功能菌剂的混合发酵研究

随着化工行业的发展，大量有毒有害难降解有机物随工业废水的排放进入环境，这些物质能够在环境中长期存在、积累和扩散，通过食物链对动植物的生存及人类的健康造成不良影响。本文以苯酚、对氯硝基苯、氯苯和十六烷为模拟污染物，以前期研制的功能菌剂为对象，经过紫外线线诱变筛选出优于出发菌株的功能菌，对诱变后功能菌的理化性能进行了研究，对菌种进行了鉴定，在此基础上，就其相互之间的微生态关系进行研究，为混合发酵提供理论基础，并就其最佳发酵条件及发酵参数进行了研究，最后对发酵产品的性能进行了检测。目前，国内外有关功能菌剂的研究还存在多方面的不足，主要包括：①由于多菌种混合发酵过程较为复杂，各菌之间存在复杂的相互作用，影响因素较多，关于菌种之间的相互关系研究得很少，环境功能菌剂的发酵方法大多采用单独发酵后混合的方式。单独发酵对原材料、设备和能源的利用率较低，对于多菌种制剂发酵，在设备、能源和原材料的方面造成的浪费更大，将会大幅增加菌剂的生产成本，影响多菌种功能菌剂的发展；②功能菌剂生产过程的质量控制方面研究得较少；③功能菌剂产品的稳定性、抗冲击性能研究得较少，对环境微生物制剂的研究主要集中在菌种选育和培养条件优化方面。 通过本论文研究，得到以下主要结论。 （1）在紫外线诱变处理中，用紫外线对发生一定程度退化的出发菌株进行诱变处理后，六株具有高效降解性能的菌株被筛选出来，诱变筛选出的菌株形态和ERIC-PCR指纹图谱与出发菌株相比发生了明显改变；而且诱变后的菌株对目标难降解底物的降解能力均得到改善，其中，FPN、FCB、F14、FEm对目标底物的降解率提高了20%以上；诱变后菌株经过7次连续传代接种后，对目标难降解底物的降解率无显著变化，具有一定的遗传稳定性。并对诱变后的功能菌进行了初步的鉴定，这6株菌都分别是芽孢杆菌。 （2）对诱变后的功能菌相互之间的微生态关系进行了研究，通过抑菌实验、生长量以及基质消耗量的比较，确定它们之间的生长关系是无害共栖关系，可以进行混合发酵。 （3）对该功能菌剂进行发酵培养条件研究，结果表明发酵培养基的最佳成分（g/L）：葡萄糖 31.0g/L、玉米粉10.0g/L、磷酸氢二钾1.0g/L、硫酸铵1.1g/L、硫酸镁0.55g/L。通过研究不同的培养条件对菌体生长和降解性能的影响，确定了最佳培养条件：培养基初始pH7.5；最适温度32℃；培养基装液量125mL（250 mL三角瓶），以及培养时间对降解性能的影响，培养20 h的产物对降解最为有利。通过研究添加不同目标污染物对菌体生长和降解性能的影响，确定了添加目标污染物的最佳量以及最佳时间：苯酚投加量：1.125 g/L，对氯硝基苯投加量：0.1 g/L；最佳投加时间为发酵培养开始后4 h。 （4）以摇瓶分批发酵最优条件为基础，对FPN、F10、FCB、FNa、F14 和 FEm进行了摇瓶分批发酵试验。以摇瓶分批发酵试验数据为依据，对功能菌剂分批发酵动力学进行了研究，建立了菌体生长和基质消耗的动力学模型，拟合模型能较好的反映功能菌剂分批发酵过程。 （5）功能菌剂和活性污泥协同作用，可以提高系统的生物降解能力，功能菌剂投加量为2%，新鲜活性污泥3500 mg/L，降解24 h条件下，功能菌剂和活性污泥的协同作用对COD的去除率和对照组相比，最多的提高了36.8%。功能菌剂和活性污泥协同作用以及活性污泥的单独作用，其生物降解过程均符合一级反应动力学过程，功能菌剂和活性污泥协同作用的生物降解动力学方程为：，相关系数97%。采用SBR运行方式，引入功能菌剂的SBR系统明显能够改善和提高生物降解的效率。与仅有活性污泥的系统相比，系统对COD的平均去除率可以提高27.1%，同时，系统的耐负荷冲击以及耐毒害冲击的性能比仅有活性污泥的SBR系统强，特别是负荷冲击对引入功能菌剂的SBR系统影响很小。仅有活性污泥的SBR系统经过负荷冲击和毒害冲击之后，不能恢复到冲击之前的水平，而且系统有效作用时间的周期比引入功能菌剂的SBR系统相比大大缩短，而引入功能菌剂的SBR系统处理效果较为稳定，恢复能力很强。 Along with the development of industries, many recalcitrant organic chemicals have been discharged into natural environments together with wastewaters and can exist in waters, soil and sediments for a long time without degradation. These haz-ardous substances, their byporducts and metabolizabilities can be highly toxic, mu-tagenic and carcinogenic, thereby threatening animals, plants and human health through food chain. Consequently the removal of these compounds is of significant interest in the area of wastewater treatment. In this dissertation, the phenol, hydro-quinone, chlorobenzene and hexadecane treated as the model pollutants, the func-tional microorganism agent was used as the starting strains, they treated with ultra-violet light, and then the mutant strains with high degradation ability were screened out and identified primarily, the relationship between these stains were studied, the medium composition and fermentation conditions were optimized, the degradation ability of the fermented production was tested. The literature survey indicates that the study of the microorganism agent is far from complete and more information is re-quired on following problems. 1, Because of the complexity of relationship in mixed fermentation and the complicated factors, the study is hardly to process.2, There is a lack of information on the quality control of the producing process .3, And there is a lack of information on the stability about the microorganism agent. In this dissertation, the main results of the present study could be summarized as follows: (1)The degenerate starting strains were treated with the ultraviolet light, and six mutant strains with high biodegradation ability were screened out by using the me-dium with selective pressure of model pollutants. The mutant strains had great changes in colonialmorphology and ERIC-PCR fingerprinting. And the mutant strains got obvious advantages over the starting strains in degradation ability and over 20% improvement of removal rates was achieved for FPN、FCB、F14 and FEm. The de-gradation ability of the mutant strains was stable after seven generations. After that, the mutant strains were primarily identified as bacillus respectively. (2) The relationship between these mutant strains was studied. By the compari-son of antibiosis effect, biomass and consumption of substrate, the relationships were neutralism and they could be mixed fermented. (3) The optimized cultivation conditions were as follows: glucose 31.0 g/L, corn power 10 g/L, K2HPO4 1.0 g/L, (NH4)2SO4 1.1 g/L, MgSO4 0.55 g/L, initial pH7.5, temperature 32℃, working volume 125 mL/250 mL, and cultivation time 20h (con-sidering the time effect on degradation ability), adding pollutants phenol (1.125 g/L) and hydroquinone (0.1 g/L) into the broth at 4 h after cultivation. (4) Based on the above optimum condition, the batch fermentation was per-formed with strains FPN, F10, FCB, FNa, F14 and FEm in shake flask. The batch fermentation kinetics was studied based on the experimental data. Two kinetic models were constructed which could reflect the regularity of growth and substrate consump-tion in the process of batch fermentation. (5) The co-operation of functional microorganism agent and activated sludge could raise biodegradation of system by adding some microorganism agent and 3500 mg/L fresh activated sludge. Bioaugumentation by the addition of high effective deg-radation culture enhanced the treatment effect of SBR system and the COD removal rate was increased by 20%-36.8%. Its biodegradation matched first-order dynamical reaction equation, and the reaction equation was ln0.2327.391ct=−+. The micro-organism agent had the effect of optimization to activated sludge micro-ecosystem. The SBR system adding 2% microorganism agent, the average COD removal rate of that was increased by 27.1% and stronger anti-shock ability to load and toxicant were achieved (compared with SBR system just adding activated sludge). Especially the load-shock has barely effect to the SBR system adding microorganism agent. After the load and toxicant shock, the SBR system just adding activated sludge couldn’t come back to original level and the activated sludge micro-ecosystem was frustrated. The applying of microorganism agent increased biological activity and system’s re-sistance ability to load shock and toxicant shock.

Two Polyketides from a Co-culture of Two Marine-derived Fungal Strains

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by anaerobic mixed culture fermentation was investigated using batch experiments at 37 degrees C. Seven varieties of typical individual components of OFMSW including rice, potato, lettuce, lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production potential. Experimental results showed that the boiling treated anaerobic sludge was effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is the most optimal substrate for fermentative hydrogen production compared with proteins, lipids and lignocelluloses. This conclusion was also substantiated by experimental results of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas produced from rice, potato and lettuce were 57-70%, 41-55% and 37-67%. 2008 International Association for Hydrogen Energy.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro

A series of in vitro studies was, conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 muL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20degreesC. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 muL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 muL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.

In vitro fermentation of mixed linkage glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

The aim of this study was to develop selectively fermented (prebiotic) carbohydrate molecules which would also result in the generation of butyric acid. Glucooligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from various types of maltodextrins were evaluated for their fermentation by mixed cultures of human colonic microflora. The selectivity of growth of desirable bacteria (bifidobacteria, lactobacilli) was studied in stirred pH-controlled (6.8) batch cultures. Bacterial populations were enumerated using fluorescent in situ hybridization (FISH). Gluco-oligosaccharides resulted in significantly (P<0.05) increased numbers of bifidobacteria and lactobacilli within 24 hours. Bacteroides, clostridial and eubacterial populations were slightly decreased at 48 h. There was very little difference in selectivity between the maltodextrin substrates and the products, although maltodextrin displayed a slightly less selective fermentation than the gluco-oligosaccharide products, also stimulating the growth of bacteroides, clostridia and eubacteria. Gluco-oligosaccharides, produced from G19 maltodextrin, resulted in the best prebiotic effect with the highest prebiotic index (PI) of 5.90 at 48 hours. Acetate, propionate and butyrate were all produced from glucooligosaccharides, derived from G19 maltodextrin, at 48 hours but no lactate or formate were detected.

In vitro fermentation of chitosan derivatives by mixed cultures of human faecal bacteria

Stirred, pH controlled batch cultures were carried out with faecal inocula and various chitosans to investigate the fermentation of chitosan derivatives by the human gut flora. Changes in bacterial levels and short chain fatty acids were measured over time. Low, medium and high molecular weight chitosan caused a decrease in bacteroides, bifidobacteria, clostridia and lactobacilli. A similar pattern was seen with chitosan oligosaccharide (COS). Butyrate levels also decreased. A three-stage fermentation model of the human colon was used for investigation of the metabolism of COS. In a region representing the proximal colon, clostridia decreased while lactobacilli increased. In the region representing the transverse colon, bacteroides and clostridia increased. Distally a small increase in bacteroides occurred. Butyrate levels increased. Under the highly competitive conditions of the human colon, many members of the microflora, are unable to compete for chitosans of low, medium or high molecular weight. COS were more easily utilised and when added to an in vitro colonic model led to increased production of butyrate, but some populations of potentially detrimental bacteria also increased. (c) 2005 Elsevier Ltd. All rights reserved.

In vitro fermentation of oat bran obtained by debranning with a mixed culture of human fecal bacteria

The prebiotic potential of oat samples was investigated by in vitro shaker-flask anaerobic fermentations with human fecal cultures. The oat bran fraction was obtained by debranning and was compared with other carbon sources such as whole oat flour, glucose, and fructo-oligosaccharide. The oat bran fraction showed a decrease in culturable anaerobes and clostridia and an increase in bifidobacteria and lactobacilli populations. A similar pattern was observed in fructo-oligosaccharide. Butyrate production was higher in oat bran compared to glucose and similar to that in fructo-oligosaccharide. Production of propionate was higher in the two oat media than in fructo-oligosaccharide and glucose, which can be used as energy source by the liver. This study suggests that the oat bran fraction obtained by debranning is digested by the gut ecosystem and increases the population of beneficial bacteria in the indigenous gut microbiota. This medium also provides an energy source preferred by colonocytes when it is metabolized by the gut flora.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.

In vitro fermentation of lactulose-derived oligosaccharides by mixed fecal microbiota

Fermentation properties of oligosaccharides derived from lactulose (OsLu) and lactose (GOS) have been assessed in pH-controlled anaerobic batch cultures using lactulose and Vivinal-GOS as reference carbohydrates. Changes in gut bacterial populations and their metabolic activities were monitored over 24 h by fluorescent in situ hybridization (FISH) and by measurement of short-chain fatty acid (SCFA) production. Lactulose-derived oligosaccharides were selectively fermented by Bifidobacterium and lactic acid bacterial populations producing higher SCFA concentrations compared to GOS. The highest total SCFA production was from Vivinal-GOS > lactulose > OsLu > GOS. Longer incubation periods produced a selective fermentation of OsLu when they were used as a carbon source reaching the highest selective index scores. The new oligosaccharides may constitute a good alternative to lactulose, and they could belong to a new generation of prebiotics to be used as a functional ingredient for improving the composition of gut microflora.

Effects of nutritional factors on growth of Lactobacillus fermentum mixed with Saccharomyces cerevisiae in alcoholic fermentation

The growth of Lactobacillus fermentum was studied in mixed culture with Saccharomyces cerevisiae during alcoholic fermentation of high test molasses (HTM). Yeast extract or a group of 17 amino acids caused a strong and fast decrease in yeast viability due to the strong increase of acidity produced by bacteria. Pure culture of Lactobacillus fermentum in dry sugar cane broth confirmed amino acids as the main nutrients needed to stimulate the growth of bacterial contaminant during alcoholic fermentation. The absence of L. fermentum growth was obtained when leucine: isoleucine or valine were not added to the medium. Phenylalanine, alanine, glutamic acid, cystine, proline, histidine, arginine, threonine, tryptophane, serine and methionine inhibited the bacterial growth at least in one of the cultures of L. fermentum tested.

Bio-hydrogen production from food waste through anaerobic fermentation

In order to protect our planet and ourselves from the adverse effects of excessive CO2 emissions and to prevent an imminent non-renewable fossil fuel shortage and energy crisis, there is a need to transform our current ‘fossil fuel dependent’ energy systems to new, clean, renewable energy sources. The world has recognized hydrogen as an energy carrier that complies with all the environmental quality and energy security, demands. This research aimed at producing hydrogen through anaerobic fermentation, using food waste as the substrate. Four food waste substrates were used: Rice, fish, vegetable and their mixture. Bio-hydrogen production was performed in lab scale reactors, using 250 mL serum bottles. The food waste was first mixed with the anaerobic sewage sludge and incubated at 37°C for 31 days (acclimatization). The anaerobic sewage sludge was then heat treated at 80°C for 15 min. The experiment was conducted at an initial pH of 5.5 and temperatures of 27, 35 and 55°C. The maximum cumulative hydrogen produced by rice, fish, vegetable and mixed food waste substrates were highest at 37°C (Rice =26.97±0.76 mL, fish = 89.70±1.25 mL, vegetable = 42.00±1.76 mL, mixed = 108.90±1.42 mL). A comparative study of acclimatized (the different food waste substrates were mixed with anaerobic sewage sludge and incubated at 37°C for 31days) and non-acclimatized food waste substrate (food waste that was not incubated with anaerobic sewage sludge) showed that acclimatized food waste substrate enhanced bio-hydrogen production by 90 - 100%.

Food waste valorization through the production of polyhydroxyalkanoates by mixed microbial cultures

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

L-Glutaminase production by marine vibrio costicola under solid state fermentation

Use of inert supports have been recommended for SSF in on ar to overcome its inherent problems and efforts are being made to search for newer and better materials to act as inert solid supports lidoo et al, 1982; Zhu et al, 1994).In the present study an attempt is made to produce L-glutaminase, which is industrially and therapeutically impo rtant, from marine bacteria under solid state fermentation using natura.l. inert and mixed substrates with a view to develop an ideal bioprocess for its large scale production.

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid., naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-D-hydroxyphenyl)propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols. (C) 2003 Elsevier Inc. All rights reserved.