Microbial production host selection for converting second-generation feedstocks into bioproducts

Background  

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.     

Cellulosic ethanol production using the naturally occurring xylose-fermenting yeast, Pichia stipitis

Rising crude oil prices and environmental concerns have renewed interest in renewable energy. Cellulosic ethanol promises to deliver a renewable fuel from non-food feedstocks. One technical challenge producing cellulosic ethanol economically is a robust organism to utilize the different sugars present in cellulosic biomass. Unlike starch where glucose is the only sugar present, cellulosic biomass has other sugars such as xylose and arabinose, usually called C5 sugars. This review examines the most promising naturally occurring C5 fermenting organism, Pichia stipitis. In this work, the properties that make P. stipitis unique from other organisms, its physiology and fermentation results on lignocellulosic substrates have been reviewed. P. stipitis can produce 41 g ethanol/l with a potential to cleanup some of the most concentrated toxins. These results coupled with the less stringent nutritional requirements, great resistance to contamination and its thick cell walls makes P. stipitis a viable organism for scale-up. However, P. stipitis has a slower sugar consumption rate compared to Saccharomyces cerevisiae and requires microaerophilic condition for ethanol production. Finally, future studies to enhance fermentation capabilities of this yeast have been discussed.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.

     

Improved ethanol production by a xylose-fermenting recombinant yeast strain constructed through a modified genome shuffling method

ABSTRACT: BACKGROUND: Xylose is the second most abundant carbohydrate in the lignocellulosic biomass hydrolysate. The fermentation of xylose is essential for the bioconversion of lignocelluloses to fuels and chemicals. However the wild-type strains of Saccharomyces cerevisiae are unable to utilize xylose. Many efforts have been made to construct recombinant yeast strains to enhance xylose fermentation over the past few decades. Xylose fermentation remains challenging due to the complexity of lignocellulosic biomass hydrolysate. In this study, a modified genome shuffling method was developed to improve xylose fermentation by S. cerevisiae. Recombinant yeast strains were constructed by recursive DNA shuffling with the recombination of entire genome of P. stipitis with that of S. cerevisiae. RESULTS: After two rounds of genome shuffling and screening, one potential recombinant yeast strain ScF2 was obtained. It was able to utilize high concentration of xylose (100 g/L to 250 g/L xylose) and produced ethanol. The recombinant yeast ScF2 produced ethanol more rapidly than the naturally occurring xylose-fermenting yeast, P. stipitis, with improved ethanol titre and much more enhanced xylose tolerance. CONCLUSION: The modified genome shuffling method developed in this study was more effective and easier to operate than the traditional protoplast fusion based method. Recombinant yeast strain ScF2 obtained in this was a promising candidate for industrial cellulosic ethanol production. In order to further enhance its xylose fermentation performance, ScF2 needs to be additionally improved by metabolic engineering and directed evolution.     

Mixed Feedstock Approach to Lignocellulosic Ethanol Production—Prospects and Limitations

Lignocellulosic ethanol is a promising alternative to fossil-derived fuels because lignocellulosic biomass is abundant, cheap and its use is environmentally friendly. However, the high costs of feedstock supply and the expensive processing requirements of lignocellulosic biomass hinder the development of the lignocellulosic biorefinery. Lignocellulosic ethanol production so far, has been based mainly on single feedstocks while the use of mixed feedstocks has been poorly explored. Previous studies from alternative applications of mixed lignocellulosic biomass (MLB) have shown that their use can bring about significant cost savings when compared to single feedstocks. Although laboratory-scale evaluations have demonstrated that mixed feedstocks give comparable or even higher ethanol yields compared to single feedstocks, more empirical studies are needed to establish the possibility of achieving significant cost savings in terms of pre-biorefinery logistics. In this review, some potential benefits of the use of MLB for ethanol production are highlighted. Some anticipated limitations of this approach have been identified and ways to surmount them have been suggested. The outlook for ethanol production from MLB is promising provided that revolutionary measures are taken to ensure the sustainability of the industry.     

Random UV-C mutagenesis of Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 to improve anaerobic growth on lignocellulosic sugars

Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124 was mutagenized using UV-C irradiation to produce yeast strains for anaerobic conversion of lignocellulosic sugars to ethanol. UV-C irradiation potentially produces large numbers of random mutations broadly and uniformly over the whole genome to generate unique strains. Wild-type cultures of S. stipitis NRRL Y-7124 were subjected to UV-C (234 nm) irradiation targeted at approximately 40% cell survival. When surviving cells were selected in sufficient numbers via automated plating strategies and cultured anaerobically on xylose medium for 5 months at 28°C, five novel mutagenized S. stipitis strains were obtained. Variable number tandem repeat analysis revealed that mutations had occurred in the genome, which may have produced genes that allowed the anaerobic utilization of xylose. The mutagenized strains were capable of growing anaerobically on xylose/glucose substrate with higher ethanol production during 250- to 500-h growth than a Saccharomyces cerevisiae yeast strain that is the standard for industrial fuel ethanol production. The S. stipitis strains resulting from this intense multigene mutagenesis strategy have potential application in industrial fuel ethanol production from lignocellulosic hydrolysates.     

Comparative study on a series of recombinant flocculent Saccharomyces cerevisiae strains with different expression levels of xylose reductase and xylulokinase

Ethanol production from xylose is important for the utilization of lignocellulosic biomass as raw materials. Recently, we reported the development of an industrial xylose-fermenting Saccharomyces cerevisiae strain, MA-R4, which was engineered by chromosomal integration to express the genes encoding xylose reductase and xylitol dehydrogenase from Pichia stipitis along with S. cerevisiae xylulokinase gene constitutively using the alcohol-fermenting flocculent yeast strain, IR-2. IR-2 has the highest xylulose-fermenting ability of the industrial diploid strains, making it a useful host strain for genetically engineering xylose-utilizing S. cerevisiae. To optimize the activities of xylose metabolizing enzymes in the metabolic engineering of IR-2 for further improvement of ethanol production from xylose, we constructed a set of recombinant isogenic strains harboring different combinations of genetic modifications present in MA-R4, and investigated the effect of constitutive expression of xylulokinase and of different levels of xylulokinase and xylose reductase activity on xylose fermentation. This strain comparison showed that constitutive expression of xylulokinase increased ethanol production from xylose at the expense of xylitol excretion, and that high activity of xylose reductase resulted in an increased rate of xylose consumption and an increased glycerol yield. Moreover, strain MA-R6, which has moderate xylulokinase activity, grew slightly better but accumulated more xylitol than strain MA-R4. These results suggest that fine-tuning of introduced enzyme activity in S. cerevisiae is important for improving xylose fermentation to ethanol.     

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> as a potent biocatalyst for the bioconversion of pentose sugars to value-added products

Corynebacterium glutamicum, the industrial microbe traditionally used for the production of amino acids, proved its value for the fermentative production of diverse products through genetic/metabolic engineering. A successful demonstration of the heterologous expression of arabinose and xylose utilization genes made them interesting biocatalysts for pentose fermentation, which are the main components in lignocellulosic hydrolysates. Its ability to withstand substantial amount of general growth inhibitors like furfurals, hydroxyl methyl furfurals and organic acids generated from the acid/alkali hydrolysis of lignocellulosics in growth arrested conditions and its ability to produce amino acids like glutamate and lysine in acid hydrolysates of rice straw and wheat bran, indicate the future prospective of this bacterium as a potent biocatalyst in fermentation biotechnology. However, the efforts so far on these lines have not yet been reviewed, and hence an attempt is made to look into the efficacy and prospects of C. glutamicum to utilize the normally non-fermentable pentose sugars from lignocellulosic biomass for the production of commodity chemicals.     

Comparative study of pyrolysis of algal biomass from natural lake blooms with lignocellulosic biomass

Pyrolysis experiments were performed with algal and lignocellulosic feedstocks under similar reactor conditions for comparison of product (bio-oil, gas and bio-char) yields and composition. In spite of major differences in component bio-polymers, feedstock properties relevant to thermo-chemical conversions, such as overall C, H and O-content, C/O and H/C molar ratio as well as calorific values, were found to be similar for algae and lignocellulosic material. Bio-oil yields from algae and some lignocellulosic materials were similar; however, algal bio-oils were compositionally different and contained several N-compounds (most likely from protein degradation). Algal bio-char also had a significantly higher N-content. Overall, our results suggest that it is feasible to convert algal cultures deficient in lipids, such as nuisance algae obtained from natural blooms, into liquid fuels by thermochemical methods. As such, pyrolysis technologies being developed for lignocellulosic biomass may be directly applicable to algal feedstocks as well.     

Improvements in ethanol production from xylose by mating recombinant xylose-fermenting Saccharomyces cerevisiae strains

To improve the ability of recombinant Saccharomyces cerevisiae strains to utilize the hemicellulose components of lignocellulosic feedstocks, the efficiency of xylose conversion to ethanol needs to be increased. In the present study, xylose-fermenting, haploid, yeast cells of the opposite mating type were hybridized to produce a diploid strain harboring two sets of xylose-assimilating genes encoding xylose reductase, xylitol dehydrogenase, and xylulokinase. The hybrid strain MN8140XX showed a 1.3- and 1.9-fold improvement in ethanol production compared to its parent strains MT8-1X405 and NBRC1440X, respectively. The rate of xylose consumption and ethanol production was also improved by the hybridization. This study revealed that the resulting improvements in fermentation ability arose due to chromosome doubling as well as the increase in the copy number of xylose assimilation genes. Moreover, compared to the parent strain, the MN8140XX strain exhibited higher ethanol production under elevated temperatures (38 °C) and acidic conditions (pH 3.8). Thus, the simple hybridization technique facilitated an increase in the xylose fermentation activity.     

Fermentative hydrogen production from pretreated biomass: A comparative study

The aim of this work was to evaluate the potential of employing biomass resources from different origin as feedstocks for fermentative hydrogen production. Mild-acid pretreated and hydrolysed barley straw (BS) and corn stalk (CS), hydrolysed barley grains (BG) and corn grains (CG), and sugar beet extract (SB) were comparatively evaluated for fermentative hydrogen production. Pretreatments and/or enzymatic hydrolysis led to 27, 37, 56, 74 and 45 g soluble sugars/100 g dry BS, CS, BG, CG and SB, respectively. A rapid test was applied to evaluate the fermentability of the hydrolysates and SB extract. The thermophilic bacterium Caldicellulosiruptor saccharolyticus showed high hydrogen production on hydrolysates of mild-acid pretreated BS, hydrolysates of BG and CG, and SB extract. Mild-acid pretreated CS showed limited fermentability, which was partially due to inhibitory products released in the hydrolysates, implying the need for the employment of a milder pretreatment method. The difference in the fermentability of BS and CS is in strong contrast to the similarity of the composition of these two feedstocks. The importance of performing fermentability tests to determine the suitability of a feedstock for hydrogen production was confirmed.     

Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.     

Strain improvement of thermotolerant Saccharomyces cerevisiae VS strain for better utilization of lignocellulosic substrates

AIM: Pentose-utilizing yeast development by protoplast fusion and sequential mutations and ethanol fermentation using lignocellulosic substrate. METHODS AND RESULTS: Protoplasts of thermotolerant Saccharomyces cerevisiae and mesophilic, xylose-utilizing Candida shehatae were fused by electrofusion. The fusants were selected based on their growth at 42 degrees C and ability to utilize xylose. The selected best fusant was mutated sequentially and 3 mutant fusants obtained were tested for their stability. The mutant fusant CP11 was found to be stable and used for lignocellulosic fermentation. The Prosopis juliflora wood material was hydrolysed with 1% sulphuric acid initially for 18 h at room temperature and then for 20 min at 121 degrees C. The acid hydrolysate was separated and used for detoxification by ethyl acetate and overliming. The hard cellulosic fraction was hydrolysed with Aspergillus niger crude cellulase enzyme for 18 h at 50 degrees C. The substrate (15% w/v) yielded 84 g l(-1) sugars, representing 80% (w/w) hydrolysis of carbohydrate content present in the lignocellulosic material. The acid and enzyme hydrolysates were then equally mixed and used for fermentation with the developed fusant yeast (CP11). The fusant yeast gave an ethanol yield of 0.459 +/- 0.012 g g(-1), productivity of 0.67 +/- 0.015 g l(-1) h(-1) and fermentation efficiency of 90%. CONCLUSIONS: Protoplast fusion followed by sequential mutations method gave a stable and good performing fusant with maximum utilization of reducing sugars in the media. SIGNIFICANCE AND IMPACT OF THE STUDY: This new method could be applied to develop fusants for better biotechnological applications.     

C_4 Plants as Biofuel Feedstocks: Optimising Biomass Production and Feedstock Quality from a Lignocellulosic Perspective

The main feedstocks for bioethanol are sugarcane (Saccharum officinarum) and maize (Zea mays),both of which are C4 grasses,highly efficient at converting solar energy into chemical energy,and both are food crops.As the systems for lignocellulosic bioethanol production become more efficient and cost effective,plant biomass from any source may be used as a feedstock for bioethanol production.Thus,a move away from using food plants to make fuel is possible,and sources of biomass such as wood from forestry and ...     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Economic impact of combined torrefaction and pelletization processes on forestry biomass supply

The cost of supplying wood biomass from forestry operations in remote areas has been an obstacle to expansion of forest‐based bioenergy in much of the western United States. Economies of scale in the production of liquid fuels from lignocellulosic biomass feedstocks favor large centralized biorefineries. Increasing transportation efficiency through torrefaction and pelletization at distributed satellite facilities may serve as a means to expand the utilization of forestry residuals in biofuel production. To investigate this potential, a mixed‐integer linear program was developed to optimize the feedstock supply chain design with and without distributed pretreatment. The model uses techno‐economic assessment of scale‐dependent biomass pretreatment processes from existing literature and multimodal biomass transportation cost evaluations derived from a spatially explicit network analysis as input. In addition, the sensitivity of the optimal system configuration was determined for variations of key input parameters including the production scale of pretreatment facilities, road and rail transportation costs, and feedstock procurement costs. Torrefaction and densification were found to reduce transportation costs by $0.84 per GJ and overall delivered costs by $0.24 per GJ, representing 14.5% and 5.2% cost reductions compared to feedstock collection without pretreatment. Significant uncertainties remain in terms of the costs associated with deploying torrefaction equipment at the scales modeled, but the level of potential cost savings suggests further analysis and development of these alternatives.     

Feasibility of filamentous fungi for biofuel production using hydrolysate from dilute sulfuric acid pretreatment of wheat straw

ABSTRACT: BACKGROUND: Lipids produced from filamentous fungi show great promise for biofuel production, but a major limiting factor is the high production cost attributed to feedstock. Lignocellulosic biomass is a suitable feedstock for biofuel production due to its abundance and low value. However, very limited study has been performed on lipid production by culturing oleaginous fungi with lignocellulosic materials. Thus, identification of filamentous fungal strains capable of utilizing lignocellulosic hydrolysates for lipid accumulation is critical to improve the process and reduce the production cost. RESULTS: The growth performances of eleven filamentous fungi were investigated when cultured on glucose and xylose. Their dry cell weights, lipid contents and fatty acid profiles were determined. Six fungal strains with high lipid contents were selected to culture with the hydrolysate from dilute sulfuric acid pretreatment of wheat straw. The results showed that all the selected fungal strains were able to grow on both detoxified liquid hydrolysate (DLH) and non-detoxified liquid hydrolysate (NDLH). The highest lipid content of 39.4% was obtained by Mortierella isabellina on NDLH. In addition, NDLH with some precipitate could help M. isabellina form pellets with an average diameter of 0.11 mm. CONCLUSION: This study demonstrated the possibility of fungal lipid production from lignocellulosic biomass. M. isabellina was the best lipid producer grown on lignocellulosic hydrolysates among the tested filamentous fungi, because it could not only accumulate oils with a high content by directly utilizing NDLH to simplify the fermentation process, but also form proper pellets to benefit the downstream harvesting. Considering the yield and cost, fungal lipids from lignocellulosic biomass are promising alternative sources for biodiesel production.     

发酵木糖生产乙醇的野生菌和转基因菌的研究进展

木糖发酵是利用植物纤维原料生物转化制取乙醇工业化生产的技术基础和关键。野生酵母中有些种属菌株可以高效利用木糖产生乙醇,其中毕赤酵母（Pichiastipim）的乙醇转化速度最高达到0．99g／L／h,转化率几乎接近理论值0．5g／g,发酵液中最高乙醇浓度可迭到（61±9）g／L。但工业生产中要达到毕赤酵母所要求的微氧最佳发酵条件比较困难。近十几年来许多研究尝试根据代谢工程原理,利用基因工程技术对酿酒酵母进行改造。从而提高其发酵木糖产生乙醇的能力。这些研究大多是将毕赤酵母的一些木糖发酵关键酶基因（XYL1、XYL2、XYL3以及ADHl、ADH2等）转入酿酒酵母细胞内,并试图得到正常转录和表达。但到目前为止,大部分的重组菌株的乙醇发酵性能还没有达到工业生产的要求。     

Trends in biotechnological production of fuel ethanol from different feedstocks

Present work deals with the biotechnological production of fuel ethanol from different raw materials. The different technologies for producing fuel ethanol from sucrose-containing feedstocks (mainly sugar cane), starchy materials and lignocellulosic biomass are described along with the major research trends for improving them. The complexity of the biomass processing is recognized through the analysis of the different stages involved in the conversion of lignocellulosic complex into fermentable sugars. The features of fermentation processes for the three groups of studied feedstocks are discussed. Comparative indexes for the three major types of feedstocks for fuel ethanol production are presented. Finally, some concluding considerations on current research and future tendencies in the production of fuel ethanol regarding the pretreatment and biological conversion of the feedstocks are presented.