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.     

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.     

Biodiesel fuel production from lipids of filamentous fungi

The main stages in the production of biodiesel fuel from lipids of filamentous fungi belonging to the order Mucorales are described. Fungi of the family Cunninghamellaceae have been screened; the lipogenic activity of the examined strains has been assessed; and a producer generating up to 50% of lipids, represented by triacylglycerols, has been found. The substitution effect of a source of carbon and nitrogen with less expensive components (in particular, various industrial wastes) has been studied, as well as their influence on the quantity and major characteristics of the final product. An ecologically friendly method for extracting lipids from fungal mycelia, utilizing supercritical technologies, has been used. A correlation between the lipid content in the spore inoculum and the maximal lipid content in biomass has been discovered; this correlation is proposed for optimizing the biotechnology and increasing the yield of final products.     

丝状真菌纤维素酶合成诱导及转录调控

利用廉价可再生木质纤维素资源水解产生的可发酵糖生产生物能源和生物基化学品是近年来国内外研究的热点。纤维素酶酶解是木质纤维素原料生物降解的重要手段,但目前纤维素酶生产成本过高,限制了纤维素生物转化和生物炼制的工业化应用。对丝状真菌纤维素酶基因表达和调控进行研究,有利于进一步选育纤维素酶高产菌株,降低纤维素酶生产成本。随着高通量测序及丝状真菌遗传操作等技术的进步,对丝状真菌纤维素酶诱导和基因表达调控机理有了更深入的认识。本文综述了近年来丝状真菌纤维素酶诱导和纤维素酶基因表达调控的最新进展,重点论述糖转运蛋白、转录因子和染色质重塑对纤维素酶表达调控的影响,并对利用人工锌指蛋白进行丝状真菌纤维素酶诱导调控研究进行了展望。     

Comparative lipid profiling of two endophytic fungal isolates--Colletotrichum sp. and Alternaria sp. having potential utilities as biodiesel feedstock

Lipid accumulation abilities of two endophytic fungal isolates - Colletotrichum sp. and Alternaria sp. grown under optimum and nutrient-stress conditions were investigated and compared. Significant variations in lipid contents, ranging from 30% to 58% of their dry biomass were found in liquid culture using various carbon sources. Since, >50% of the total lipid was estimated to be neutral lipid for both the fungal species, predicted biodiesel properties were theoretically calculated based upon the determined fatty acid profiles; and the values were found to be comparable to those of commonly used plant oils for biodiesel production. The two endophytes grew successfully on the combined rice straw and wheat bran as substrate that was degraded by their secretory enzymes including cellulase [1.21-2.51 FPU/g dry substrate (gds)] in solid state fermentation and produced substantial amount of lipid (60.32-84.30 mg/gds). Our study highlights the potential utilities of these two novel endophytic fungi as biodiesel feedstock.     

Exploring the Natural Fungal Biodiversity of Tropical and Temperate Forests toward Improvement of Biomass Conversion

In this study, natural fungal diversity in wood-decaying species was explored for biomass deconstruction. In 2007 and 2008, fungal isolates were collected in temperate forests mainly from metropolitan France and in tropical forests mainly from French Guiana. We recovered and identified 74 monomorph cultures using morphological and molecular identification tools. Following production of fungal secretomes under inductive conditions, we evaluated the capacity of these fungal strains to potentiate a commercial Trichoderma reesei cellulase cocktail for the release of soluble sugars from biomass. The secretome of 19 isolates led to an improvement in biomass conversion of at least 23%. Of the isolates, the Trametes gibbosa BRFM 952 (Banque de Ressources Fongiques de Marseille) secretome performed best, with 60% improved conversion, a feature that was not universal to the Trametes and related genera. Enzymatic characterization of the T. gibbosa BRFM 952 secretome revealed an unexpected high activity on crystalline cellulose, higher than that of the T. reesei cellulase cocktail. This report highlights the interest in a systematic high-throughput assessment of collected fungal biodiversity to improve the enzymatic conversion of lignocellulosic biomass. It enabled the unbiased identification of new fungal strains issued from biodiversity with high biotechnological potential.     

加快微生物油脂研究为生物柴油产业提供廉价原料

当前国内外致力于发展生物柴油,因其性能优良,成为化石柴油的替代品。由于以植物油脂生产生物柴油原料成本占总成本的70％-85％,所以亟待开发廉价油脂资源。微生物油脂主要是微生物利用碳水化合物合成的甘油脂,其脂肪酸组成和植物油相近。产油微生物具有资源丰富、油脂含量高、碳源利用谱广等特点,开发潜力大。然而,目前微生物油脂生产成本偏高,研究工作仍以富含多不饱和脂肪酸的高附加值菌油为目标。随着现代分子生物学和生物化工技术的发展,对产油微生物菌种筛选、改良、代谢调控和发酵工程的研究日趋深入,将降低微生物油脂生产成本,为未来生物柴油产业提供廉价原料。     

Endophytic Cephalotheca sulfurea AGH07 reprograms soybean to higher growth

Abstract

Gibberellins (GAs) are well known for plant growth promotion. GAs production by fungi has received little attention, although substantial work has been carried out on other aspects of plant growth-promoting fungi (PGPF). We investigated GAs production and plant growth-promoting capacity of an endophytic fungus isolated from the roots of soil grown soybean plants. The endophytic fungus is reported as GAs producer and as PGPF for the first time in this study. Nine endophytic isolates were collected from the roots of soybean, and culture filtrates (CFs) obtained from their pure cultures were screened on Waito-C, a dwarf rice cultivar, for the presence of GAs. Of these, seven fungal isolates promoted shoot length as compared to control (distilled water), while one inhibited it. Three fungal isolates were selected on the basis of higher shoot elongation as compared to wild type Gibberella fujikuroi, which was used as positive control. The growth-prompting capacity of selected fungal isolates SB5-1, SB3-2, and SB3-3 was bio-assayed on soybean cv. Hwangkeumkong. Fungal isolate SB5-1 provided maximum plant height (31.6 cm), shoot length (21.1 cm), whole plant fresh biomass (2.41 g), shoot fresh biomass (1.99 g), and leaf area (24.37 cm²). The CF of isolate SB5-1 was analyzed for the presence of GAs, and it was found that all physiologically active GAs were present (GA₁, 0.15 ng/ml, GA₃, 1.2 ng/ml, GA₄, 7.37 ng/ml, and GA₇, 3.18 ng/ml) in conjunction with physiologically inactive GA₅, GA₉, GA₁₅, GA₁₉, GA₂₀, and GA₂₄. The fungal isolate SB5-1 was identified as a new strain of Cephalotheca sulfurea through molecular and phylogenetic approaches.     

Production of extracellular hydrolase enzymes by fungi from King George Island

Fungi are known to produce a range of extracellular enzymes and other secondary metabolites. Investment in extracellular enzyme production may be an important element of the survival strategy of these fungi in maritime Antarctic soils. This study focuses on fungi that were isolated from ornithogenic, undisturbed and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica, during the austral summer in February 2007. We (1) describe fungal diversity based on molecular approaches, (2) describe the thermal characteristics of the fungal isolates, and (3) screen extracellular hydrolase enzyme production (amylase and cellulase) by the isolates. Soil samples were cultured using the Warcup soil plating technique and incubated at 4 and 25 °C to allow basic thermal classification. In total, 101 isolates were obtained. All the isolates were screened at culture temperatures of 4 and 25 °C in order to detect activity of extracellular hydrolase enzymes. At 25 °C, ornithogenic penguin rookery soils recorded the lowest diversity of fungi, with little difference in diversity apparent between the other soils examined. At 4 °C, an undisturbed site recorded the lowest and a human-impacted site the highest diversity of fungi. The majority of the fungi identified in this study were in the mesophilic thermal class. Six strains possessed significant activity for amylase and 13 for cellulase at 25 °C. At 4 °C, four strains showed significant amylase and 22 significant cellulase activity. The data presented increase our understanding of microbial responses to environmental temperature.     

Hydrolytic enzymes and ability of arbuscular mycorrhizal fungi to colonize roots

The production of hydrolytic enzymes from external mycelia associated with roots and colonized soybean roots (Glycine max L.) inoculated with different arbuscular-mycorrhizal (AM) fungi of the genus GLOMUS:, and the possible relationship between these activities and the capacity of the AM fungi to colonize plant roots was studied. There were differences in root colonization and plant growth between the GLOMUS: strains, and also between two isolates of G. mosseae. Hydrolytic activities in the root and external mycelia associated with roots differed in the AM fungi tested. Correlations were only found between the endoxyloglucanase activity of the external mycelia associated with roots of the AM fungi tested and the percentage root colonization or plant growth. However, hydrolytic activities of roots colonized by the different endophytes correlated with those of external mycelia. The hydrolytic activities were not qualitatively different because the endoxyloglucanase from AM colonized roots and the external mycelia did not show a high degree of polymorphism in the different species of fungus tested. The possible role of the hydrolytic activity of external hyphae of AM fungi was discussed as a factor affecting fungal ability to colonize the root and influence plant growth.     

Single cell oil production from rice hulls hydrolysate

Rice hull hydrolysate was used as feedstock for microbial lipids production using the oleaginous fungus Mortierella isabellina. Kinetic experiments were conducted in C/N ratios 35, 44 and 57 and the oil accumulation into fungal biomass was 36%, 51.2% and 64.3%, respectively. A detailed mathematical model was used in order to describe the lipid accumulation process. This model was able to predict reducing sugar and nitrogen consumption, fat-free biomass synthesis and lipid accumulation. Neutral lipids constitute the predominant lipid fraction, while the major fatty acids were oleic, palmitic and linoleic acid. Fatty acids of long aliphatic chain were not detected, thus the microbial oil produced is a promising feedstock for biodiesel production.     

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.     

中国如何突破生物柴油产业的原料瓶颈

因应我国日益严峻的能源资源、能源环境和能源安全形势,国家大力倡导发展可再生能源。生物柴油是最重要的液体可再生燃料之一,在能源性质方面可以完全替代化石柴油,而且还具有安全环保等其它优良特性。当前利用动植物油脂生产生物柴油,原料成本偏高,而且稳定、充足的油脂原料供应体系尚未形成。我国是油脂资源短缺国家,近年来植物油进口量逐年增加。同时,我国耕地资源匮乏,粮食供应形势不容乐观,扩大油料作物种植的潜力非常有限。但是,我国宜林地丰富,农林废弃生物质资源量巨大。综合以上因素,我国应重点发展木本油料植物规模化种植和推广,加快微生物油脂发酵技术创新和产业化进程;同时,利用植物遗传育种技术提高油料作物产量以及选择性发展不与粮争地的油料作物。依靠各方面的进步,发展创新的油脂生产技术,保障我国生物柴油产业和油脂化工行业健康发展。     

Antagonism between Bacteria and Fungi on Decomposing Aquatic Plant Litter

Bacterial and fungal decomposers of aquatic plant litter may exhibit either synergistic or antagonistic interactions, which are likely to influence microbial growth as well as the decomposition of litter and, eventually, the carbon metabolism of aquatic systems. To elucidate such interactions, we inoculated decomposing Phragmites culms in microcosms with fungal isolates and with natural communities of bacteria and fungi in different combinations. The development of fungal and bacterial biomass and the carbon dynamics were studied during several months of degradation. The results show a bilateral antagonistic relationship between bacteria and fungi. After 3 months, fungal biomass accumulation was approximately 12 times higher in the absence than in the presence of bacteria. Bacterial biomass accumulation was about double in the absence of fungi compared to when fungi were present. Similar interactions developed between a natural assemblage of bacteria and five different fungal strains isolated from Phragmites litter (three identified hyphomycetes and two unidentified strains). Despite the great difference in biomass development between the treatments, the carbon metabolism was similar regardless of whether fungi and/or bacteria were present alone or in coexistence. We suggest that the antagonism between bacteria and fungi is an important controlling factor for microbial colonization and growth on aquatic plant litter.     

The growth of external AM fungal mycelium in sand dunes and in experimental systems

We estimated the biomass and growth of arbuscular mycorrhizal (AM) mycelium in sand dunes using signature fatty acids. Mesh bags and tubes, containing initially mycelium-free sand, were buried in the field near the roots of the dune grass Ammophila arenaria L. AM fungal mycelia were detected at a distance of about 8.5 cm from the roots after 68 days of growth by use of neutral lipid fatty acid (NLFA) 16:1ω5. The average rate of mycelium extension during September and October was estimated as 1.2 mm day⁻¹. The lipid and fatty acid compositions of AM fungal mycelia of isolates and from sand dunes were analysed and showed all to be of a similar composition. Phospholipid fatty acids (PLFAs) can be used as indicators of microbial biomass. The mycelium of G. intraradices growing in glass beads contained 8.3 nmol PLFAs per mg dry biomass, and about 15% of the PLFAs in G. intraradices, G. claroideum and AM fungal mycelium extracted from sand dunes, consisted of the signature PLFA 16:1ω5. We thus suggest a conversion factor of 1.2 nmol PLFA 16:1ω5 per mg dry biomass. Calculations using this conversion factor indicated up to 34 μg dry AM fungal biomass per g sand in the sand dunes, which was less than one tenth of that found in an experimental system with Glomus spp. growing with cucumber as plant associate in agricultural soil. The PLFA results from different systems indicated that the biomass of the AM fungi constitutes a considerable part of the total soil microbial biomass. Calculations based on ATP of AM fungi in an experimental growth system indicated that the biomass of the AM fungi constituted approximately 30% of the total microbial biomass. This revised version was published online in June 2006 with corrections to the Cover Date.     

Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%–133% and 35.22%–59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.     

Efficient oleaginous yeasts for lipid production from lignocellulosic sugars and effects of lignocellulose degradation compounds on growth and lipid production

Microbial lipid production using lignocellulosic biomass is considered an alternative for biodiesel production. In this study, 418 yeast strains were screened to find efficient oleaginous yeasts which accumulated large quantities of lipid when cultivated in lignocellulosic sugars. Preliminary screening by Nile red staining revealed that 142 strains contained many or large lipid bodies. These strains were selected for quantitative analysis of lipid accumulation by shaking flask cultivation in nitrogen-limited medium II containing 70 g/L glucose or xylose or mixture of glucose and xylose in a ratio of 2:1. Rhodosporidium fluviale DMKU-SP314 produced the highest lipid concentration of 7.9 g/L when cultivated in the mixture of glucose and xylose after 9 days of cultivation, which was 55.0% of dry biomass (14.3 g/L). The main composition of fatty acids were oleic acid (40.2%), palmitic acid (25.2%), linoleic acid (17.9%) and stearic acid (11.1%). Moreover, the strain DMKU-SP314 could grow and produce lipid in a medium containing predominantly lignocellulose degradation products, namely, acetic acid, formic acid, furfural, 5-hydroxymethylfurfural (5-HMF) and vanillin, with however, some inhibitory effects. This strain showed high tolerance to acetic acid, 5-HMF and vanillin. Therefore, R. fluviale DMKU-SP314 is a promising strain for lipid production from lignocellulosic hydrolysate.     

培养条件对产油微生物生长的影响

为了筛选出高产油菌株, 首先采用细胞形态学方法与细胞化学方法(苏丹III染色法)对4株高产油脂菌株进行初筛, 并通过索氏提取法对初筛菌株油脂含量进行分析, 确定M2菌株为实验菌株, 其油脂含量达53.09%。为了增加产油微生物油脂产量, 本试验考察了不同发酵条件对其细胞生长和油脂积累的影响。优化工艺参数为: 10° Bx玉米皮渣水解液为培养基质, 0.2% NaNO3为氮源, pH 6.0、28oC下发酵培养6 d, 微生物油脂含量75.21%, 菌体生物量30.40 g/L, 油脂产量22.86 g/L。气相色谱分析表明该油脂的脂肪酸组成与植物油相似, 主要含有16碳和18碳系脂肪酸, 可作为生物柴油的原料, 不饱和脂肪酸含量达68%, 可应用于医药化工领域。     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。脂肪酸组成分析结果表明,菌油富含饱和及低度不饱和长链脂肪酸,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

Fluorescein Diacetate Hydrolysis as a Measure of Fungal Biomass in Soil

The fatty acid methyl esters of lipids extracted from an agricultural soil in the preharvest period of soybean or middle growth cycle from wheat were characterized and quantified by gas-liquid chromatography. The fatty acids 18:2ω6 and 16:1ω5 were used as markers of saprotrophic and arbuscular mycorrhizal fungi. In parallel, biomass estimation through plate counts in selective media for cellulolytic and saprotrophic fungi was also performed all throughout a soybean crop or middle growth cycle of wheat. As an enzymatic method, the fluorescein diacetate (FDA) hydrolytic activity of the samples was determined. Owing to the high relationship exhibited by FDA hydrolysis with organic carbon and total nitrogen content of soil, the enzymatic activity was correlated with the microbial biomass estimated through marker lipids or plate counts. The results obtained point out that FDA hydrolysis may be used as a rapid, cheap, and reliable estimator of fungal biomass. Received: 3 August 2000 / Accepted: 13 October 2000