Evaluation of the xylan breakdown potential of eight mesophilic endoxylanases

In biomass degradation using simultaneous saccharification and fermentation (SSF), there is a need for efficient biomass degrading enzymes that can work at lower temperatures suitable for yeast fermentation. As xylan is an important lignocellulosic biomass constituent, this study aimed at investigating the possible differences in xylan breakdown potential of endoxylanases using eight different endoxylanases at conditions relevant for SSF. Both solubilising and degrading capacities of the endoxylanases were investigated using water-insoluble and water-soluble oat spelt xylan as model substrates for biomass xylan. Results showed that selecting for combinations of endoxylanases that are efficient at solubilising xylan on the one hand and degrading it to large extent on the other hand, coupled to high specific activities, seems the best option for complete xylan breakdown in lignocellulosic biomass conversion using SSF.     

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.     

Complete genome sequence of Lactobacillus buchneri NRRL B-30929, a novel strain from a commercial ethanol plant

Lactobacillus buchneri strain NRRL B-30929 was a contaminant obtained from a commercial ethanol fermentation. This facultative anaerobe is unique because of its rapid growth on xylose and simultaneous fermentation of xylose and glucose. The strain utilizes a broad range of carbohydrate substrates and possesses a high tolerance to ethanol and other stresses, making it an attractive candidate for bioconversion of biomass substrates to various bioproducts. The genome sequence of NRRL B-30929 will provide insight into the unique properties of this lactic acid bacterium.     

A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol

Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of S. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase. Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose. With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight). h(-1) under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.     

L-丙氨酸厌氧发酵关键技术及产业化

传统氨基酸制造主要是通过化学合成或好氧发酵实现。相对于化学合成,微生物发酵可以实现以可再生资源为原料直接生产氨基酸,减少了对石油基原料的依赖,解决了化学合成高污染、高能耗等问题。好氧发酵具有生长快、产量高等特点,但好氧发酵中大量碳源用于细胞生长容易造成糖酸转化率低、能耗高等问题。厌氧发酵是近年来新出现的氨基酸生产模式,具有操作简单、无需通氧、糖酸转化率高容易接近理论最大值等优势。L-丙氨酸是国际上首个实现厌氧发酵产业化生产的氨基酸。本文以L-丙氨酸为例,综述了氨基酸厌氧发酵过程中的关键问题及其在产业化实施中的应用。未来,随着厌氧发酵关键技术在更多化合物生物制造技术中的突破,这种低成本、高效、低碳环保型发酵方式将会带来更大的经济价值和社会效益。     

Modelling fungal solid-state fermentation: the role of inactivation kinetics

The theoretical mathematical models described in this paper are used to evaluate the effects of fungal biomass inactivation kinetics on a non-isothermal tray solid-state fermentation (SSF). The inactivation kinetics, derived from previously reported experiments done under isothermal conditions and using glucosamine content to represent the amount of biomass, are described in different ways leading to four models. The model predictions show only significant effects of inactivation kinetics on temperature and biomass patterns in the tray SSF after long fermentation periods. The models in which inactivation is triggered by low specific growth rates can predict restricted biomass evolution in combination with a fast temperature increase followed by a slower temperature decrease. Such inactivation might occur when substrate is limiting or products are formed in toxic concentrations. Temperature is predicted to be the key parameter. Oxygen concentration is predicted to become limiting only at high heat conduction and low oxygen diffusion rates. Desiccation of the substrate is predicted not to occur.     

Improvement of Y-values of Hansenula polymorpha growth on methanol by simultaneous utilization of glucose

The simultaneous utilization of methanol and glucose by Hansenula polymorpha MH20 was investigated in chemostat (C-limited) cultivation. The mixed-substrate utilization results in biomass yields which are greater up to 20 to 25% as expected assuming an additive growth on both substrates. This is referred to as an auxiliary-substrate effect. Additionally, methanol can be utilized at higher growth rates in the presence of glucose compared to those obtained on this substrate alone. The extend of the auxiliary-substrate effect and the optimum ratio of substrates to reach this effect depend on dilution rate. The greatest stimulation in yield is obtained at D approximately 0.1 h-1, after raising the dilution rate this effect diminishes. At a rate of 0.1 h-1 the optimum mixed-substrate ratio of methanol: glucose is 7:1 (g). By increasing the growth rate the ratio changes toward glucose and reached a value of 1:1 (g) at D = 0.3 h-1. This change in the optimum ratio correlates with diminution in yield coefficient of methanol accompanying an increase in growth rate greater than 0.15 h-1. Energy balances of the utilization of the single substrates are used for interpretation of these results. From this it is evident that methanol does not play the role of an energy-rich substrate in the metabolism of yeast. Rather glucose is the energy-providing substrate in this combination.     

Influence of the ethanol and glucose supply rate on the rate and enantioselectivity of 3-oxo ester reduction by baker's yeast

Baker's-yeast-mediated reductions of ketones hold great potential for the industrial production of enantiopure alcohols. In this article we describe the stoichiometry and kinetics of asymmetric ketone reduction by cell suspensions of bakers' yeast (Saccharomyces cerevisiae). A system for quantitative analysis of 3-oxo ester reduction was developed and allowed construction of full mass and redox balances as well as determination of the influence of different process parameters on aerobic ketone reduction. The nature of the electron donor (ethanol or glucose) and its specific consumption rate by the biomass (0-1 mol.kg dw(-1).h(-1)) affected the overall stoichiometry and rate of the process and the final enantiomeric excess of the product. Excess glucose as the electron donor, i.e. a very high consumption rate of glucose, resulted in a high rate of alcoholic fermentation, oxygen consumption, and biomass formation and therefore causing low efficiency of glucose utilization. Controlled supply of the electron donor at the highest rates applied prevented alcoholic fermentation but still resulted in biomass formation and a high oxygen requirement, while low rates resulted in a more efficient use of the electron donor. Low supply rates of ethanol resulted in biomass decrease while low supply rates of glucose provided the most efficient strategy for electron donor provision and yielded a high enantiomeric excess of ethyl (S)-3-hydroxybutanoate. In contrast to batchwise conversions with excess glucose as the electron donor, this strategy prevented by-product formation and biomass increase, and resulted in a low oxygen requirement.     

Modeling bisubstrate removal by biofilms

A bisubstrate secondary utilization model is based on the concept that an individual substrate can be utilized not only by the biomass by its utilization but also by the biomass made from the utilization of the other substrate. When substrate concentrations are low, a key factor is having sufficient substrate to initiate biofilm growth. Modeling results for three characteristic cases demonstrate that satisfying a total S(min) concentration for a bisubstrate system is the necessary condition for initiating biofilm growth and simultaneous utilization of both substrates. Because having more than one substrate supporting biofilm growth enhances the removal of each compound, the utilization rate of a specific compound can be increased by the concentration of other compounds, and the total S(min) concentration can be less than the weighted average of individual S(min) values.     

Bioenergetic aspects of aerobic glucose metabolism of Escherichia coli K-12 under varying specific growth rates and glucose concentrations.

An attempt was made to find a bioenergetical explanation for the differential effect of specific growth rate and glucose concentration on glucose metabolism of Escherichia coli K-12 with the help of 2,4-dinitrophenol (DNP). The effect of DNP on biomass occurred only at high glucose concentrations. The presence of this uncoupler strongly stimulated glucose uptake rates and oxygen uptake rates, but repressed severly Yg values. Increase in glucose concentration, however, sharply decreased QO2. The amount of oxygen required for maintenance was not affected by DNP, but Yomax values were much lower in the presence of DNP. The results are discussed and it is suggested that aerobic fermentation is caused by a severe reduction of site 1 of the respiratory chain region, whereas biomass formation is affected by repression of the terminal cytochrome a2. In comparing the effect of glucose on biomass formation at similar Qglucose levels aerobic and anaerobic fermentation, repression occurred in both cases at glucose concentrations of 0.3% and above. Although the analyses of 15 enzymes established the metabolic differences, the repression of growth was common to both fermentation types.     

Ethanol fermentation from biomass resources: current state and prospects

In recent years, growing attention has been devoted to the conversion of biomass into fuel ethanol, considered the cleanest liquid fuel alternative to fossil fuels. Significant advances have been made towards the technology of ethanol fermentation. This review provides practical examples and gives a broad overview of the current status of ethanol fermentation including biomass resources, microorganisms, and technology. Also, the promising prospects of ethanol fermentation are especially introduced. The prospects included are fermentation technology converting xylose to ethanol, cellulase enzyme utilized in the hydrolysis of lignocellulosic materials, immobilization of the microorganism in large systems, simultaneous saccharification and fermentation, and sugar conversion into ethanol.     

Inhibition of <Emphasis Type="Italic">Pichia stipitis</Emphasis> fermentation of hydrolysates from olive tree cuttings

The ethanolic fermentation of liquid fractions (hydrolysates) issued from dilute acid pre-treatment of olive tree biomass by Pichia stipitis is reported for the first time. On the one side, P. stipitis has been reported as the most promising naturally occurring C5 fermenting microorganism; on the other side, olive tree biomass is a renewable, low cost, and lacking of alternatives agricultural residue especially abundant in Mediterranean countries. The study was performed in two steps. First, the fermentation performance of P. stipitis was evaluated on a fermentation medium also containing the main inhibitors found in these hydrolysates (acetic acid, formic acid, and furfural), as well as glucose and xylose as carbon sources. The effect of inhibitors, individually or in a mixture, on kinetic and yield parameters was calculated. In a second step, hydrolysates obtained from 1% (w/w) sulfuric acid pre-treatment of olive tree biomass at 190°C for 10 min were used as a real fermentation medium with the same microorganism. Due to inhibition, effective fermentation required dilution of the hydrolysate and either overliming or activated charcoal treatment. Results show that ethanol yields obtained from hydrolysates, ranging from 0.35 to 0.42 g/g, are similar to those from synthetic medium, although the process proceeds at lower rates. Inhibiting compounds affect the fermentation performance in a synergistic way. Furfural is rapidly assimilated by the yeast; acetic acid and formic acid concentrations decrease slowly during the process. Activated charcoal or overliming detoxification improve the fermentability of diluted hydrolysates.     

Conversion of rice straw to bio-based chemicals: an integrated process using Lactobacillus brevis

Commercialization of lignocellulosic biomass as a feedstock for bio-based chemical production is problematic due to the high processing costs of pretreatment and saccharifying enzymes combined with low product yields. Such low product yield can be attributed, in large part, to the incomplete utilization of the various carbohydrate sugars found in the lignocellulosic biomass. In this study, we demonstrate that Lactobacillus brevis is able to simultaneously metabolize all fermentable carbohydrates in acid pre-processed rice straw hydrolysate, thereby allowing complete utilization of all released sugars. Inhibitors present in rice straw hydrolysate did not affect lactic acid production. Moreover, the activity of exogenously added cellulases was not reduced in the presence of growing cultures of L. brevis. These factors enabled the use of L. brevis in a process termed simultaneous saccharification and mixed sugar fermentation (SSMSF). In SSMSF with L. brevis, sugars present in rice straw hydrolysate were completely utilized while the cellulase maintained its maximum activity due to the lack of feedback inhibition from glucose and/or cellobiose. By comparison to a sequential hydrolysis and fermentation process, SSMSF reduced operation time and the amount of cellulase enzyme necessary to produce the same amount of lactic acid.     

Dispersion optimization to enhance PHB production in fed-batch cultures of Ralstonia eutropha

Despite its many useful properties, microbial production of poly-beta-hydroxybutyrate (PHB) is not yet commercially competitive with synthetic polymers. One reason is inadequate optimization of the fermentation under industrial conditions. In this study, a physiologically reasonable and experimentally validated kinetic model for PHB synthesis by Ralstonia eutropha was incorporated into a dispersion model to simulate a large fed-batch bioreactor. Solutions of the model indicated that cell growth and PHB synthesis were maximum at Peclet numbers (Pe) between 20 and 30, representing limited finite dispersion. At these Peclet numbers, the optimum feed rates also showed lower consumptions of the substrates than at Pe=0. Since complete dispersion was also difficult to achieve in production-scale bioreactors, these results pointed to the possibility of exploiting controlled dispersion for productivity enhancement.     

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.     

Transient sensitivity analysis of a cybernetic model of microbial growth on two substrates

When a microbe has a choice of two substrates for its growth in a fermentation medium, its preference varies with the substrates and with time. This "informed" choice is conveniently expressed by cybernetic models. For the growth of Klebsiella oxytoca in a medium of glucose and lactose, a one-parameter cybernetic model of growth has been employed in a batch bioreactor to analyse sensitivity of the fermentation to perturbations in the parameter, ! (0 h ! h 1). The sensitivity surfaces in the (!-time) space show interesting variations which are discussed. An important observation is that while growth is best promoted with !=1, i.e. sequential consumption of the substrates [8], low sensitivities require smaller values of !, i.e. simultaneous utilisation. Thus, in a realistic operation it may be necessary to compromise between high growth (with subsequent high productivity) and low sensitivity.     

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

Improving the fermentation performance of saccharomyces cerevisiae by laccase during ethanol production from steam‐exploded wheat straw at high‐substrate loadings

Operating the saccharification and fermentation processes at high‐substrate loadings is a key factor for making ethanol production from lignocellulosic biomass economically viable. However, increasing the substrate loading presents some disadvantages, including a higher concentration of inhibitors (furan derivatives, weak acids, and phenolic compounds) in the media, which negatively affect the fermentation performance. One strategy to eliminate soluble inhibitors is filtering and washing the pretreated material. In this study, it was observed that even if the material was previously washed, inhibitory compounds were released during the enzymatic hydrolysis step. Laccase enzymatic treatment was evaluated as a method to reduce these inhibitory effects. The laccase efficiency was analyzed in a presaccharification and simultaneous saccharification and fermentation process at high‐substrate loadings. Water‐insoluble solids fraction from steam‐exploded wheat straw was used as substrate and Saccharomyces cerevisiae as fermenting microorganism. Laccase supplementation reduced strongly the phenolic content in the media, without affecting weak acids and furan derivatives. This strategy resulted in an improved yeast performance during simultaneous saccharification and fermentation process, increasing significantly ethanol productivity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Determination of maintenance coefficients of Saccharomyces cerevisiae cultures with cell recycle by cross-flow membrane filtration

The fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single-stage process with recycle of the cells via cross-flow micro-filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.     

Effect of fermentation broth and biosurfactants on mass transfer during liquid-liquid extraction

Mass transfer rates in liquid-liquid extraction processes can be seriously affected by the presence of surface-active contaminants. This is especially true of applications of a biotechnological origin, where the microorganism used in the process may produce the surface-active contaminants. An investigation into the effects of soluble and insoluble fermentation broth components on mass transfer using chloramphenicol extraction into octanol as the model system was conducted. Soluble components produced during fermentation were found to adsorb to the interface, where they reduced the overall mass transfer coefficient by up to 70%. After fractionation it was found that components in the weight range from 10-30 kDa had the greatest effect on mass transfer. Protein and phospholipid compounds of similar size were found to reduce the overall mass transfer coefficient to a similar extent to the broth components at concentrations around 0.001mg/l. The biomass produced during the fermentation also reduced mass transfer substantially, and it is likely that this was due to physical blockage of the interface.