 g/l, it was able to support the fermentation of glucose (80.0 g/l) to ethanol, with both ethanol yield and volumetric productivity comparable to those obtained with fermentation media containing tryptone and yeast extract. Hemicellulose hydrolysate of sugar cane bagasse supplemented with tryptone and yeast extract was also readily fermented to ethanol within 48 h, and ethanol yield achieved 91.5% of the theoretical maximum conversion efficiency. However, fermentation of bagasse hydrolysate supplemented with 12.5 g of CSP/l took twice as long to complete. This revised version was published online in November 2006 with corrections to the Cover Date.     

Improved bioethanol production through simultaneous saccharification and fermentation of lignocellulosic agricultural wastes by <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> 6556

The combined effect of simultaneous saccharification and fermentation and separate hydrolysis and fermentation (SHF) for ethanol production by Kluyveromyces marxianus 6556 was studied using two lignocellulosic feedstocks viz., corncob and soybean cake. The ethanologenic efficiency of K. marxianus 6556 was observed as 28% (theoretical yield) in a fermentation medium containing glucose, but, there was no ethanol production by cells grown on xylose. A maximum sugar release of 888 mg/g corncob and 552 mg/g soybean cake was achieved through acid hydrolysis pretreatment. Furthermore, corncob and soybean cake treated with commercial cellulase (100 IU for 48 h) from Trichoderma reesei yielded reducing sugars of 205 and 100 mg/g, respectively. Simultaneous saccharification and fermentation resulted in highest ethanol production of 5.68 g/l on corncob and 2.14 g/l on soybean cake after 48 h of incubation. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the hydrolysates obtained through SHF of corncob and soybean cake.     

Fermentation of lignocellulosic sugars to acetic acid by <Emphasis Type="Italic">Moorella thermoacetica</Emphasis>

A systematic study of bioconversion of lignocellulosic sugars to acetic acid by Moorella thermoacetica (strain ATCC 39073) was conducted. Four different water-soluble fractions (hydrolysates) obtained after steam pretreatment of lignocellulosic biomass were selected and fermented to acetic acid in batch fermentations. M. thermoacetica can effectively ferment xylose and glucose in hydrolysates from wheat straw, forest residues, switchgrass, and sugarcane straw to acetic acid. Xylose and glucose were completely utilized, with xylose being consumed first. M. thermoacetica consumed up to 62 % of arabinose, 49 % galactose and 66 % of mannose within 72 h of fermentation in the mixture of lignocellulosic sugars. The highest acetic acid yield was obtained from sugarcane straw hydrolysate, with 71 % of theoretical yield based on total sugars (17 g/L acetic acid from 24 g/L total sugars). The lowest acetic acid yield was observed in forest residues hydrolysate, with 39 % of theoretical yield based on total sugars (18 g/L acetic acid from 49 g/L total sugars). Process derived compounds from steam explosion pretreatment, including 5-hydroxymethylfurfural (0.4 g/L), furfural (0.1 g/L) and total phenolics (3 g/L), did not inhibit microbial growth and acetic acid production yield. This research identified two major factors that adversely affected acetic acid yield in all hydrolysates, especially in forest residues: (i) glucose to xylose ratio and (ii) incomplete consumption of arabinose, galactose and mannose. For efficient bioconversion of lignocellulosic sugars to acetic acid, it is imperative to have an appropriate balance of sugars in a hydrolysate. Hence, the choice of lignocellulosic biomass and steam pretreatment design are fundamental steps for the industrial application of this process.     

Bioethanol from fermentation of cassava pulp in a fibrous-bed bioreactor using immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense

There is an increasing worldwide interest in bioethanol production from agricultural, industrial, and urban residues for both ecological and economic reasons. The acid hydrolysis of cassava pulp to reducing sugars and their fermentation to ethanol were evaluated in a fibrousbed bioreactor with immobilized Δldh, a genetically engineered Thermoanaerobacterium aotearoense. A maximum yield of total reducing sugars of 53.5% was obtained after 8 h of hydrolysis at 85oC in 0.4 mol/L hydrochloric acid with a solid-to-liquid ratio of 1:20, which was optimized by using an orthogonal design based on preliminary experiments. In the FBB, the fed-batch fermentation, using glucose as the sole carbon source, gave a maximum ethanol production of 38.3 g/L with a yield of 0.364 g/g in 100 h; whereas the fed-batch fermentation, using xylose as the sole carbon source, gave 34.1 g/L ethanol with a yield of 0.342 g/g in 135 h. When cassava pulp hydrolysate was used as a carbon source, 39.1 g/L ethanol with a yield of 0.123 g/g cassava pulp in185 h was observed, using the fed-batch fermentation model. In addition, for repeated batch fermentation of cassava pulp hydrolysate carried out in the fibrous-bed bioreactor, long-term operation with high ethanol yield and volumetric productivity were achieved. The above results show that the acid hydrolysate of cassava pulp can be used for ethanol production in a fibrous-bed bioreactor, although some inhibition phenomena were observed during the process of fermentation.     

Fermentation of a wheat straw acid hydrolysate by Bacillus stearothermophilus T-13 in continuous culture with partial cell recycle

The effects of pretreatment by overliming and addition of nutrients (yeast extract, tryptone and ammonium chloride) on fermentation of mixed sugars derived by acid hydrolysis of the hemicellulose fraction of wheat straw with Bacilllus stearothermophilus strain T-13, an l-lactate dehydrogenase deficient mutant was investigated in continuous culture with partial cell recycle. Pretreatment and addition of nutrients to the hydrolysate improved the fermentation considerably. Sugar utilization, ethanol yields and productivities obtained in the treated hydrolysate with added nutrients were comparable to those obtained in a synthetic medium. Sugar utilization in the synthetic medium and treated and crude hydrolysates with added nutrients were 86%, 89% and 56%, respectively, compared with 45% in the treated hydrolysate without extra nutrients. Ethanol yields obtained were 0.32 g g⁻¹ sugars and 0.38 g g⁻¹ sugars in the treated hydrolysate with and without extra nutrients, respectively, compared with 0.24 g g⁻¹ sugars in the crude hydrolysate with added nutrients. Continuous culture with partial cell recycle significantly increased the rate of ethanol production (0.60–1.02 g litre⁻¹ h⁻¹) in the various media and the stability of the mutant strain compared with conventional continuous culture.     

Effects of dilution rate and pH on the ruminal cellulolytic bacterium Fibrobacter succinogenes S85 in cellulose-fed continuous culture

The ruminal cellulolytic bacterium Fibrobacter succinogenes S85 was grown in cellulose-fed continuous culture at 22 different combinations of dilution rate (D, 0.014–0.076 h^-1) and extracellular pH (6.11–6.84). Effects of pH and D on the fermentation were determined by subjecting data on cellulose consumption, cell yield, product yield (succinate, acetate, formate), and soluble sugar concentrationto response surface analysis. The extent of cellulose conversion decreased with increasing D. First-order rate constants at rapid growth rates were estimated as 0.07–0.11 h^-1, and decreased with decreasing pH. Apparent decreases in the rate constant with increasing D was not due to inadequate mixing or preferential utilization of the more amorphous regions of the cellulose. Significant quantities of soluble sugars (0.04–0.18 g/l, primarily glucose) were detected in all cultures, suggesting that glucose uptake was rather inefficient. Cell yields (0.11–0.24 g cells/g cellulose consumed) increased with increasing D. Pirt plots of the predicted yield data were used to determined that maintenance coefficient (0.04–0.06 g cellulose/g cells · h) and true growth yield (0.23–0.25 g cells/g cellulose consumed) varied slightly with pH. Yields of succinate, the major fermentation endproduct, were as high as 1.15 mol/mol anhydroglucose fermented, and were slightly affected by dilution rate but were not affected by pH. Comparison of the fermentation data with that of other ruminal cellulolytic bacteria indicates that F. succinogenes S85 is capable of rapid hydrolysis of crystalline cellulose and efficient growth, despite a lower _max on microcrystalline cellulose.     

Comparative fermentability of enzymatic and acid hydrolysates of steam-pretreated aspenwood hemicellulose by Pichia stipitis CBS 5776

Summary Enzymatic hydrolysates of hemicellulose from steam-pretreated aspenwood were more fermentable than the acid hydrolysate after rotoevaporation or ethyl acetate extraction treatments to remove acetic acid and sugar- and lignin-degradation products prior to fermentation by Pichia stipitis CBS 5776. Total xylose and xylobiose utilization from 5.0% (w/v) ethyl acetate extracted enzymatic hydrolysate was observed with an ethanol yield of 0.47 g ethanol/g total available substrate and an ethanol production rate of 0.20 g·l^-1 per hour in 72 h batch fermentation.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

ABE production from yellow poplar through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation

ABE (acetone-butanol-ethanol) was produced through alkaline pre-hydrolysis, enzymatic saccharification, and fermentation using yellow poplar as a raw material. In alkaline pre-hydrolysis, 51.1% of the biomass remained as a residue. In the main woody components, the degrees of lignin and xylan removal were 94.3 and 62.0%, respectively. A yield of 80.9% for cellulose-to-glucose and 81.2% for xylan-to-xylose were obtained by enzymatic hydrolysis. The sugar composition of enzymatic hydrolysate was 95.1 g/L of glucose and 21.4 g/L of xylose. The enzymatic hydrolysate also contained 0.5 g/L of acetic acid and 0.5 g/L of total phenolics. Furfural and 5-hydroxymethylfurfural (5-HMF) were not detected in this hydrolysate. The yellow poplar hydrolysate (YPH) from enzymatic saccharification was used for the production of ABE using Clostridium acetobutylicum and C. beijerinckii. In YPH fermentation, C. acetobutylicum produced 18.1 g/L total ABE (productivity 0.38 g/L h, and yield 0.42), and C. beijerinckii produced 12.1 g/L (productivity 0.25 g/L h, and yield 0.37). Although the ABE productivity by C. beijerinckii was slightly low, the general performance of ABE fermentation in YPH was similar to or higher than those reported previously. Therefore, alkaline pre-hydrolysis could be a very effective pretreatment step prior to enzymatic hydrolysis.     

Butanol production by Clostridium beijerinckii ATCC 55025 from wheat bran

Wheat bran, a by-product of the wheat milling industry, consists mainly of hemicellulose, starch and protein. In this study, the hydrolysate of wheat bran pretreated with dilute sulfuric acid was used as a substrate to produce ABE (acetone, butanol and ethanol) using Clostridium beijerinckii ATCC 55025. The wheat bran hydrolysate contained 53.1 g/l total reducing sugars, including 21.3 g/l of glucose, 17.4 g/l of xylose and 10.6 g/l of arabinose. C. beijerinckii ATCC 55025 can utilize hexose and pentose simultaneously in the hydrolysate to produce ABE. After 72 h of fermentation, the total ABE in the system was 11.8 g/l, of which acetone, butanol and ethanol were 2.2, 8.8 and 0.8 g/l, respectively. The fermentation resulted in an ABE yield of 0.32 and productivity of 0.16 g l⁻¹ h⁻¹. This study suggests that wheat bran can be a potential renewable resource for ABE fermentation.     

拜氏梭菌Clostridium beijerinckii NCIMB 8052发酵玉米皮生产丁醇

玉米皮作为玉米淀粉加工的副产物,是一种可用于生产液体燃料的潜在廉价优质的生物质资源。本文以玉米皮为原料,对拜氏梭菌发酵生产丁醇进行了研究。实验结果表明,玉米皮首先在最优的预处理温度140℃下使用0.5%硫酸水溶液以固液比1∶8处理20 min,再添加200 IU/g底物糖化酶、1.0 IU/g底物木聚糖酶进行酶解,可以使原料中的淀粉和半纤维素转化为可发酵糖,此时水解液中的总糖浓度为50.46 g/L。然后使用1.0%的活性炭对水解液进行脱毒处理以去除发酵抑制物,再进行丁醇发酵,丁醇产量为9.72 g/L,总溶剂产量可达14.09 g/L,糖醇转化率为35.1%。上述研究结果证明玉米皮作为一种粮食加工废弃物用于液体燃料丁醇的生产在技术上是完全可行的。     

Ethanol production from rice straw using optimized aqueous-ammonia soaking pretreatment and simultaneous saccharification and fermentation processes

Rice straw was pretreated using aqueous-ammonia solution at moderate temperatures to enable production of the maximum amount of fermentable sugars from enzymatic hydrolysis. The effects of various operating variables including pretreatment temperature, pretreatment time, the concentration of ammonia and the solid-to-liquid ratio on the degree of lignin removal and the enzymatic digestibility were optimized using response surface methodology. The optimal reaction conditions, which resulted in an enzymatic digestibility of 71.1%, were found to be 69 °C, 10 h and an ammonia concentration of 21% (w/w). The effects of different commercial cellulases and the additional effect of a non-cellulolytic enzyme, xylanase, were also evaluated. Additionally, simultaneous saccharification and fermentation was conducted with rice straw to assess the ethanol production yield and productivity.