Enhanced enzymatic hydrolysis of hydrothermally pretreated empty fruit bunches at high solids loadings by the synergism of hemicellulase and polyethylene glycol

In enzymatic hydrolysis, high lignocellulose loadings are required to obtain high sugar titers. However, the high solids loadings limit enzymatic hydrolysis. In this study, to overcome this limitation, the promoting and synergistic effects of the accessory agents of hemicellulase (i.e., Cellic HTec2) and polyethylene glycol (PEG) 8000 were investigated in the enzymatic hydrolysis of hydrothermally pretreated empty fruit bunches (EFBs). After the optimal addition of Cellic HTec2 and PEG, high enzymatic digestion of the pretreated EFBs was achieved owing to their synergistic effects, even at high solids loadings. For example, the enzymatic digestibility of pretreated EFBs at a 21.7% (w/v) solids loading with 10 FPU of Cellic CTec2/g glucan reached 72.5% when 2.7 mg of Cellic HTec2/g glucan and 62.5 mg of PEG/g glucan were used as the accessory agents. These results suggested that the optimal addition of accessory agents is effective for the enhanced hydrolysis of lignocellulose using even a commercial cellulase preparation.     

Co‐utilization of acidified glycerol pretreated‐sugarcane bagasse for microbial oil production by a novel Rhodosporidium strain

Acidified glycerol pretreatment is very effective to deconstruct lignocellulosics for producing glucose. Co‐utilization of pretreated biomass and residual glycerol to bioproducts could reduce the costs associated with biomass wash and solvent recovery. In this study, a novel strain Rhodosporidium toruloides RP 15, isolated from sugarcane bagasse, was selected and tested for coconversion of pretreated biomass and residual glycerol to microbial oils. In the screening trails, Rh. toruloides RP 15 demonstrated the highest oil production capacity on glucose, xylose, and glycerol among the 10 strains. At the optimal C:N molar ratio of 140:1, this strain accumulated 56.7, 38.3, and 54.7% microbial oils based on dry cell biomass with 30 g/L glucose, xylose, and glycerol, respectively. Furthermore, sugarcane bagasse medium containing 32.6 g/L glucose from glycerol‐pretreated bagasse and 23.4 g/L glycerol from pretreatment hydrolysate were used to produce microbial oils by Rh. toruloides RP 15. Under the preliminary conditions without pH control, this strain produced 7.7 g/L oil with an oil content of 59.8%, which was comparable or better than those achieved with a synthetic medium. In addition, this strain also produced 3.5 mg/L carotenoid as a by‐product. It is expected that microbial oil production can be significantly improved through process optimization.     

Culturable and metagenomic approaches of wheat bran and wheat straw phyllosphere’s highlight new lignocellulolytic microorganisms

The phyllosphere, defined as the aerial parts of plants, is one of the most prevalent microbial habitats on earth. The microorganisms present on the phyllosphere can have several interactions with the plant. The phyllosphere represents then a unique niche where microorganisms have evolved through time in that stressful environment and may have acquired the ability to degrade lignocellulosic plant cell walls in order to survive to oligotrophic conditions. The dynamic lignocellulolytic potential of two phyllospheric microbial consortia (wheat straw and wheat bran) has been studied. The microbial diversity rapidly changed between the native phyllospheres and the final degrading microbial consortia after 48 h of culture. Indeed, the initial microbial consortia was dominated by the Ralstonia (35·8%) and Micrococcus (75·2%) genera for the wheat bran and wheat straw whereas they were dominated by Candidatus phytoplasma (59%) and Acinetobacter (31·8%) in the final degrading microbial consortia respectively. Culturable experiments leading to the isolation of several new lignocellulolytic isolates (belonging to Moraxella and Atlantibacter genera) and metagenomic reconstruction of the microbial consortia highlighted the existence of an unpredicted microbial diversity involved in lignocellulose fractionation but also the existence of new pathways in known genera (presence of CE2 for Acinetobacter, several AAs for Pseudomonas and several GHs for Bacillus in different metagenomes-assembled genomes). The phyllosphere from agricultural co-products represents then a new niche as a lignocellulolytic degrading ecosystem.     

黑曲霉木质纤维素降解酶基因的可变剪接分析及验证

黑曲霉Aspergillus niger因能够产生大量的木质纤维素降解酶而在木质纤维素资源利用中发挥重要作用。目前,有关黑曲霉基因组中与木质纤维素降解相关的基因是否存在可变剪接的情况尚不清楚。本研究以黑曲霉CBS513.88菌株为研究对象,采用rMATS和ABLas两种方法对黑曲霉在葡萄糖为唯一碳源(G组)和小麦秸秆为唯一碳源(WS组)下的56个木质纤维素降解酶基因的可变剪接事件进行分析,并通过RT-PCR扩增和内含子特异性扩增对3个典型基因的可变剪接体进行了验证。结果表明,ABLas可变剪接分析算法相较于rMATS分析算法更为准确,ABLas分析算法显示G组和WS组共有21个木质纤维素降解酶基因出现了可变剪接,可变剪接类型以内含子保留(IR)为主,占所有可变剪接事件的82.85%。另外,G组和WS组发生可变剪接的木质纤维素降解酶基因也有所不同：G组发生可变剪接的基因为13个,WS组发生可变剪接的基因为14个,两组都发生可变剪接的基因为6个,这表明黑曲霉木质纤维素降解酶基因的可变剪接在不同生长条件下存在差异,另一方面,黑曲霉中众多可变剪接体的存在也为开发新型的木质纤维素降解酶资源提供基础。     

一株产酶真菌Phaeophlebiopsis sp.转录组分析

对从药渣中分离的真菌ZYJHYZ254进行鉴定及产酶活性研究,从转录组分析菌株不同生长时期差异表达基因对其生长发育及产酶调控的影响,以筛选高产木质纤维素水解酶真菌,寻找调控关键基因。鉴定ZYJHYZ254为拟暗射脉菌Phaeophlebiopsis sp.,产酶在第5-7天最高。从生长3 d与7 d的菌丝中共检测到1 232个差异基因,以3 d的菌丝为对照,显著上调、下调基因分别有826及406个,基因注释和GO、KEGG功能富集分析结果表明差异表达基因主要与蛋白质合成、代谢及酶合成相关。此外,共有387个CAZymes基因表达,GH数量最多,约占49.61%,其次为AA (97)与GT (62),约占25.06%与16.02%。GH16 (24个)占GH的12.50%,含量最多,主要编码葡萄糖苷酶、木聚糖酶等,AA中AA3 (37个)占比38.14%,编码氧化酶、脱氢酶等。结果表明ZYJHYZ254中生长3 d与7 d的菌丝经功能富集分析发现差异表达基因主要与蛋白质合成、代谢,以及酶合成相关。进一步研究发现在两个生长时期中CAZymes基因表达最多的是GH16与AA3,预示了该菌葡萄糖苷酶、木聚糖酶、β-半乳糖苷酶、氧化酶与脱氢酶含量最丰富,对降解特殊生物质中的木质纤维素具有重要意义。     

发酵性丝孢酵母HWZ004利用水稻秸秆水解液发酵产油脂

为高效利用水稻秸秆中的纤维素和半纤维素产油脂,采用稀酸预处理和酶水解两步法对水稻秸秆进行水解,然后以水解液为碳源,培养发酵性丝孢酵母Trichosporon fermentans HWZ004产微生物油脂。结果表明,经简单overliming法脱毒后水稻秸秆水解液中乙酸、糠醛和5-羟甲基糠醛的浓度分别为0.4 g/L、0.1 g/L和0.05 g/L。只需添加少量氮源和微量CuSO4?5H2O,该水解液即可满足T. fermentans HWZ004发酵产油脂的要求。发酵最适接种量、初始pH和温度分别是5.0％、7.0和25 ℃。T. fermentans HWZ004在优化条件下培养7 d的生物量、油脂含量和油脂产量分别是26.4 g/L,52.2％和13.8 g/L;油脂得率系数为17.0,大大高于驯化前菌株T. fermentans CICC 1368在脱毒水稻秸秆半纤维素水解液中的对应值 (11.9)。所产油脂的脂肪酸组成与植物油相似,不饱和脂肪酸含量达70％以上,宜作为生物柴油的生产原料。     

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest ¹. In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification ². These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90°C. The pretreatment solution is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme mixture for a variable length of time at 50°C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH colorimetric method.     

一株高效降解木糖的耐酸、嗜热厌氧杆菌的生理特性及产物分析

【目的】分离高效降解木糖的嗜热厌氧杆菌菌株,用于发酵生产生物燃料乙醇,为后继的构建基因工程菌株及联合生物工艺提供材料。【方法】运用亨盖特厌氧操作技术从胜利油田油层采出液两年的富集样中分离到一株嗜热厌氧杆菌xyl-d。采用形态学观察、生理生化指标鉴定及基于16S rRNA的系统发育学分析确定其分类地位。【结果】菌株xyl-d为革兰氏阴性厌氧杆菌,菌体大小为(1.35-5.08)μm×(0.27-0.40)μm,单生、成对或成簇生长,芽胞圆形,端生。温度生长范围30-85℃(最适温度65℃);pH范围3.0-10.0(最适pH 7.5);NaCl浓度范围0%-4%(最适NaCl浓度2.0%)。发酵D-木糖的产物是乙醇、乙酸、CO2及少量的异丁醇、丙酸。菌株xyl-d的(G+C)mol%含量为45.6%,与热厌氧杆菌属模式菌株威吉利热厌氧杆菌(Thermoanaerobacter wiegelii)DSM10319T及嗜热乙醇杆菌(Thermoanaerobacter ethanolicus)DSM2246T的16S rRNA序列相似性均为99.3%。菌株利用D-木糖产乙醇的最佳初始pH为8.5;少量酵母粉能刺激生长并显著提高发酵D-木糖的产醇率,使乙醇成为主要的发酵产物;培养基中乙醇浓度达到7%(V/V)时菌体生长受到抑制,最佳生长条件下D-木糖的降解率可达91.37%,最佳产醇条件下发酵1摩尔D-木糖可产生1.29摩尔的乙醇。【结论】菌株xyl-d是从特殊生境(油藏)中分离到的一株高效降解D-木糖的耐酸、嗜热的厌氧杆菌,其为半纤维素降解产乙醇的联合生物工艺提供了菌源。