The bioconversion of wood hydrolyzates to butanol and butanediol

Steam-exploded aspenwood chips were acid hydrolysed to their component sugars. Near theoretical solvent yields were achieved in both the acetone-butanol-ethanol (ABE) fermentation and 2,3-butanediol fermentation of these liberated sugars. When Clostridium acetobutylicum was grown on wood hydrolysates, final butanol yields of 9.0 g/L (0.26 g of butanol per g of sugar consumed) were obtained. When Klebsiella pneumoniae was grown on the wood hydrolysates, final butanediol concentrations exceeded 20 g/L, resulting in a bioconversion efficiency approaching 0.5 g of butanediol per g of sugar utilised.     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

Fed-batch approach to production of 2,3-butanediol by Klebsiella pneumoniae grown on high substrate concentrations.

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol by Klebsiella pneumoniae grown on high sugar concentrations was investigated. When K. pneumoniae was grown under finite air conditions in the presence of added acetic acid, 50 g of D-glucose and D-xylose per liter could be converted to 25 and 27 g of butanediol per liter, respectively. The efficiency of bioconversion decreased with increasing sugar substrate concentrations (up to 200 g/liter). Butanediol production at low sugar substrate concentrations was less efficient when the organism was grown under aerobic conditions; however, final butanediol values were higher for cultures grown on an initial sugar concentration of 150 g/liter, particularly when the inoculum was first acclimatized to high sugar levels. When a double fed-batch approach (daily additions of sugars together with yeast extract) was used under aerobic conditions, up to 88 and 113 g of combined butanediol and acetyl methyl carbinol per liter could be obtained from the utilization of 190 g of D-xylose and 226 g of D-glucose per liter, respectively.     

Butanediol production by Aerobacter aerogenes NRRL B199: effects of initial substrate concentration and aeration agitation

Butanediol production by Aerobacter aerogenes NRRL B199 grown on glucose requires an optimal rate of aeration for the obtention of butanediol 2, 3. In the absence of air, Aerobacter aerogenes NRRL B199 growth and production are weak. Agitation-aeration is necessary for producing the biomass, but an excess of oxygen proves to be toxic with regard to metabolite production. Oxygen is a limiting substrate with regard to growth and an inhibitor with regard to the specific metabolite productivity. This observation is discussed from a kinetic stand point and in relation to the search for the optimum oxygen transfer coefficient (K(L)a), which is found to be in the range of 50-100h(-1). It has also been observed that K(L)a increases during the fermentation cycle. The initial substrate concentration effects the yield production of biomass and butanediol production. Low yields of butanediol are obtained at low initial sugar concentrations, but good yields of butanediol are obtained (0.45 g/g) at high concentrations of glucose (195 g/L). Carbon substrates and butanediol are inhibitors of cell growth while butanediol is not quite an inhibitor of the specific rate of butanediol production for the range of butanediol of 0-100 g/L.     

Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3 I: production of xylitol and ethanol

An endophytic yeast, Rhodotorula mucilaginosa strain PTD3, that was isolated from stems of hybrid poplar was found to be capable of production of xylitol from xylose, of ethanol from glucose, galactose, and mannose, and of arabitol from arabinose. The utilization of 30 g/L of each of the five sugars during fermentation by PTD3 was studied in liquid batch cultures. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. PTD3 demonstrated an exceptional ability to ferment the hydrolysate, overcome hexose repression of xylose utilization with a short lag period of 10 h, and tolerate sugar degradation products. In direct comparison, PTD3 had higher xylitol yields from the mixed sugar hydrolysate compared with the widely studied and used xylitol producer Candida guilliermondii.     

The combined enzymatic hydrolysis and fermentation of hemicellulose to 2,3-butanediol

Summary Hemicellulose-rich fractions from several agricultural residues were converted to 2,3-butanediol by a combined enzymatic hydrolysis and fermentation process. Culture filtrates from Trichoderma harzianum E58 were used to hydrolyze the substrates while Klebsiella pneumoniae fermented the liberated sugars to 2,3-butanediol. Approximately 50–60% of a 5% (w/v) xylan preparation could be hydrolyzed and quantitatively converted to 2,3-butanediol using this procedure. Although enzymatic hydrolysis was optimal at pH 5.0 and 50° C, the combined hydrolysis and fermentation was most efficient at pH 6.5 and 30° C. Combined hydrolysis and fermentation resulted in butanediol levels that were 20–40% higher than could be obtained with a separate hydrolysis and fermentation process. The hemicellulose-rich water-soluble fractions obtained from a variety of steam-exploded agricultural residues could be readily used by the combined hydrolysis and fermentation approach resulting in butanediol yields of 0.4–0.5 g/g of reducing sugar utilized.     

Characterization of the impact of acetate and lactate on ethanolic fermentation by Thermoanaerobacter ethanolicus

Ethanolic fermentation of simple sugars is an important step in the production of bioethanol as a renewable fuel. Significant levels of organic acids, which are generally considered inhibitory to microbial metabolism, could be accumulated during ethanolic fermentation, either as a fermentation product or as a by-product generated from pre-treatment steps. To study the impact of elevated concentrations of organic acids on ethanol production, varying levels of exogenous acetate or lactate were added into cultures of Thermoanaerobacter ethanolicus strain 39E with glucose, xylose or cellobiose as the sole fermentation substrate. Our results found that lactate was in general inhibitory to ethanolic fermentation by strain 39E. However, the addition of acetate showed an unexpected stimulatory effect on ethanolic fermentation of sugars by strain 39E, enhancing ethanol production by up to 394%. Similar stimulatory effects of acetate were also evident in two other ethanologens tested, T. ethanolicus X514, and Clostridium thermocellum ATCC 27405, suggesting the potentially broad occurrence of acetate stimulation of ethanolic fermentation. Analysis of fermentation end product profiles further indicated that the uptake of exogenous acetate as a carbon source might contribute to the improved ethanol yield when 0.1% (w/v) yeast extract was added as a nutrient supplement. In contrast, when yeast extract was omitted, increases in sugar utilization appeared to be the likely cause of higher ethanol yields, suggesting that the characteristics of acetate stimulation were growth condition-dependent. Further understanding of the physiological and metabolic basis of the acetate stimulation effect is warranted for its potential application in improving bioethanol fermentation processes.     

Fermentation of Xylan,Corn Fiber,or Sugars to Acetoin and Butanediol by Bacillus polymyxa Strains

Bacillus polymyxa can produce levo-butanediol, a potential biogradable anti-freeze, and ethanol, a fuel additive, using starch-based fermentations. To explore use of less expensive biomass fermentation substrates, we screened B. polymyxa strains for good growth on xylans. During aerobic growth on glucose, six selected xylanolytic strains produced mainly acetoin and butanediol plus lesser amounts of acetaldehyde and ethanol. Undesirable acetoin formation was eliminated by anaerobic growth on glucose, but substrate usage, butanediol, and other fermentation products were greatly reduced. High xylanase activity occurred with growth on xylans or corn fiber, and about 50–65% of oatspelt xylan and 25–35% of the corn fiber were used during aerobic growth, but unexpectedly no butanediol and only small levels of acetoin were produced. Aerobic growth on arabinose, arabinose plus glucose, or xylose plus glucose resulted in both acetoin and butanediol formation. Little or no butanediol was made from xylose alone. Growth on an acid hydrolysate of corn fiber that contained a mixture of these sugars resulted in the formation of acetoin, acetaldehyde, and ethanol, but very little butanediol. The data suggest B. polymyxa is limited in conversion of xylan-rich biomass sources or their hydrolysates to butanediol. This limitation might be overcome by using better cultivation conditions and/or genetically engineered strains.     

Studies of the Utilization of Coconut Water Waste for the Production of the Food Yeast Saccharomyces fragilis

The accepted food yeast Saccharomyces fragilis was grown in batch and chemostat culture on coconut water and on a simulated coconut-water medium containing glucose, fructose, sucrose and sorbitol, to provide kinetic data for a feasibility study of microbial protein production. Analyses of growth on individual and mixed carbon substrates were made to determine sugar assimilation patterns in batch and chemostat cultures on coconut water. Growth on the polyol produced a much reduced specific growth rate, assimilation rate, growth yield and productivity compared to growth on the sugars. In mixed substrate fermentations a sequential utilization of the carbohydrates occurred. Both the monosaccharides repressed invertase synthesis and all three sugars repressed sorbitol assimilation. Complete carbon assimilation was only obtained by prolonged batch fermentation or in chemostat cultures at low dilution rates (<0.10 h^-1). Supplementation of coconut water with biotin and nicotinic acid increased biomass yields in chemostat cultures.     

Metabolism of glucose and cellobiose by cellulolytic mesophilic Clostridium sp. strain H10.

The metabolism of strain H10, a cellulolytic mesophilic Clostridium sp., was studied on glucose and cellobiose as energy and carbon sources. The main products of fermentation of both sugars were acetate, lactate, and ethanol. At low sugar levels, molar growth yields were better for cellobiose than for glucose. In both cases, an inhibition of growth was observed between 1 and 2 g/liter and a total inhibition after the latter concentration. Inhibition was not the result of low pH due to acid formation; growth under static pH conditions was limited in the same way. On the other hand, acetate and lactate had no inhibitory effect when added at concentrations equal to the final titers. Concomitant with the inhibition of growth was a change in metabolic pathways for sugar concentrations between 1 and 2 g/liter, i.e., the production of lactate was higher. After complete inhibition of growth, an accumulation of carbohydrates which were neither glucose nor cellobiose was observed.     

The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger

Summary The influence of various carbon sources and their concentration on the production of citrate by Aspergillus niger has been investigated. The sugars maltose, sucrose, glucose, mannose and fructose (in the given order) were carbon sources giving high yields of citric acid. Optimal yields were observed at sugar concentrations of 10% (w/v), with the exception of glucose (7.5%). No citric acid was produced on media containing less than 2.5% sugar. Precultivation of A. niger on 1% sucrose and transference to a 14% concentration of various other sugars induced citrate accumulation. This could be blocked by the addition of cycloheximide, an inhibitor of de novo protein synthesis. This induction was achieved using maltose, sucrose, glucose, mannose and fructose, and also by some other carbon sources (e.g. glycerol) that gave no citric acid accumulation in direct fermentation. Precultivation of A. niger at high (14%) sucrose concentrations and subsequent transfer to the same concentrations of various other carbohydrates, normally not leading to citric acid production, led to formation of citrate. Endogenous carbon sources were also converted to citrate under these conditions. A 14%-sucrose precultivated mycelium continued producing some citrate upon transfer to 1% sugar. These results indicate that high concentrations of certain carbon sources are required for high citrate yields, because they induce the appropriate metabolic imbalance required for acidogenesis.     

The influence of iron,sulphite and paraformaldehyde on the formation of 2.3 butanediol in the cultures of bacillus polymyxa

Summary InB. polymyxa cultures containing sucrose as the substrate the amount of 2.3 butanediol decreases between 10 and 30 days of fermentation but after 30 days, the formation of the glycol increases. If iron or sulphite ions are present in the culture ofB. polymyxa the yield of 2.3 butanediol is small in the first 10 days, as compared to the control culture but it gradually increases with the increase of the period of fermentation. This increase was observed upto 50 days. In the culture containing paraformaldehyde 2.3 butanediol is not formed. The sugar consumption in the cultures is high only during the first ten days of fermentation and after that though there is enough sugar present, it is not utilised and the amount of 2.3 butanediol goes on increasing. It appears that 2.3 butanediol is formed from some intermediate material formed during the period of high sugar consumption. Later the increase of 2.3 butanediol is due to its formation from the intermediate compound.     

Kinetics of ethanol production by immobilized Kluyveromyces marxianus cells at varying sugar concentrations of Jerusalem artichoke juice

Summary Kinetics of ethanol fermentation at varying sugar concentrations of Jerusalem artichoke tuber extract has been studied using Kluyveromyces marxianus cells immobilized in calcium alginate gel beads. A maximum ethanol concentration of 111 g/l was achieved at an initial sugar concentration of 260 g/l in 20 hours, when the immobilized cell concentration in the calcium alginate beads was 53.3 g dry wt./l bead volume. Ethanol yield remained almost unaffected by initial sugar concentration up to 250 g/l and was found to be about 88% of the theoretical. Maximum rate of ethanol production decreased from 22.5 g ethanol/l/h to 10.5 g ethanol/l/h while the maximum rate of total sugars utilization decreased from 74.9 g sugars/l/h to 28.5 g sugars/l/h as the initial substrate concentration was increased from 100 to 300 g/l. The concentration of free cells in the fermentation broth was low.     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

Bacterial synthesis of poly(hydroxybutyrate- co-hydroxyvalerate) using carbohydrate-rich mahua (Madhuca sp.) flowers

AIMS: The objective of the present work was to utilize an unrefined natural substrate namely mahua (Madhuca sp.) flowers, as a carbon source for the production of bacterial polyhydroxyalkanoate (PHA) copolymer by Bacillus sp-256. METHODS AND RESULTS: In the present work, three bacterial strains were tested for PHA production on mahua flower extract (to impart 20 g l(-1) sugar) amongst which, Bacillus sp-256 produced higher concentration of PHA in its biomass (51%) compared with Rhizobium meliloti (31%) or Sphingomonas sp (22%). Biosynthesis of poly(hydroxybutyrate-co-hydroxyvalerate) - P(HB-co-HV)--of 90 : 10 mol% by Bacillus sp-256 was observed by gas chromatographic analysis of the polymer. Major component of the flower is sugars (57% on dry weight basis) and additionally it also contains proteins, vitamins, organic acids and essential oils. The bacterium utilized malic acid present in the substrate as a co-carbon source for the copolymer production. The flowers could be used in the form of aqueous extract or as whole flowers. PHA content of biomass (%) and yield (g l(-1)) in a 3.0-l stirred tank fermentor after 30 h of fermentation under constant pH (7) and dissolved oxygen content (40%) were 54% and 2.7 g l(-1), respectively. Corresponding yields for control fermentation with sucrose as carbon source were 52% and 2.5 g l(-1). The polymer was characterized by proton NMR. CONCLUSIONS: Utilization of mahua flowers, a natural substrate for bacterial fermentation aimed at PHA production, had additional advantage, as the sugars and organic acids present in the flowers were metabolized by Bacillus sp-256 to synthesize P(HB-co-HV) copolymer. SIGNIFICANCE AND IMPACT OF THE STUDY: Literature reports on utilization of suitable cheaper natural substrate for PHA copolymer production is scanty. Mahua flowers used in the present experiment is a cheaper carbon substrate compared with several commercial substrates and it is rich in main carbon as well as co-carbon sources that can be utilized by bacteria for PHA copolymer production.     

Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids

A number of previous studies determined dilute acid pretreatment conditions that maximize xylose yields from pretreatment or glucose yields from subsequent digestion of the pretreated cellulose, but our emphasis was on identifying conditions to realize the highest yields of both sugars from both stages. Thus, individual xylose and glucose yields are reported as a percentage of the total potential yield of both sugars over a range of sulfuric acid concentrations of 0.22%, 0.49% and 0.98% w/w at 140, 160, 180 and 200 degrees C. Up to 15% of the total potential sugar in the substrate could be released as glucose during pretreatment and between 15% and 90+% of the xylose remaining in the solid residue could be recovered in subsequent enzymatic hydrolysis, depending on the enzyme loading. Glucose yields increased from as high as 56% of total maximum potential glucose plus xylose for just enzymatic digestion to 60% when glucose released in pretreatment was included. Xylose yields similarly increased from as high as 34% of total potential sugars for pretreatment alone to between 35% and 37% when credit was taken for xylose released in digestion. Yields were shown to be much lower if no acid was used. Conditions that maximized individual sugar yields were often not the same as those that maximized total sugar yields, demonstrating the importance of clearly defining pretreatment goals when optimizing the process. Overall, up to about 92.5% of the total sugars originally available in the corn stover used could be recovered for coupled dilute acid pretreatment and enzymatic hydrolysis. These results also suggest that enhanced hemicellulase activity could further improve xylose yields, particularly for low cellulase loadings.     

Ethanol Inhibition Kinetics of Kluyveromyces marxianus Grown on Jerusalem Artichoke Juice

The kinetics of ethanol inhibition on cell growth and ethanol production by Kluyveromyces marxianus UCD (FST) 55-82 were studied during batch growth. The liquid medium contained 10% (wt/vol) inulin-type sugars derived from an extract of Jerusalem artichoke (Helianthus tuberosus) tubers, supplemented with small amounts of Tween 80, oleic acid, and corn steep liquor. Initial ethanol concentrations ranging from 0 to 80 g/liter in the liquid medium were used to study the inhibitory effect of ethanol on the following parameters: maximum specific growth rate (μ_max), cell and ethanol yields, and sugar utilization. It was found that as the initial ethanol concentration increased from 0 to 80 g/liter, and maximum specific growth rate of K. marxianus cells decreased from 0.42 to 0.09 h⁻¹, whereas the ethanol and cell yields and sugar utilization remained almost constant. A simple kinetic model was used to correlate the μ_max results and the rates of cell and ethanol production, and the appropriate constants were evaluated.     

亚硫酸盐甘蔗渣浆酶解液发酵制备乙醇

以亚硫酸盐甘蔗渣浆酶解液作为原料,利用C. shehatae发酵制取燃料乙醇。结果表明：还原糖最适初始质量浓度为葡萄糖140 g/L、木糖60 g/L、酶解液总糖80 g/L。利用初始葡萄糖55.06 g/L、木糖11.18 g/L、纤维二糖4.51 g/L的亚硫酸盐甘蔗渣浆酶解液发酵,经18 h获得乙醇22.98 g/L。乙醇得率为67.23%,葡萄糖利用率为99.27%,木糖利用率为32.96%,C. shehatae适合作为蔗渣为原料的乙醇发酵菌株。     

Comparative evaluations of cellulosic raw materials for second generation bioethanol production

Aims: To evaluate sugar recoveries and fermentabilities of eight lignocellulosic raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis. Methods and Results: Dilute acid pretreatment (2% H₂SO₄) with 10% (w/v) substrate loading was performed at 134°C for 60 min followed by enzyme hydrolysis at 60°C. The results demonstrated that hydrolysis of herbaceous raw materials resulted in higher sugar recoveries (up to 60–75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 (pZB5) demonstrated that final ethanol concentrations and yields were also higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50°C compared to 60°C following acid pretreatment. Conclusions: Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates. Significance and Impact of the Study: The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural/forestry biomass rather than food‐based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.