Stability Analysis of a Lignocellulose Degrading Microbial Consortium

Introduction

Rice straw is an important carbon resource for the biorefinery industry, its use for bio-energy-biogas is also a very promising way, and is thus considered a sustainable and environmentally friendly alternative to the fossil energy (Kamm and Kamm, 2004; Edward et al., 2007).The use of microbial populations in decomposing lignocellulosic biomass to produce highcalorific value products is an important research area in alleviating energy crisis and effectively utilizing the cellulose resources (Barkha et al., 2016;Zhou et al., 2009).

农药在喷施过程中的雾滴飘移是造成环境污染、农药流失和农药有效利用率低的重要原因。常规喷雾方式下，减小雾滴直径可以提供良好的覆盖率，却增大了飘移；而粗雾滴降低飘移的同时，也降低了雾滴的附着率。风助式喷雾是一种利用专用设施产生定向气流辅助的喷雾方式，能够在雾滴直径较小的情况下提高雾滴在靶标上的沉积率，减少雾滴飘移。风助式喷雾技术的应用提高了农药的生物效果、拓宽了喷雾设备的应用条件，有利于提高农药有效利用率，减少化学污染[1-3]。

In natural, lignocellulosic biomass is decomposed under aerobic or anaerobic conditions due to synergy of various microorganisms, which will avoid feedback regulation and metabolic inhibition in contrast to single strain (Kumar et al., 2008; Mohamed et al.,2015; Miqueleto et al., 2010).In recent years, many lignocellulose degradation complex consortia were successfully obtained by limiting-culture and directional construction (Tang et al., 2015; Barkha 2016;Li et al., 2009; Gao et al., 2009).Haruta et al.(2002)obtained a structural stable and complex lignocellulolytic microbial consortium with high degradable activity on various cellulosic materials from rice straw compost by successive enrichment culture.Study on the dynamic changes of genetic components and functional stability of microbial consortia could provide necessary basis for subsequent theoretical and applied researchs (Bandal, 2003).The application of molecular ecology technology can avoid the cumbersome process in traditional separation and cultivation, directly to the relationships between microbial consortia structure and environment, DGGE technology is one of the commonly used molecular ecology technology (Gong et al., 2004).Liu (2009)studied the microbial community dynamics and determined the dominant bacteria in different rice stalk fermentation stages with PCR-DGGE technology.

The functional and structural stabilities of microbial consortia are considered to be important factors in biomass degradation capability and potential for biotechnological application (Wongwilaiwalin et al.,2010).In this study, the functional and structural stability of five different generations of reserved microbial consortium LZF-12 capable of degrading rice straw were evaluated through batch experiments and a molecular culture-independent approach.This study provided a valuable platform for the subsequent targeted regulation and control of fermentation system.The microbial consortia have potential in biotechnological application on lignocellulosic biomass degradation.

Materials and Methods

Culture and medium

文丘里管采用缩扩形喷管结构，主要结构见图1,主要组成部分分别为收缩喷嘴段、喉口混合段和扩压段。在收缩段气流实现膨胀，流速上升，马赫数变大，流体压力、密度和温度均会降低；而在扩压段中，流体压力逐步上升，温度和密度均逐渐恢复，流速减慢。在膨胀过程中，流体的压力逐渐变小，当到喉口部位时，形成低压区，最终使得废气压力与进气压力之间形成一定的压力差，保证废气从涡轮前引射入进气管。此外，扩压段的混合气流动是一种压缩过程，压力上升，降低压力损失带来的不利影响。对比其余辅助混合技术，文丘里管可在不损失机械功的状况下实现进排气的混合。

The mesophilic microbial consortium LZF-12 capable of effectively degrading lignocelluloses was established by the Biomass Laboratory of Northeast Agricultural University (Chong et al., 2011).Rice straw was collected from local farm, pretreated with 1% (w/v) NaOH and dried at 50℃, used as the sole carbon source (Zheng, 2013).The microbial consortium was grown in peptone cellulose selection(PCS) medium (peptone, 5 g; yeast powder, 0.8 g;calcium carbonate, 2.0 g; sodium chloride, 5.0 g; dry straw, 10.0 g; distilled water, 1 000 mL) autoclaved at 121℃ for 15 min using standard methods (Wang et al., 2005).

Analytical methods

⑤整个闸门由多组强化钢闸门和气袋单元连接而成，长度不受限制，也不需要中间墩座，可最大程度地提高过水面，即使在失电的情况下也可以手动排气倒伏，不影响泄洪。

摄影的情况是，歧义的坍缩常常借助于——有时是不恰当地受制于——标题，文字试图解决不确定性，告诉我们薛定谔的猫到底是死是活，却不管正确与否。（没有这些说明，基于照片所显示的证据，猫很可能被认为是睡着了以及/或者死了，如量子一般。）标题是为了将照片限定在单一的状态里，而非开启意义的扩展。如果一张照片被认为值得用一千个词来说明，在标题告诉读者这张照片所谓的内容之后，通常人们不会记得多少。[1]190

The preserved 15th, 20th, 25th, 30th and 35th generations microbial consortia were inoculated in 500 mL petone cellulose substrate (PCS) medium containing 1% rice straw at a 5% inoculation amount,incubated at 35℃ under static condition.pH, dissolved oxygen (DO) value and liquid end products of the fermentation broth were monitored daily, the residual solid cellulosic substrates were determined gravimetrically incubated for 7 days with uninoculated media as the contrast group.

如何发挥产业转移的雾霾治理效应？——基于长三角地区的实证研究..................................................................................................................................................刘曦彤（83）

DO and pH were monitored regularly using the Portable Dissolved Oxygen Analyzer (WTW oxi315i)and the portable pH meter (Accuracy: 0.01).The methods referred to Li et al (2006).

长江河道采砂管理已形成以《长江河道采砂管理条例》为核心，以地方政府规章和各种规范性文件为补充的采砂管理法规体系。而沂沭泗地区江苏境内的河道采砂管理，虽然出台了《徐州市河道采砂管理条例》，但由于没有配套的实施细则，操作性不强，在没有国家法规或配套规章的情况下，很难执行。因此，在呼吁“中华人民共和国河道采砂管理条例”及配套制度出台的同时，对于采砂管理中遇到的迫切和关键问题急需权力机关作出相应的、可具操作性的立法解释。

通过对配方B的感官评价可知，配方B的得分为8.41，硫代薄荷酮的加入增强了百香果香气的特征性，乙基麦芽酚增加了香精的留香值，橙油圆和了果香酯类原料的化学气息，使香气天然感突出。玫瑰精油的加入给百香果香精带进一些花香，使百香果香精的香气得到很好的修饰。总之，配方B的果香主题突出并带有热带水果气息，整个香气头、体、尾香过渡自然且有层次感，能让人联想到自然的百香果的味道，符合食品香精的质量要求。

Where, Mt was the total weight of the cellulosic materials before degradation and Mr was the weight of the residual substrates after degradation.The reactions were performed in triplicate and the averages of the results were reported.Volatile fat acid (VFA) was determined by a gas chromatography (GC-6890N,Agilent Inc., USA) equipped with a flame ionization detector and a 30 m×0.25 mm×0.25 μm fused-silica capillary column.Nitrogen was used as the carrier gas with split injection method, split ratio of 20 : 1.The details as described by Zheng et al ( 2013).Retention time of ethanol, acetic acid, propionate and butyrate(analytically pure) were determined to obtain the peak sequence.A standard solution containing ethanol,acetic acid, propionic acid, butyric acid was diluted with deionized water to five different concentrations and measured to plot the standard curve.The concentrations of unknown alcohols and acids in the samples were determined according to external standard method (Chong, 2011).

The total genomic DNA was extracted from the microbial consortia using bacterial genomic DNA extraction kit.The purified DNA was used as a template for amplification of the partial 16S rDNA fragment using Taq DNA polymerase (Fermentas,Vilnius, Lithunia) according to the manufacturer's protocol with 338GC-F forward primer, which was attached to a GC clamp at the 5-terminus (5'-cgcccgc cgcgcgcggcgggcggggcgggggcacggggggactcctacggga ggca-3', GC clamp sequence is underlined), and 518R reverse primer (5'-attaccgcggctgctgg-3').Reactions were performed in a My Cycler thermal cycler (Bio-Rad Laboratories, Hercules, CA).The temperature profile consisted of 94℃ for 3 min, followed by 30 cycles of denaturation at 94℃ for 1 min, annealing at 55℃ for 1 min and extension at 72℃ for 2 min,followed by a final extension step at 72℃ for 3 min.

Stability analysis of microbial consortium LZF-12

Results

The pro files of amplified 16S rDNA fragments were analyzed by DGGE technology on DCodeTM system(Bio-Rad).The target bands were sequenced after cloning.Sequences were initially compared to the available databases using basic local alignment search tool (BLAST) server to determine their approximate phylogeny and analyze the genetic relationship and similarity.

The degradation rate of rice straw, the types and concentration of fermentation metabolites and genetic stability of the reaction system were used as measurable indicators to screen high efficient microbial consortia.After subculture, transfer experiments were conducted respectively to investigate the fermentation stability of rice straw microbial consortium LZF-12 using the degradation efficiency, pH, DO and terminal liquid products as key indicators.

Fig.1 showed pH changes of fermentation broth were similar in the 15th, 20th, 25th, 30th and 35th generation cultures with time.At the initial phase of fermentation (1 day), pH of different generations decreased rapidly from the initial 6.8 to 6.1, 6.2, 6.3,and 6.4, respectively.This could be explained by the production of organic acids from the degradation of easily compounds, as acids accumulate, the medium becomes acidified.After 1 day, pH increased from 6.3,6.1, 6.2, 6.4 and 6.3 to 7.0, 7.1, 7.2, 7.0 and 7.1 for each generation respectively, could be explained by the degradation of organic acids along with the growth and fermentation of microbial consortium LZF-12.On the 7th day, the rice straw was mostly degraded and pH was kept 7.0-7.2, it could be inferred that pH changes of the fermentation were closely related to the decomposition process of cellulose.There was no obvious difference in five pH change trends,suggesting the stability of the fermentation system in different generations of LZF-12.

DO in the 15th, 20th, 25th, 30th and 35th generation cultures were determined.Table 1 showed the changing trends of DO were basically same in five experimental groups.After inoculated 1 day, the decrease in DO significantly from initial 2.3 mg · L-1 to 0.07-0.08 mg · L-1, which could be explained by the consumption of dissolved oxygen, due to the growth of facultative anaerobes.In 2-7 days, pH fluctuated slightly (0.07≤DO≤0.09), on the 7th day of the fermentation, DO were 0.07, 0.08, 0.08, 0.07 and 0.09,respectively, indicated that the fermentation of the microbial consortium adapted to micro-aerobic environment.

The microbial consortium was incubated in PCS medium containing 1% rice straw.Residual solid cellulosic substrates were analyzed after incubation with the microbial consortia for 7 days at 35℃ under the defined facultative anaerobic static condition.The residual substrates were then determined gravimetrically after dried at 105℃ for 2 days with uninoculated medium as a contrast group.Percent of residual weight was reported based on the total holocellulose content.Degradation ratio was described by the following equation (Kluzek-Turpeinen B,2000).

The component and concentration of the liquid end products could provide very important reference for controlling the normal operation of the reaction system.In view of the previous studies,the fermentation products of LZF-12 were mainly ethanol, acetic acid, butyric acid and a small amount of propionic acid, and the acetic acid made up about 70% of the total liquid product volume (Chong,2011).Therefore, in this experiment, the changes of acetic acid contents in the fermentation broth of different generations were analyzed dynamically(Fig.2).The main types of liquid end products did not change (the data was not listed) in different generation fermentation systems which were still composed of alcohol, acetic acid, butyric acid and propionic acid.The acetic acid showed small difference and slight variations in different generations.The increase of acetic acid concentrations from 0 to 2.2-2.4 g · L-1 appeared to correspond to cellulose degradation amount within 6 days of the incubation.Then gradually decreased to 2.01-2.15 g · L-1, and the concentration of acetic acid in five experimental groups were all above 70% of the total liquid phase end product concentration.

Fig.1　Variation of pH of different subcultures of microbial system

Table 1　DO variation of different subcultures of microbial consortium LZF-12

Generation DO value (Days of incubation)0 1 2 3 4 5 6 7 15　2.3　0.07　0.08　0.07　0.09　0.07　0.08　0.07 20　2.3　0.07　0.07　0.08　0.07　0.08　0.08　0.08 25　2.3　0.07　0.07　0.08　0.09　0.08　0.07　0.08 30　2.3　0.08　0.07　0.08　0.09　0.08　0.09　0.07 35　2.3　0.07　0.08　0.09　0.08　0.08　0.07　0.09

Fig.2　Variation of acetic acid content of different subcultures of microbial system

The cryopreserved 15th, 20th, 25th, 30th and 35th generations of the microbial consortium were activated and inoculated to PCS mediums.The results are shown in Fig.3, the degradation rates of the five cultivate generations were all more than 70% under the same culture conditions, and there was no obvious difference in rice straw degradation rate of each generation,indicated that the microbial consortium LZF-12 had strong stability in decomposing lignocellulosic substrates effectively.

Fig.3　% Residual weight of 15th, 20th, 25th, 30th and 35th generations of microbial consortium LZF-12

Microbial community structure

In order to assess the structural stability of the microbial consortium during subcultivation, 16S rDNA PCR products amplified with primer pairs collected from different cultured generations were analyzed by denaturing gradient gel electrophoresis to contrast to the community DGGE profiles.Sixteen bands were observed in DGGE pro files (Fig.4).

Comparison of the sequences originating from the numbered bands with GenBank (Table 2) revealed that LZF-12 was composed of five major bacteria.Bands 5, 9 and 10 originated from Clostridium sp.AP81, Clostridium cellobioparum strain DSM 1351 and Clostridium sp.T241, respectively.

Clostridium strains had a strong adaptation to ambient temperature and pH, and could produce extracellular hydrolytic enzymes decomposing proteins,sugars and lignocellulose to small molecular substances.Bands 7 and 8 originated from Clostridiumcellulolyticum H10 and C.cellulolyticum, gram negative and spore-forming, bacterium with flagella,could produce lignin modifying enzymes including extracellular oxidase, laccase, tyrosinase and peroxidase with a strong ability to degrade cellulose into acetic acid, H2, CO2 and lactic acid (Wiegel et al., 2006).The sequences of bands 2, 3, 4, 6, 11 and 15 were identical to some uncultured bacteria which stably co-existed in the consortia, respectively.

Fig.4　Structural stability of composite microbes in microbial consortium LZF-12 by denaturing gradient gel electrophoresis

Serial numbers 1-16 indicate DGGE bands in 15th, 20th, 25th, 30th and 35th generations.

Bands 13, 14 and 16 originated from uncultured Verrucomicrobia were presented, in addition to Pseudomonas sp.F5OHPNU07IE0B8 (Band 1) and Acetivibrio sp.WSC-27 (Band 12).Being one of the dominant bacterium, Verrucomicrobia appeared in the later stage owing to oxygen depletion in reaction system and were good for lignocellulosic substrate degradation (Kuang, 2010).Pseudomonas was rodshaped or slightly curved, gram negative, no spores and aerobic bacterium, used the dissolved oxygen at the initial stage of fermentation to create a microaerobic environment for the system, thus accelerated the lignocellulose degradation by anaerobic and facultative anaerobes.Furthermore, Acetivibrio sp.WSC-27 was a mesophilic, chemoorganotrophic bacterium isolated from the waste sludge or pig manure, its main end products were acetates, in additional to a few of ethanol, CO2 and H2.DGGE pattern pro file dynamics from different LZF-12 generations were reproducible,suggesting the relative stabilities of the microbial community structure and succession mechanism in the established consortia.

Table 2　Sequence similarity analysis of bands 1-16

Phylotype　Closet relative　Accession number　Identity (%)Band-1　Pseudomonas sp.F5OHPNU07IE0B8　HQ083441.1　92 Band-2　Uncultured soil bacterium clone TB5　DQ248270.1　95 Band-3　Uncultured bacterium clone Fec185　FJ911492.1　92 Band-4　Uncultured soil bacterium clone bacterium clone TB5　DQ248270.1　95 Band-5　Clostridium sp.AP81　AB273730.1　98 Band-6　Uncultured Lachnospiraceae bacterium clone GE7GXPU01AFOLV　HM974554.1　99 Band-7　Clostridium cellulolyticum H10　CP001348.1　98 Band-8　C.cellulolyticum　X71847.1　96 Band-9　Clostridium cellobioparum strain DSM 1351　NR026104.1　95 Band-10　Clostridium sp.T241　AB277865.1　93 Band-11　Azovibrio sp.RV1　DQ833392.1　97 Band-12　Acetivibrio sp.WSC-27　HM635208.1　92 Band-13　Uncultured Verrucomicrobia QEDT2BA10　CU920803.1　99 Band-14　Uncultured Verrucomicrobia QEDT2CD06　CU920805.1　99 Band-15　Uncultured Coprothermobacteria QEDT3BA02　CU920461.1　95 Band-16　Uncultured Verrucomicrobia QEDT2CD06　CU920805.1　99

Conclusions

These experimental results showed that efficient degradation of rice straw (＞70%) could be achieved in batch cultures inoculated with microbial consortium LZF-12 from different generations.Acetic acid was the major aqueous products of five generations,ethanol, butyric acid and a small amount of propionic acid were also formed, but in low levels, the difference in liquid end products showed slightly variation and was the same with DO and pH in the whole fermentation process.Similar DGGE patterns among five generations (from generation 15 to 35) were observed, indicated that the structural and functional stability of the composite microbial co-existed in the consortium, which comprised mainly of approx.Five major composite members including Clostridium,Pseudomonas, Acetivibrio, Verrucomicrobia and some uncultured unidentified bacteria.Their coexistence was assumed to be important for effective lignocellulose degradation by complex metabolic interaction.The balance of various types of metabolic relationships was considered to be essential for the stable co-existence of the composite members in the community, which resulted in efficient biomass degradation.The results also illustrated the high stability of the microbial community LZF-12 and allowed long-term storage of the seed culture for further experimental studies and application.

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