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.     

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.     

Enhanced Production of 2,3-Butanediol by Klebsiella pneumoniae Grown on High Sugar Concentrations in the Presence of Acetic Acid

The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol (hereafter referred to as butanediol) by Klebsiella pneumoniae grown on high initial concentrations (up to 10%) of sugars was investigated. Initial fermentation studies with a chemically defined medium suggested that sugar levels in excess of 2% could not be utlized even when a higher inoculum size (5 to 10%) was used. The addition of nutrient supplements, viz., yeast extract, urea, ammonium sulfate, and trace elements resulted in a 10 to 50% increase in butanediol yields, although sugar utilization remained incomplete. The concentration of end products normally found at the termination of fermentation was shown to be noninhibitory to growth and substrate utilization. Acetic acid was inhibitory at concentrations above 1%, although growth and butanediol yield were stimulated in cultures supplemented with lower levels of acetic acid. The efficient utilization of 4% substrate concentrations of d-glucose and d-xylose was achieved, resulting in butanediol yields of 19.6 and 22.0 g/liter, respectively.     

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.     

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.     

The Effect of Gas Sparging in Cross‐Flow Microfiltration of 2,3‐Butanediol Fermentation Broth

Recovery of 2,3‐butanediol from a fermentation broth entails the separation of cells and other suspended solids as the initial step for subsequent separation stages. The aim of this work was to study the cross‐flow filtration of broth in the fermentation of 2,3‐butanediol from blackstrap molasses by Klebsiella oxytoca (NRRL B‐199). A plate type laboratory scale cross‐flow microfiltration unit with a 0.2‐μm cellulose acetate membrane was employed for this purpose. Preliminary results showed that the permeate flux would decline rapidly due to fouling caused by the natural impurities of blackstrap molasses, and modifications of the conventional cross‐flow filtration would be essential to achieve a filtration rate appropriate for practical purposes. In this work, the permeate flux was enhanced by air sparging, which scoured the membrane surface of colloidal deposits and allowed a practical filtration rate to be maintained. The average permeate flux increased by 39 % and 54 % for an air sparging rate of 0.5 L/min and 1.0 L/min respectively, in the case of an initial biomass concentration of 4.66 g/L. For an initial biomass concentration of 14.2 g/L, the flux increased by 105 % and 146 % for the gas rate of 0.5 and 1.0 L/min, respectively. It may be concluded that gas sparging is beneficial in cross‐flow filtration of thick suspensions like a fermentation broth.     

A simulation model for the continuous production of acetoin and butanediol using Bacillus subtilis with integrated pervaporation separation

The potential for producing acetoin and butanediol with a Bacillus subtilis strain was investigated with continuous culture using molasses as carbon substrate. The steady-state results were influenced by both oxygen and undetermined limiting compounds. Employing the known metabolic pathways, four overall stoichiometry relations were used with an energetic assumption on the energy requirements for biomass formation to establish a linear relations were used with an energetic assumption on the energy requirements for biomass formation to establish a linear relation between the overall rates, whose parameters were determined by linear regression. This provided a relationship for the product formation rate. The chemostat culture data were described with a growth kinetics model, which included limitation by molasses and oxygen as well as diauxic effects and product inhibition. The biokinetics model was combined with an experimentally verified model for the membrane Pervaporation. From this combined model were determined the influence of the membrane characteristics (enrichment factors and membrane area) and the dilution rate on the performance of the integrated process. Simulations revealed that an increase of the enrichment factor, possible by membrane improvement, would have counteracting influences, owing to decreased product inhibition but with lower biomass concentration. (c) 1993 Wiley & Sons, Inc.     

Microbial production of 2,3-butanediol from whey permeate

Summary Of four organisms tested in semi-synthetic medium for the production of 2,3-butanediol from lactose, Klebsiella pneumoniae N.C.I.B. 8017 proved to be the most promising. When tested using rennet whey permeate as substrate, a butanediol concentration of 7.5 g/l, representing a yield of 0.46 g/g lactose utilized, was observed after 96 h incubation. In whey permeate where the lactose had been hydrolysed enzymatically prior to the fermentation, a butanediol concentration of 13.7 g/l, representing a yield of 0.39 g/g sugar utilized was obtained. These results indicate that lactose utilization may be a limiting step in the fermentation process.     

Engineering the Pichia pastoris methanol oxidation pathway for improved NADH regeneration during whole-cell biotransformation

Industrial biocatalytic reduction processes require the efficient regeneration of reduced cofactors for the asymmetric reduction of prochiral compounds to chiral intermediates which are needed for the production of fine chemicals and drugs. Here, we present a new engineering strategy for improved NADH regeneration based on the Pichia pastoris methanol oxidation pathway. Studying the kinetic properties of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) and using the derived kinetic data for subsequent kinetic simulations of NADH formation rates led to the identification of FLD activity to constitute the main bottleneck for efficient NADH recycling via the methanol dissimilation pathway. The simulation results were confirmed constructing a recombinant P. pastoris strain overexpressing P. pastoris FLD and the highly active NADH-dependent butanediol dehydrogenase from S. cerevisiae. Employing the engineered strain, significantly improved butanediol production rates were achieved in whole-cell biotransformations.     

Fed-batch with biomass recycle and batch production of 2,3-butanediol from glucose by Bacillus polymyxa

The production of 2,3-butanediol from glucose by Bacillus polymyxa in batch was sensitive to both protein concentration and aeration rate. Two fed-batch experiments which were resupplied from a reservoir containing urea as the sole source of nitrogen, and incorporated total biomass recycle resulted in yields of 65.84 mM and 69.66 mM of 2,3-butanediol per 100 mM of glucose utilized. No massive sporulation events were detected. In addition, fed-batch with recycle yielded more butanediol and less acetate than any batch run.     

Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724

It is known that 2,3-butanediol is a potentially valuable chemical feedstock that can be produced from the sugars present in hemicellulose and celluose hydrolysates. Klebsiella oxytoca is able to ferment most pentoses, hexoses, and disaccharides. Butanediol appears to be a primary metabolite, excreted as a product of energy methabolism. The theoretical maximum yield of butanediol from monosaccharides is 0.50 g/g. This article describes the effects of pH, xylose concentration, and the oxygen transfer rate on the bioconversion of D-xylose to 2,3-butanediol. Product inhibition by butanediol is also examined. The most important variable affecting the kinetics of this system appears to be the oxygen transfer rate. A higher oxygen supply favors the formation of cell mass at the expense of butanediol. Decreasing the oxygen supply rate increases the butanediol yield, but decreases the overall conversion rate due to a lower cell concentration.     

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.     

Myxococcus xanthus biomass as biosorbent for lead

This paper deals with lead biosorption by Myxococcus xanthus biomass in which dry biomass, accumulating up to 1.28 mmol of lead g(-1), is demonstrated to be a more efficient biosorbent than wet biomass. Dry biomass biosorption was found to be very rapid, reaching equilibrium after 5-10 min. Culture age, the initial lead concentration and pH affected this process, but temperature did not. Furthermore, by using sodium citrate as a desorbent agent, 92.17% of the biosorbed lead could be recovered. It was also established that the biosorbed lead is located on the cellular wall and within the characteristic extracellular polysaccharide of this micro-organism.     

Kinetic and equilibrium studies on the biosorption of reactive black 5 dye by Aspergillus foetidus

An isolated fungus, Aspergillus foetidus had the ability to decolourize growth unsupportive medium containing 100 mg L(-1) of reactive black 5 (RB5) dye with >99% efficiency at acidic pH (2-3). Pre-treatment of fungal biomass by autoclaving or exposure to 0.1M sodium hydroxide facilitated more efficient uptake of dye as compared to untreated fungal biomass. Pre-equilibrium biosorption of RB5 dye onto fungus under different temperatures followed pseudo-second-order kinetic model with high degree of correlation coefficients (R(2)>0.99). Biosorption isotherm data fitted better into Freundlich model for lower concentrations of dye probably suggesting the heterogeneous nature of sorption process. Based on the Langmuir isotherm plots the maximum biosorption capacity (Q(0)) value was calculated to be 106 mg g(-1) at 50 degrees C for fungal biomass pre-treated with 0.1M NaOH. Thermodynamic studies revealed that the biosorption process was favourable, spontaneous and endothermic in nature. Recovery of both adsorbate (dye) and adsorbent (fungal biomass) was possible using sodium hydroxide. Recovered fungal biomass could be recycled number of times following desorption of dye using 0.1M NaOH. Fungal biomass pre-treated with NaOH was efficient in decolourizing solution containing mixture of dyes as well as composite raw industrial effluent generated from leather, pharmaceutical and dye manufacturing company.     

Myxococcus xanthus biomass as biosorbent for lead

This paper deals with lead biosorption by Myxococcus xanthus biomass in which dry biomass, accumulating up to 1·28 mmol of lead g⁻¹, is demonstrated to be a more efficient biosorbent than wet biomass. Dry biomass biosorption was found to be very rapid, reaching equilibrium after 5–10 min. Culture age, the initial lead concentration and pH affected this process, but temperature did not. Furthermore, by using sodium citrate as a desorbent agent, 92·17% of the biosorbed lead could be recovered. It was also established that the biosorbed lead is located on the cellular wall and within the characteristic extracellular polysaccharide of this micro-organism.     

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.     

Lessons from the cow: What the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass

Consolidated bioprocessing (CBP) of cellulosic biomass is a promising source of ethanol. This process uses anaerobic bacteria, their own cellulolytic enzymes and fermentation pathways that convert the products of cellulose hydrolysis to ethanol in a single reactor. However, the engineering and economics of the process remain questionable. The ruminal fermentation is a very highly developed natural cellulose-degrading system. We propose that breakthroughs developed by cattle and other ruminant animals in cellulosic biomass conversion can guide future improvements in engineered CBP systems. These breakthroughs include, among others, an elegant and effective physical pretreatment; operation at high solids loading under non-aseptic conditions; minimal nutrient requirements beyond the plant biomass itself; efficient fermentation of nearly all plant components; efficient recovery of primary fermentation end-products; and production of useful co-products. Ruminal fermentation does not produce significant amounts of ethanol, but it produces volatile fatty acids and methane at a rapid rate. Because these alternative products have a high energy content, efforts should be made to recover these products and convert them to other organic compounds, particularly transportation fuels.     

The influence of minerals on the formation of 2,3 butanediol by Aerobacter aerogenes

Summary The presence of a small concentration of nickel or zinc ions in the cultures ofAerobacter aerogenes increases the formation of 2,3 butanediol in the cultures. The presence of 1 per cent of milk increases the formation of 2,3 butanediol in the cultures and decreases the consumption of sugar. Copper ions inhibit the formation of 2,3 butanediol.The combination of zinc and nickel ions decrease the formation of 2,3 butanediol in the cultures ofAerobacter aerogenes and even with addition of milk this combination is not beneficial to the formation of the diol. Nickel in presence of milk increases the formation of 2,3 butanediol.     

Online monitoring of the respiratory quotient reveals metabolic phases during microaerobic 2,3‐butanediol production with Bacillus licheniformis

Microaerobic cultivation conditions are often beneficial for the biotechnological production of reduced metabolites like 2,3‐butanediol. However, due to oxygen limitation, process monitoring based on oxygen transfer rate, or dissolved oxygen measurement provides only limited information. In this study, online monitoring of the respiratory quotient is used to investigate the metabolic activity of Bacillus licheniformis DSM 8785 during mixed acid‐2,3‐butanediol production under microaerobic conditions. Thereby, the respiratory quotient provides valuable information about different metabolic phases. Based on partial reaction stoichiometries, the metabolic activity in each phase of the cultivation was revealed, explaining the course of the respiratory quotient. This provides profound information on the formation or consumption of glucose, 2,3‐butanediol, ethanol and lactate, both, in shake flasks and stirred tank reactor cultivations. Furthermore, the average respiratory quotient correlates with the oxygen availability during the cultivation. Carbon mass balancing revealed that this reflects the increased formation of reduced metabolites with increasing oxygen limitation. The results clearly demonstrate that the respiratory quotient is a valuable online signal to reveal and understand the metabolic activity during microaerobic cultivations. The approach of combining respiratory quotient monitoring with stoichiometric considerations can be applied to other organisms and processes to define suitable cultivation conditions to produce the desired product spectrum.