From wheat straw to bioethanol: integrative analysis of a separate hydrolysis and co-fermentation process with implemented enzyme production

BACKGROUND: Lignocellulosic ethanol has a high potential as renewable energy source. In recent years, much research effort has been spent to optimize parameters involved in the production process. Despite that, there is still a lack of comprehensive studies on process integration. Single parameters and process configurations are, however, heavily interrelated and can affect the overall process efficiency in a multitude of ways. Here, we present an integrative approach for bioethanol production from wheat straw at a representative laboratory scale using a separate hydrolysis and co-fermentation (SHCF) process. The process does not rely on commercial (hemi-) cellulases but includes enzyme production through Hypocrea jecorina (formerly Trichoderma reesei) on the pre-treated feedstock as key unit operation. Hydrolysis reactions are run with high solid loadings of 15% dry mass pre-treated wheat straw (DM WS), and hydrolyzates are utilized without detoxification for mixed glucose-xylose fermentation with the genetically and evolutionary engineered Saccharomyces cerevisiae strain IBB10B05. RESULTS: Process configurations of unit operations in the benchtop SHCF were varied and evaluated with respect to the overall process ethanol yield (Y(Ethanol-Process)). The highest Y(Ethanol-Process) of 71.2 g ethanol per kg raw material was reached when fungal fermentations were run as batch, and the hydrolysis reaction was done with an enzyme loading of 30 filter paper units (FPU)/g(DM WS). 1.7 ± 0.1 FPU/mL were produced, glucose and xylose were released with a conversion efficiency of 67% and 95%, respectively, and strain IBB10B05 showed an ethanol yield of 0.4 g/g(Glc + Xyl) in 15% hydrolyzate fermentations. Based on the detailed process analysis, it was further possible to identify the enzyme yield, the glucose conversion efficiency, and the mass losses between the unit operations as key process parameters, exhibiting a major influence on Y(Ethanol-Process). CONCLUSIONS: Y(Ethanol-Process) is a measure for the efficiency of the lignocellulose-to-bioethanol process. Based on mass balance analysis, the correlations between single process parameters and Y(Ethanol-Process) were elucidated. The optimized laboratory scale SHCF process showed efficiencies similar to pilot scale plants. The herein presented process analysis can serve as effective and simple tool to identify key process parameters, bottlenecks, and future optimization targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0232-0) contains supplementary material, which is available to authorized users

Stepwise metabolic adaption from pure metabolization to balanced anaerobic growth on xylose explored for recombinant Saccharomyces cerevisiae

BACKGROUND: To effectively convert lignocellulosic feedstocks to bio-ethanol anaerobic growth on xylose constitutes an essential trait that Saccharomyces cerevisiae strains normally do not adopt through the selective integration of a xylose assimilation route as the rate of ATP-formation is below energy requirements for cell maintenance (m(ATP)). To enable cell growth extensive evolutionary and/or elaborate rational engineering is required. However the number of available strains meeting demands for process integration are limited. In this work evolutionary engineering in just two stages coupled to strain selection under strict anaerobic conditions was carried out with BP10001 as progenitor. BP10001 is an efficient (Y(ethanol) = 0.35 g/g) but slow (q(ethanol) = 0.05 ± 0.01 g/g(BM)/h) xylose-metabolizing recombinant strain of Saccharomyces cerevisiae that expresses an optimized yeast-type xylose assimilation pathway. RESULTS: BP10001 was adapted in 5 generations to anaerobic growth on xylose by prolonged incubation for 91 days in sealed flasks. Resultant strain IBB10A02 displayed a specific growth rate μ of 0.025 ± 0.002 h(-1) but produced large amounts of glycerol and xylitol. In addition growth was strongly impaired at pH below 6.0 and in the presence of weak acids. Using sequential batch selection and IBB10A02 as basis, IBB10B05 was evolved (56 generations). IBB10B05 was capable of fast (μ = 0.056 ± 0.003 h(-1); q(ethanol) = 0.28 ± 0.04 g/g(BM)/h), efficient (Y(ethanol) = 0.35 ± 0.02 g/g), robust and balanced fermentation of xylose. Importantly, IBB10A02 and IBB10B05 displayed a stable phenotype. Unlike BP10001 both strains displayed an unprecedented biphasic formation of glycerol and xylitol along the fermentation time. Transition from a glycerol- to a xylitol-dominated growth phase, probably controlled by CO(2)/HCO(3)(-), was accompanied by a 2.3-fold increase of m(ATP) while Y(ATP) (= 87 ± 7 mmol(ATP)/g(BM)) remained unaffected. As long as glycerol constituted the main by-product energetics of anaerobic growth on xylose and glucose were almost identical. CONCLUSIONS: In just 61 generation IBB10B05, displaying ~530% improved strain fitness, was evolved from BP10001. Its excellent xylose fermentation properties under industrial relevant conditions were proven and rendered it competitive. Based on detailed analysis of growth energetics we showed that m(ATP) was predominantly determined by the type of polyol formed rather than, as previously assumed, substrate-specific

Fermentation of mixed glucose-xylose substrates by engineered strains of Saccharomyces cerevisiae: role of the coenzyme specificity of xylose reductase, and effect of glucose on xylose utilization

<p>Abstract</p> <p>Background</p> <p>In spite of the substantial metabolic engineering effort previously devoted to the development of <it>Saccharomyces cerevisiae </it>strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from <it>Candida tenuis </it>in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate <it>q</it>_xylose(g xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit <it>q</it>_xyloseof BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose.</p> <p>Results</p> <p>BP10001 and BP000, expressing <it>C. tenuis </it>xylose reductase in NADPH-preferring wild-type form, were used. Glucose and xylose (each at 10 g/L) were converted sequentially, the corresponding <it>q</it>_substratevalues being similar for each strain (glucose: 3.0; xylose: 0.05). The distribution of fermentation products from glucose was identical for both strains whereas when using xylose, BP10001 showed enhanced ethanol yield (BP10001 0.30 g/g; BP000 0.23 g/g) and decreased yields of xylitol (BP10001 0.26 g/g; BP000 0.36 g/g) and glycerol (BP10001 0.023 g/g; BP000 0.072 g/g) as compared to BP000. Increase in xylose concentration from 10 to 50 g/L resulted in acceleration of substrate uptake by BP10001 (0.05 - 0.14 g/g CDW/h) and reduction of the xylitol yield (0.28 g/g - 0.15 g/g). In mixed substrate batches, xylose was taken up at low glucose concentrations (< 4 g/L) and up to fivefold enhanced xylose uptake rate was found towards glucose depletion. A fed-batch process designed to maintain a "stimulating" level of glucose throughout the course of xylose conversion provided a <it>q</it>_xylosethat had an initial value of 0.30 ± 0.04 g/g CDW/h and decreased gradually with time. It gave product yields of 0.38 g ethanol/g total sugar and 0.19 g xylitol/g xylose. The effect of glucose on xylose utilization appears to result from the enhanced flux of carbon through glycolysis and the pentose phosphate pathway under low-glucose reaction conditions.</p> <p>Conclusions</p> <p>Relative improvements in the distribution of fermentation products from xylose that can be directly related to a change in the coenzyme preference of xylose reductase from NADPH in BP000 to NADH in BP10001 increase in response to an increase in the initial concentration of the pentose substrate from 10 to 50 g/L. An inverse relationship between xylose uptake rate and xylitol yield for BP10001 implies that xylitol by-product formation is controlled not only by coenzyme regeneration during two-step oxidoreductive conversion of xylose into xylulose. Although xylose is not detectably utilized at glucose concentrations greater than 4 g/L, the presence of a low residual glucose concentration (< 2 g/L) promotes the uptake of xylose and its conversion into ethanol with only moderate xylitol by-product formation. A fed-batch reaction that maintains glucose in the useful concentration range and provides a constant <it>q</it>_glucosemay be useful for optimizing <it>q</it>_xylosein processes designed for co-fermentation of glucose and xylose.</p

Process intensification through microbial strain evolution: mixed glucose-xylose fermentation in wheat straw hydrolyzates by three generations of recombinant Saccharomyces cerevisiae

BACKGROUND: Lignocellulose hydrolyzates present difficult substrates for ethanol production by the most commonly applied microorganism in the fermentation industries, Saccharomyces cerevisiae. High resistance towards inhibitors released during pretreatment and hydrolysis of the feedstock as well as efficient utilization of hexose and pentose sugars constitute major challenges in the development of S. cerevisiae strains for biomass-to-ethanol processes. Metabolic engineering and laboratory evolution are applied, alone and in combination, to adduce desired strain properties. However, physiological requirements for robust performance of S. cerevisiae in the conversion of lignocellulose hydrolyzates are not well understood. The herein presented S. cerevisiae strains IBB10A02 and IBB10B05 are descendants of strain BP10001, which was previously derived from the widely used strain CEN.PK 113-5D through introduction of a largely redox-neutral oxidoreductive xylose assimilation pathway. The IBB strains were obtained by a two-step laboratory evolution that selected for fast xylose fermentation in combination with anaerobic growth before (IBB10A02) and after adaption in repeated xylose fermentations (IBB10B05). Enzymatic hydrolyzates were prepared from up to 15% dry mass pretreated (steam explosion) wheat straw and contained glucose and xylose in a mass ratio of approximately 2. RESULTS: With all strains, yield coefficients based on total sugar consumed were high for ethanol (0.39 to 0.40 g/g) and notably low for fermentation by-products (glycerol: ≤0.10 g/g; xylitol: ≤0.08 g/g; acetate: 0.04 g/g). In contrast to the specific glucose utilization rate that was similar for all strains (q(Glucose) ≈ 2.9 g/g(cell dry weight (CDW))/h), the xylose consumption rate was enhanced by a factor of 11.5 (IBB10A02; q(Xylose) = 0.23 g/g(CDW)/h) and 17.5 (IBB10B05; q(Xylose) = 0.35 g/g(CDW)/h) as compared to the q(Xylose) of the non-evolved strain BP10001. In xylose-supplemented (50 g/L) hydrolyzates prepared from 5% dry mass, strain IBB10B05 displayed a q(Xylose) of 0.71 g/g(CDW)/h and depleted xylose in 2 days with an ethanol yield of 0.30 g/g. Under the conditions used, IBB10B05 was also capable of slow anaerobic growth. CONCLUSIONS: Laboratory evolution of strain BP10001 resulted in effectively enhanced q(Xylose) at almost complete retention of the fermentation capabilities previously acquired by metabolic engineering. Strain IBB10B05 is a sturdy candidate for intensification of lignocellulose-to-bioethanol processes