Xylitol production from a mutant strain of Candida tropicalis

Aims: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed‐batch cultures for a xylitol assimilation‐deficient strain of Candida tropicalis isolated via chemical mutagenesis. Methods and Results: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol‐assimilation deficient strain C. tropicalis SS2. Shake‐flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l^?1 glucose and 25 g l^?1 xylose. With 20 g l^?1 glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed‐batch fermentation with repeated addition of glycerol and xylose resulted in 3·3 g l^?1 h^?1 xylitol volumetric productivity with the final concentration of 220 g l^?1 and overall yield of 0·93 g g^?1 xylitol. Conclusions: The xylitol assimilation‐deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low‐cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. Significance and Impact of the Study: The study confirms the advantage of using a xylitol assimilation‐deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass‐based bio‐refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.     

Xylitol Production from D‐Xylose at Different Oxygen Transfer Coefficients in a Batch Bioreactor

Conversion of D‐xylose to xylitol by Candida boidinii NRRL Y‐17213 was studied under anaerobic and oxygen limited conditions by varying the oxygen transfer coefficient k_La. Shake flask experiments were used to provide the preliminary information required to perform experiments in a bioreactor. The yeast did not grow under fully anaerobic conditions, but anaerobic formations of xylitol, ethanol, ribitol, and glycerol were observed as well as D‐xylose assimilation of 11 %. In shake flasks, with an initial D‐xylose concentration of 50 g/L, an increase in k_La from 8 to 46 h^–1 resulted in a faster growth, higher rate of substrate uptake and lower yields of products. The highest xylitol productivity (0.052 g/L h) was attained at k_La = 8 h^–1. At k_La = 46 h^–1, 98.6 % of D‐xylose was consumed and mainly converted to biomass. Using 130 g/L D‐xylose, k_La was varied in the fermenter from 26 to 78 h^–1. The percentage of consumed D‐xylose increased from 31 % at k_La = 26 h^–1 to 93–94 % at all other aeration levels. Biomass yield increased with k_La, whereas ethanol, ribitol, and glycerol yields exhibited an opposite dependence on the oxygenation level. The most favorable oxygen transfer coefficient for xylitol formation, in the fermenter, was k_La = 47 h^–1 when its concentration (57.5 g/L) surpassed ethanol accumulation by 3.6‐fold, and the glycerol plus ribitol by 10‐fold. Concurrently, xylitol yield and productivity reached 0.45 g/g and 0.26 g/L h, respectively. The volumetric xylitol productivity was affected more by changes in the aeration than the corresponding yield.     

Fermentation behavior of an osmotolerant yeast D. hansenii for Xylitol production

Realizing the importance of xylitol as a high‐valued compound that serves as a sugar substitute, a new, one step thin layer chromatographic procedure for quick, reliable, and efficient determination of xylose and xylitol from their mixture was developed. Two hundred and twenty microorganisms from the laboratory stock cultures were screened for their ability to produce xylitol from D ‐xylose. Amongst these, an indigenous yeast isolate no.139 (SM‐139) was selected and identified as Debaryomyces hansenii on the basis of morphological and biochemical characteristics and (26S) D1/D2 r DNA region sequencing. Debaryomyces hansenii produced 9.33 gL^?1 of xylitol in presence of 50.0 gL^?1 of xylose in 84 h at pH 5.5, 30°C, 200 rpm. In order to utilize even higher concentrations of xylose for maximum xylitol production, a xylose enrichment technique was developed. The strain of Debaryomyces hansenii was obtained through xylose enrichment technique in a statistically optimized medium containing 0.3% yeast extract, 0.2% peptone, 0.03% MgSO₄.7H₂O along with 1% methanol. The culture was inoculated with 6% inoculum and incubated at 30°C and 250 rpm. A yield of 0.6 gg^?1 was obtained with a xylitol volumetric productivity of 0.65 g/L h^?1 in the presence of 200 gL^?1 of xylose although up to 300 gL^?1 of xylose could be tolerated through batch fermentation. Through this technique, even higher concentrations of xylose as substrate could be potentially utilized for maximum xylitol production. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Effect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate byCandida guilliermondii

The effect of inoculum level on xylitol production byCandida guilliermondii was evaluated in a rice straw hemicellulose hydrolysate. High initial cell density did not show a positive effect in this bioconversion since increasing the initial cell density from 0.67 g L^–1 to 2.41 g L^–1 decreased both the rate of xylose utilization and xylitol accumulation. The maximum xylitol yield (0.71 g g^–1) and volumetric productivity (0.56 g L^–1 h^–1) were reached with an inoculum level of 0.9 g L^–1. These results show that under appropriate inoculum conditions rice straw hemicellulose hydrolysate can be converted into xylitol by the yeastC. guilliermondii with efficiency values as high as 77% of the theoretical maximum.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

Analysis and optimization of a two-substrate fermentation for xylitol production using Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose using Candida tropicalisATCC 13803. A two-substrate fermentation was designed in order to increase xylitol yield and volumetric productivity. Glucose was used initially for cell growth followed by conversion of xylose to xylitol without cell growth and by-product formation after complete depletion of glucose. High glucose concentrations increased volumetric productivity by reducing conversion time due to high cell mass, but also led to production of ethanol, which, in turn, inhibited cell growth and xylitol production. Computer simulation was undertaken to optimize an initial glucose concentration using kinetic equations describing rates of cell growth and xylose bioconversion as a function of ethanol concentration. Kinetic constants involved in the equations were estimated from the experimental results. Glucose at 32 g L⁻¹ was estimated to be an optimum initial glucose concentration with a final xylose concentration of 86 g L⁻¹ and a volumetric productivity of 5.15 g-xylitol L⁻¹ h⁻¹. The two-substrate fermentation was performed under optimum conditions to verify the computer simulation results. The experimental results were in good agreement with the predicted values of simulation with a xylitol yield of 0.81 g-xylitol g-xylose⁻¹ and a volumetric productivity of 5.06 g-xylitol L⁻¹ h⁻¹. Received 16 June 1998/ Accepted in revised form 28 February 1999     

Continuous multistep versus fed‐batch production of ethanol and xylitol in a simulated medium of sugarcane bagasse hydrolyzates

Co‐cultures for simultaneous production of ethanol and xylitol were studied under different operation bioreactor modes using Candida tropicalis IEC5‐ITV and Saccharomyces cerevisiae ITV01‐RD in a simulated medium of sugarcane bagasse hydrolyzates. Xylitol and ethanol tolerance by S. cerevisiae and C. tropicalis, respectively, was evaluated. The results showed that C. tropicalis was sensitive to ethanol concentrations up to 30 g/L, while xylitol had no effect on S. cerevisiae viability and metabolism. The best condition found for simultaneous culture was S. cerevisiae co‐culture and C. tropicalis sequential cultivation at 24 h. Under these conditions, productivity and yield for ethanol were Q_EtOH = 0.72 g L^?1 h^?1 and Y_EtOH/s = 0.37 g/g, and for xylitol, Q_XylOH = 0.10 g L^?1 h^?1 and Y_XylOH/S = 0.31 g/g, respectively; using fed‐batch culture, the results were Q_EtOH = 0.87 g L^?1 h^?1 and Y_EtOH/s = 0.44 g L^?1 h^?1, and Q_EtOH = 0.27 g L^?1 h^?1 and Y_EtOH/s = 0.57 g/g, respectively. Maximum volumetric productivity in continuous multistep cultures of ethanol and xylitol was at dilution rates of 0.131 and 0.074 h^?1, respectively. Continuous multistep production, Q_EtOH increased up to 50% more than in fed‐batch culture, even though xylitol yield remained unchanged.     

Evaluation of cotton stalk hydrolysate for xylitol production

Cotton stalk is a widely distributed and abundant lignocellulosic waste found in Turkey. Because of its rich xylose content, it can be a promising source for the production of xylitol. Xylitol can be produced by chemical or biotechnological methods. Because the biotechnological method is a simple process with great substrate specificity and low energy requirements, it is more of an economic alternative for the xylitol production. This study aimed to use cotton stalk for the production of xylitol with Candida tropicalis Kuen 1022. For this purpose, the combined effects of different oxygen concentration, inoculum level and substrate concentration were investigated to obtain high xylitol yield and volumetric xylitol production rate. Candida tropicalis Kuen 1022 afforded different concentrations of xylitol depending on xylose concentration, inoculum level, and oxygen concentration. The optimum xylose, yeast concentration, and airflow rate for cotton stalk hydrolysate were found as 10.41 g L^?1, 0.99 g L^?1, and 1.02 vvm, respectively, and under these conditions, xylitol yield and volumetric xylitol production rate were obtained as 36% and 0.06 g L^?1 hr^?1, respectively. The results of this study show that cotton stalk can serve as a potential renewable source for the production of xylitol.     

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l⁻¹ h⁻¹ at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h. Received: 20 April 1998 / Received revision: 29 May 1998 / Accepted: 19 June 1998     

Batch xylitol production by<Emphasis Type="Italic"> Candida guilliermondii</Emphasis> FTI 20037 from sugarcane bagasse hemicellulosic hydrolyzate at controlled pH values

Batch fermentation of sugarcane bagasse hemicellulosic hydrolyzate by the yeast Candida guilliermondii FTI 20037 was performed using controlled pH values (3.5, 5.5, 7.5). The maximum values of xylitol volumetric productivity (Q _p=0.76 g/l h) and xylose volumetric consumption (Q _s=1.19 g/l h) were attained at pH 5.5. At pH 3.5 and 7.5 the Q _p value decreased by 66 and 72%, respectively. Independently of the pH value, Y _x/s decreased with the increase in Y _p/s suggesting that the xylitol bioconversion improves when the cellular growth is limited. At the highest pH value (7.5), the maximum specific xylitol production value was the lowest (q _pmax=0.085 g/l h.), indicating that the xylose metabolism of the yeast was diverted from xylitol formation to cell growth.List of symbols P _max xylitol concentration (g/l) - Q _x volumetric cell production rate (g/l h) - Q _s volumetric xylose uptake rate (g/l h) - Q _p volumetric xylitol production rate (g/l h) - q _pmax specific xylitol production (g/g h) - q _smax specific xylose uptake rate (g/g h) - _max specific cell growth rate (h^–1) - Y _p/s xylitol yield coefficient, g xylitol per g xylose consumed (g/g) - Y _p/x xylitol yield coefficient, g xylitol per g dry cell mass produced (g/g) - Y _x/s cell yield coefficient, g dry cell mass per g xylose consumed (g/g) - _cell percentage of the cell yield from the theoretical value (%) - _xylitol percentage of xylitol yield from the theoretical value (%)     

Xylitol Production by a Culture ofCandida guilliermondii2581

The yeast strain Candida guilliermondii2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Xylitol production from D-xylose byCandida guillermondii: Fermentation behaviour

Summary The ability ofCandida guillermondii to produce xylitol from xylose and to ferment individual non xylose hemicellulosic derived sugars was investigated in microaerobic conditions. Xylose was converted into xylitol with a yield of 0,63 g/g and ethanol was produced in negligible amounts. The strain did not convert glucose, mannose and galactose into their corresponding polyols but only into ethanol and cell mass. By contrast, fermentation of arabinose lead to the formation of arabitol. On D-xylose medium,Candida guillermondii exhibited high yield and rate of xylitol production when the initial sugar concentration exceeded 110 g/l. A final xylitol concentration of 221 g/l was obtained from 300 g/l D-xylose with a yield of 82,6% of theoretical and an average specific rate of 0,19 g/g.h.Nomenclature Q_p average volumetric productivity of xylitol (g xylitol/l per hour) - q_p average specific productivity of xylitol (g xylitol/g of cells per hour) - So initial xylose concentration (g/l) - tf incubation time (hours) - Y_P/S xylitol yield (g of xylitol produced/g of xylose utilized) - Y_E/S ethanol yield (g of ethanol produced/g of substrate utilized) - Y_X/S cells yield (g of cells/g of substrate utilized) - specific growth rate coefficient (h^–1) - _max maximum specific growth rate coefficient (h^–1)     

Reduced Oxidative Pentose Phosphate Pathway Flux in Recombinant Xylose-Utilizing Saccharomyces cerevisiae Strains Improves the Ethanol Yield from Xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD⁺. In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g⁻¹) and the lowest xylitol yield (0.05 g g⁻¹) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Xylitol formation by Candida boidinii in oxygen limited chemostat culture

Summary Production of xylitol by Candida boidinii NRRL Y-17213 occurs under conditions of an oxygen limitation. The extent to which substrate is converted to xylitol and its coproducts (ethanol, other polyols, acetic acid), and the relative flow rates of substrate to energetic and biosynthetic pathways is controlled by the degree of oxygen limitation.With decrease in oxygen concentration in the inlet gas, for a constant dilution rate of 0.05 1/h. the specific oxygen uptake rate decreased from 1.30 to 0.36 mmol/gh Xylitol was not produced at specific oxygen uptake rates above 0.91 mmol/gh. Upon shift to lower oxygen rates, specific xylitol production rate increased more rapidly than specific ethanol production rate:Nomenclature D dilution rate (1/h) - DOT dissolved oxygen tension (%) - m_o2 maintenance coefficient (mmol O₂/g cell mass h) - q_o2 specific oxygen uptake rate (mmol O₂/g cell mass h) - q_s specific xylose uptake rate (g xylose/g cell mass h) or (mmol xylose/g cell mass h) - q_x specific xylitol production rate (g xylitol/ g cell mass h) or (mmol xylitol/ g cell mass h) - q_e specific ethanol production rate (g ethanol/ g cell mass h) or (mmol ethanol/ g cell mass h) - q_CO2 specific carbon dioxide production rate (mmol CO₂/g cell mass h) - S xylose concentration (g/1) - Y_cm/s cell mass yield coefficient, (g cell mass/mmol xylose) or (g cell mass/ g xylose consumed) - Y_cm/O2 cell mass yield coefficient, (g cell mass/mmol O₂) - Y_X/S xylitol yield coefficient (g xylitol/g xylose consumed) - Y_x/O2 xylitol yield coefficient (g xylitol/mmol O₂) - Y_e/s ethanol yield coefficient (g ethanol/g xylose consumed) - OUR oxygen uptake rate (mmol O₂/1h) - specific growth rate (1/h)     

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

Summary The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.Nomenclature max maximum specific growth rate (per hour) - qSmax maximum specific rate of xylose consumption (g xylose per g dry biomass per hour) - qpmax maximum specific productivity of xylitol (g xylitol per g dry biomass per hour) - Qp average volumetric productivity of xylitol (g xylitol per liter per hour) - Y_P/S xylitol yield (g xylitol per g substrate utilized) - Y_P'/S glycerol yield (g glycerol per g substrate utilized) - Y_X/S biomass yield (g dry biomass per g substrate utilized)     

可同化阿拉伯糖的木糖还原酿酒酵母菌株构建

[目的] 以秸秆等木质纤维素类生物质为原料生产液体生物燃料乙醇,目前生产成本高,大规模工业化生产尚有较大难度。构建能同化阿拉伯糖进行木糖还原生产木糖醇的重组酿酒酵母菌株,以实现原料中全糖利用、生产高附加值产品,实现产品多元化。[方法] 首先,利用CRISPR/Cas9基因编辑技术依次向出发菌株中导入阿拉伯糖代谢途径和木糖还原酶基因,使菌株获得代谢阿拉伯糖和将木糖转化为木糖醇的能力;其次,通过适应性驯化的进化工程手段,提高重组菌株对阿拉伯糖的利用效率;最后,通过混合糖发酵验证重组菌株利用阿拉伯糖和还原木糖产木糖醇的能力。[结果] 通过导入植物乳杆菌的阿拉伯糖代谢途径,酿酒酵母菌株获得了较好的利用阿拉伯糖生长繁殖的能力;进一步导入假丝酵母的木糖还原酶基因后,重组菌株在葡萄糖作为辅助碳源条件下可高效还原木糖产木糖醇,但阿拉伯糖的利用能力下降。利用以阿拉伯糖为唯一碳源的培养基进行反复批次驯化,阿拉伯糖的利用能力得以恢复和提升,得到表型较好的重组菌株KAX3-2。该菌株在木糖（50 g/L）和阿拉伯糖（20 g/L）混合糖发酵条件下发酵72 h时,对阿拉伯糖和木糖利用率分别达到42.1%和65.9%,木糖醇的收率为64%。[结论] 本研究成功构建了一株能有效利用阿拉伯糖并能将木糖转化为木糖醇的重组酿酒酵母菌株KAX3-2,为后续构建、获得阿拉伯糖代谢能力更强、木糖醇积累效率更高菌株的工作奠定了基础。     

SHIFT FROM CHLOROPHYTES TO CYANOBACTERIA IN BENTHIC MACROALGAE ALONG A GRADIENT OF NITRATE DEPLETION1

A survey of the spatial distribution of benthic macroalgae in a fluvial lake of the St. Lawrence River (Lake Saint‐Pierre, Quebec, Canada) revealed a shift in composition from chlorophytes to cyanobacteria along the flow path of nutrient‐rich waters originating from tributaries draining farmlands. The link between this shift and changes in water quality characteristics was investigated by sampling at 10 sites along a 15 km transect. Conductivity, current, light extinction, total phosphorus (TP; >25 μg P · L^?1), and ammonium (8–21 μg N · L^?1) remained fairly constant along the transect in contrast to nitrate concentrations, which fell sharply. Filamentous and colonial chlorophytes [Cladophora sp. and Hydrodictyon reticulatum (L.) Bory] dominated in the first 5 km where nitrate concentrations were >240 μg N · L^?1. A mixed assemblage of chlorophytes and cyanobacteria characterized a 1 km transition zone where nitrate decreased to 40–80 μg N · L^?1. In the last section of the transect, nitrate concentrations dropped below 10 μg N · L^?1, and cyanobacteria (benthic filamentous mats of Lyngbya wollei Farl. ex Gomont and epiphytic colonies of Gloeotrichia) dominated the benthic community. The predominance of nitrogen‐fixing, potentially toxic cyanobacteria likely resulted from excessive nutrient loads and may affect nutrient and trophic dynamics in the river.     

Increasing reducing power output (NADH) of glucose catabolism for reduction of xylose to xylitol by genetically engineered Escherichia coli AI05

Anaerobic homofermentative production of reduced products requires additional reducing power (NADH and/or NADPH) output from glucose catabolism. Previously, with an anaerobically expressed pyruvate dehydrogenase operon (aceEF-lpd), we doubled the reducing power output to four NADH per glucose (or 1.2 xylose) catabolized anaerobically, which satisfied the NADH requirement to establish a non-transgenic homoethanol pathway (1 glucose or 1.2 xylose ? 2 acetyl-CoA + 4 NADH ? 2 ethanol) in the engineered strain, Escherichia coli SZ420 (?frdBC ?ldhA ?ackA ?focA-pflB ?pdhR::pflBp6-pflBrbs-aceEF-lpd). In this study, E. coli SZ420 was further engineered for reduction of xylose to xylitol by (1) deleting the alcohol dehydrogenase gene (adhE) to divert NADH from the ethanol pathway; (2) deleting the glucose-specific PTS permease gene (ptsG) to eliminate catabolite repression and allow simultaneous uptake of glucose and xylose; (3) cloning the aldose reductase gene (xylI) of Candida boidinii to reduce xylose to xylitol. The resulting strain, E. coli AI05 (pAGI02), could in theory simultaneously uptake glucose and xylose, and utilize glucose as a source of reducing power for the reduction of xylose to xylitol, with an expected yield of four xylitol for each glucose consumed (Y_RPG = 4) under anaerobic conditions. In resting cell fermentation tests using glucose and xylose mixtures, E. coli AI05 (pAGI02) achieved an actual Y_RPG value of ~3.6, with xylitol as the major fermentation product and acetate as the by-product.     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

Background

Ethanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.

Results

The specific aerobic arabinose growth rate was identical, 0.03 h^-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h^-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)^-1 h^-1 than the xylose isomerase strain, which only reached 0.03 h^-1 and 0.02 g (g cells)^-1h^-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g^-1 compared with 0.18 g g^-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g^-1 compared with 0.32 g g^-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l^-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l^-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)^-1 h^-1 compared with 0.01 g (g cells)^-1 h^-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.

Conclusion

The combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.