Methanogenic community dynamics in anaerobic co-digestion of fruit and vegetable waste and food waste

A lab-scale continuously-stirred tank reactor (CSTR), used for anaerobic co-digestion of fruit and vegetable waste (FVW) and food waste (FW) at different mixture ratios, was operated for 178 days at the organic loading rate of 3 kg VS (volatile solids)/(m³.day). The dynamics of the Archaeal community and the correlations between environmental variables and methanogenic community structure were analyzed by polymerase chain reactions - denaturing gradient gel electrophoresis (PCR-DGGE) and redundancy analysis (RDA), respectively. PCR-DGGE results demonstrated that the mixture ratio of FVW to FW altered the community composition of Archaea. As the FVW/FW ratio increased, Methanoculleus, Methanosaeta and Methanosarcina became the predominant methanogens in the community. Redundancy analysis results indicated that the shift of the methanogenic community was significantly correlated with the composition of acidogenic products and methane production yield. Different mixture ratios of substrates led to different compositions of intermediate metabolites, which may affect the methanogenic community. These results suggested that the analysis of microbial communities could be used to diagnose anaerobic processes.     

Determination of the archaeal and bacterial communities in two-phase and single-stage anaerobic systems by 454 pyrosequencing

2-Phase anaerobic digestion (AD), where the acidogenic phase was operated at 2 day hydraulic retention time (HRT) and the methanogenic phase at 10 days HRT, had been evaluated to determine if it could provide higher organic reduction and methane production than the conventional single-stage AD (also operated at 12 days HRT). 454 pyrosequencing was performed to determine and compare the microbial communities. The acidogenic reactor of the 2-phase system yielded a unique bacterial community of the lowest richness and diversity, while bacterial profiles of the methanogenic reactor closely followed the single-stage reactor. All reactors were predominated by hydrogenotrophic methanogens, mainly Methanolinea. Unusually, the acidogenic reactor contributed up to 24% of total methane production in the 2-phase system. This could be explained by the presence of Methanosarcina and Methanobrevibacter, and their activities could also help regulate reactor alkalinity during high loading conditions through carbon dioxide production. The enrichment of hydrolytic and acidogenic Porphyromonadaceae, Prevotellaceae, Ruminococcaceae and unclassified Bacteroidetes in the acidogenic reactor would have contributed to the improved sludge volatile solids degradation, and ultimately the overall 2-phase system''s performance. Syntrophic acetogenic microorganisms were absent in the acidogenic reactor but present in the downstream methanogenic reactor, indicating the retention of various metabolic pathways also found in a single-stage system. The determination of key microorganisms further expands our understanding of the complex biological functions in AD process.     

R-PFR与CSTR厌氧消化餐厨垃圾运行效率及微生物群落结构对比

以中温餐厨垃圾(FW)厌氧消化反应器为研究对象,结合稳定运行过程中的微生物群落结构特征,考察了两种常见的厌氧消化反应器—完全混合式反应器(CSTR)和有回流的推流式反应器(R-PFR)的运行效率和稳定性.结果表明,两种构型的反应器均能在3.0 kg·m-3·d-1(以VS计)负荷下稳定高效地运行.R-PFR具有更稳定的稳定性指数,如总碱度(TA)和挥发性脂肪酸比TA值(VFA/TA),以及更低的氨氮(TAN)和游离氨(FAN)浓度,但产气效率(如比甲烷产率(SMP))不及CSTR.R-PFR中以Chloroflexi门(37.35%)和Firmicutes门(31.22%)为优势细菌,而CSTR中以Bacteroidetes门(31.14%)与Firmicutes门(44.41%)为优势细菌.CSTR和R-PFR均以Methanosaeta属为优势产甲烷菌(98.72%和84.90%),乙酸型产甲烷途径为主要的产甲烷途径.但CSTR中除Methanosaeta属以外还有一定丰度的混合营养型的Methanosarcina,以及氢营养型Methanospirillum和Methanolinea.CSTR中具有对VFA和TAN更具耐受性的产甲烷菌群,更有利于餐厨垃圾产甲烷过程的稳定进行.由于R-PFR的敏感性较低,有利于处理剧烈变化的底物,但R-PFR容易出现严重的局部酸化现象,不利于长期处理过高负荷的易降解底物(如餐厨垃圾).这些结果可为优化餐厨垃圾厌氧消化工艺设计提供基础依据.     

新型两相一体厌氧消化反应器处理餐厨垃圾中试研究

针对餐厨垃圾厌氧消化过程易发生挥发性脂肪酸（VFAs）的积累,系统难以稳定高效运行等问题,本研究设计制造了一套中试规模的新型双环嵌套式两相一体厌氧反应器,采用双环嵌套式结构,在反应器内部将内环的产酸阶段与外环的产甲烷阶段分离.反应器共运行184d,初始负荷为60kg/d,运行阶段阶梯式提高负荷,最高达到180kg/d.结果表明,该新型反应器产气与有机固体去除效率高,稳定性好.在稳定运行阶段,有机负荷为3.0kgVS/（m³·d）,容积产气率平均达到1.69m³/（m³·d）,最高为2.72m³/（m³·d）,平均负荷产气率为0.57m³/（kgVS·d）,有机固体去除率达77.2%;由于内外环相分离作用,外环pH值相比内环高约0.3~0.4,外环挥发性脂肪酸（VFAs）和溶解性化学需氧量（sCOD）平均浓度相比内环分别降低25%和23%.双环嵌套式反应器实现了产酸和产甲烷阶段分离的作用,内环产酸相很好地完成了水解和酸化步骤,同时减轻产甲烷相受到酸积累的冲击,对反应器稳定运行起到关键作用.     

Impact of undissociated volatile fatty acids on acidogenesis in a two-phase anaerobic system

This study investigated the degradation and production of volatile fatty acids(VFAs)in the acidogenic phase reactor of a two-phase anaerobic system.20 mmol/L bromoethanesulfonic acid(BESA)was used to inhibit acidogenic methanogens(which were present in the acidogenic phase reactor)from degrading VFAs.The impact of undissociated volatile fatty acids(un VFAs)on"net"VFAs production in the acidogenic phase reactor was then evaluated,with the exclusion of concurrent VFAs degradation."Net"VFAs production from glucose degradation was partially inhibited at high un VFAs concentrations,with 59%,37% and 60% reduction in production rates at 2190 mg chemical oxygen demand(COD)/L undissociated acetic acid(un HAc),2130 mg COD/L undissociated propionic acid(un HPr)and 2280 mg COD/L undissociated n-butyric acid(un HBu),respectively.The profile of VFAs produced further indicated that while an un VFA can primarily affect its own formation,there were also un VFAs that affected the formation of other VFAs.     

结合态磷化氢在厌氧微生物产酸过程中的释放行为

通过动态实验分别研究了在产甲烷和酸化阶段厌氧污泥中结合态磷化氢的变化,并采用静态厌氧实验进一步阐明在酸化阶段有机酸的积累对磷化氢的影响及消失速率.结果表明,产甲烷阶段,厌氧颗粒污泥磷化氢浓度随高度升高呈下降趋势;酸化阶段,当pH达到4～5时会导致厌氧反应器颗粒污泥中结合态磷化氢消失.静态模拟实验研究表明,随着酸化程度的增加,厌氧污泥中结合态磷化氢消失速率加快.厌氧污泥在葡萄糖浓度2000mg·L^-1培养2d后,结合态磷化氢浓度从最初的1.76ng·kg^-1降低到0.09ng·kg^-1,磷化氢消失速率最高达到0.84ng·(kg·d)^-1;而葡萄糖浓度400mg·L^-1培养2d后,结合态磷化氢消失速率仅为0.27ng·(kg·d)^-1;随着进一步培养,当磷化氢浓度降低到一定程度后,消失速率也相应下降.     

两相厌氧处理有机废水快速启动试验研究

针对试验设计的两相厌氧反应器(TPAD)进行启动研究,寻求快速启动的方法. 试验采用人工配水,以淀粉、葡萄糖作为主要碳源. 采用低负荷启动方式,快速提高进水ρ(COD_Cr),缩短水力停留时间(HRT),使产酸相尽快维持在酸性最佳条件下. 间歇投加粉末状CaO调节产酸相出水pH在7.0±0.3,从而保证产甲烷相在最佳条件下. 经过36 d的启动过程,在产酸反应器和产甲烷反应器中均出现了性能良好的厌氧颗粒污泥. 启动后第36天,当两相厌氧反应器的水力停留时间(HRT)为17.03 h,COD_Cr负荷(VLR)为8.46 kg/(m3·d),水力负荷为0.059 m3/(m2·h)时,系统整体COD_Cr去除率达到最优,为96.66%.      

热水解时间对污泥厌氧消化系统微生物群落结构影响分析

为评估热水解时间对北京市大兴区某污水处理厂污泥厌氧消化系统微生物群落结构的影响,利用Illumina MiSeq高通量测序方法,分析了不同热水解时间（15、30和45 min）对初沉污泥和剩余活性污泥厌氧消化系统中微生物群落结构及其多样性的影响因素.结果表明,消化污泥优势类群主要分布在厚壁菌门（Firmicutes）、阴沟单胞菌门（Cloacimonadota）、绿弯菌门（Chloroflexi）和同力菌门（Synergistota）,相对丰度之和超过60%,相对丰度最高菌属为W5,占比为20.8%~54.5%,表现为少数优势物种的高丰度特征.热水解污泥厌氧消化过程中,高挥发性脂肪酸和氨氮浓度导致嗜乙酸产甲烷菌相对丰度减少,嗜氢产甲烷途径多于嗜乙酸产甲烷途径.环境因子关联分析结果显示,消化进泥可溶蛋白质、消化进泥pH值、消化出泥氨氮和热水解时间是影响微生物群落结构的4个主要环境因子,其中消化出泥氨氮对产甲烷菌属的影响最大,呈负相关关系.热水解时间与Chao指数和Shannon指数均呈负相关,较长热水解时间不利于提高厌氧消化过程微生物菌群丰富度和多样性.     

A comparative study on the alternating mesophilic and thermophilic two-stage anaerobic digestion of food waste

An alternating mesophilic and thermophilic two stage anaerobic digestion(AD) process was conducted. The temperature of the acidogenic(A) and methanogenic(M) reactors was controlled as follows: System 1(S1) mesophilic A-mesophilic M;(S2) mesophilic A-thermophilic M; and(S3)thermophilic A-mesophilic M. Initially, the AD reactor was acclimatized and inoculated with digester sludge. Food waste was added with the soluble chemical oxygen demand(SCOD) concentrations of41.4–47.0 g/L and volatile fatty acids of 2.0–3.2 g/L. Based on the results, the highest total chemical oxygen demand removal(86.6%) was recorded in S2 while S3 exhibited the highest SCOD removal(96.6%). Comparing S1 with S2, total solids removal increased by 0.5%; S3 on the other hand decreased by 0.1 % as compared to S1. However, volatile solids(VS) removal in S1, S2, and S3was 78.5%, 81.7%, and 79.2%, respectively. S2 also exhibited the highest CH4 content, yield, and production rate of 70.7%, 0.44 L CH4/g VSadded, and 1.23 L CH4/(L·day), respectively. Bacterial community structure revealed that the richness, diversity, evenness, and dominance of S2 were high except for the archaeal community. The terminal restriction fragments dendrogram also revealed that the microbial community of the acidogenic and methanogenic reactors in S2 was distinct. Therefore,S2 was the best among the systems for the operation of two-stage AD of food waste in terms of CH4production, nutrient removal, and microbial community structure.     

Anaerobic digestion of kitchen wastes in a single-phased anaerobic sequencing batch reactor(ASBR) with gas-phased absorb of CO2

IntroductionThereisanever increasingquantityofkitchenwasteswiththeincreasingpopulationandlivingconditionsinChina .Kitchenwasteshaveahighproportionof 5 0 %— 70 %amongmunicipalsolidwastes.TakingthecityofShanghaiforexample,thegenerationofkitchenwastesamount…     

Waste activated sludge treatment based on temperature staged and biologically phased anaerobic digestion system

The concept of temperature staged and biological phased （TSBP） was proposed to enhance the performance of waste-activated sludge anaerobic digestion. Semi-continuous experiments were used to investigate the effect of temperature （35 to 70℃） as well as the hydraulic retention time （HRT） （2, 4 and 6 days） on the acidogenic phase. The results showed that the solubilization degree of waste- activated sludge increased from 14.7% to 30.1% with temperature increasing from 35 to 70℃, while the acidification degree was highest at 45℃ （17.6%）, and this was quite different from the temperature impact on hydrolysis. Compared with HRT of 2 and 6 days, 4 days was chosen as the appropriate HRT because of its relatively high solubilization degree （24.6%） and acidification degree （20.1%） at 45℃. The TSBP system combined the acidogenic reactor （45℃, 4 days） with the methanogenic reactor （35℃, 16 days） and the results showed 84.8% and 11.4% higher methane yield and volatile solid reduction, respectively, compared with that of the single-stage anaerobic digestion system with HRT of 20 days at 35℃. Moreover, different microbial morphologies were observed in the acidogenic- and methanogenic-phase reactors, which resulted from the temperature control and HRT adjustment. All the above results indicated that 45℃ was the optimum temperature to inhibit the activity of methanogenic bacteria in the acidogenic phase, and temperature staging and phase separation was thus accomplished. The advantages of the TSBP process were also confirmed by a full-scale waste-activated sludge anaerobic digestion project which was an energy self-sufficient system.     

Eubacteria and Archaea community of simultaneous methanogenesis and denitrification granular sludge

Based on the successful performance of a lab-scale upflow anaerobic sludge blanket (UASB) reactor with the capacity of simultaneous methanogenesis and denitrification (SMD), the specific phylogenetic groups and community structure of microbes in the SMD granule in the UASB reactor were investigated by the construction of the Eubacteria and Archaea 16S rDNA clone libraries, fragment length polymorphism, and sequence blast. Real time quantitative-polymerase chain reaction (RTQ-PCR) technique was used to quantify the contents of Eubacteria and Archaea in the SMD granule. The contents of some special predominant methanogens were also investigated. The results indicated that the Methanosaeta and Methanobacteria were the predominant methanogens in all Archaea in the SMD granule, with contents of 71. 59% and 22. 73% in all 88 random Archaea clones, respectively. The diversity of Eubacteria was much more complex than that of Archaea. The low GC positive gram bacteria and Б-Protebacteria were the main predominant Eubacteria species in SMD granule, their contents were 49. 62% and 12. 03% in all 133 random Eubacteria clones respectively. The results of RTQ-PCR indicated that the content of Archaea was less than Eubacteria, the Archaea content in total microorganisms in SMD granule was about 27. 6%.     

NZVI对低有机质污泥厌氧消化系统及微生物群落的影响

采用投加不同剂量纳米零价铁（NZVI）（0,0.1,0.3,0.5 g/g VS）的低有机质污泥进行半连续中温厌氧消化试验,研究了反应器在不同NZVI投加量下的产甲烷特性及微生物群落变化。研究表明:NZVI的投加前期对产甲烷具有促进作用,随着反应器中NZVI累积逐渐对产甲烷产生抑制,且投加量越高抑制效果产生越早,抑制作用也越强。投加NZVI反应器未造成酸化或氨抑制,厌氧消化系统稳定性较好,系统上清液中磷酸盐去除率最高达到97.78%。NZVI的投加可以促进Firmicutes、Proteobacteria等水解产酸菌和Methanosarcina等多数氢营养型产甲烷菌的生长,而对严格的乙酸型产甲烷菌Methanosaeta生长有一定抑制作用。     

游离氨对热水解联合中温厌氧消化处理剩余污泥的影响

热水解预处理强化了污泥厌氧消化过程,但同时增加了微生物受游离氨抑制的风险.对比传统工艺,在有机容积负荷率1.74~4.27gVS/(L·d)条件下,研究了游离氨(FA)对热水解联合中温厌氧消化工艺性能的影响.结果表明,组合工艺甲烷产量较传统反应器提高89%~121%,有机去除率提高1.21~1.46倍.但同时导致系统中游离氨浓度达89~382mgN/L,显著高于传统工艺中37~84mgN/L的范围.游离氨毒性测试显示,传统工艺中FA浓度从43mgN/L升至84mgN/L,没有抑制解乙酸产甲烷途径,而组合工艺中FA升至264mgN/L时,表现出抑制作用,尽管厌氧菌对高浓度FA表现更强的驯化趋势.结合氮负荷试验, FA浓度与基质利用能力,解乙酸产甲烷途径抑制程度和挥发性有机酸积累的关系表明,高有机负荷条件下组合工艺效率可通过控制氮负荷优化.     

两相厌氧系统中产甲烷相有机酸转化规律

研究了两相厌氧系统产甲烷相中有机酸转化规律.以产酸相、产甲烷相COD负荷分别为41.5 kg/(m³·d)、6.05kg/(m³·d)的连续流试验装置培养种泥,运行稳定后,进行静态试验采用气相色谱仪(GC-122型)分析有机酸组成及含量结果表明,反应器相同位置的微生物降解转化有机酸速率快慢依次为:乙酸＞乙醇＞丁酸＞丙酸;利用乙醇产乙酸的微生物与产甲烷菌所需最佳pH值范围相近;乙醇型发酵是充分发挥两相厌氧系统功能的最佳发酵类型;在产甲烷反应器中,大部分挥发酸的最终降解都要经历乙酸降解途径才能完成,造成乙酸的降解转化相对缓慢,成为厌氧工艺中的限速步骤.     

Response of anaerobes to methyl fluoride, 2-bromoethanesulfonate and hydrogen during acetate degradation

To use the selective inhibition method for quantitative analysis of acetate metabolism in methanogenic systems,the responses of microbial communities and metabolic activities,which were involved in anaerobic degradation of acetate,to the addition of methyl fluoride(CH3F),2-bromoethanesulfonate(BES)and hydrogen were investigated in a thermophilic batch experiment.Both the methanogenic inhibitors,i.e.,CH3F and BES,showed their effectiveness on inhibiting CH4 production,whereas acetate metabolism other than acetoclastic methanogenesis was stimulated by BES,as reflected by the fluctuated acetate concentration.Syntrophic acetate oxidation was thermodynamically blocked by hydrogen(H2),while H2-utilizing reactions as hydrogenotrophic methanogenesis and homoacetogenesis were correspondingly promoted.Results of PCR-DGGE fingerprinting showed that,CH3F did not influence the microbial populations significantly.However,the BES and hydrogen notably altered the bacterial community structures and increased the diversity.BES gradually changed the methanogenic community structure by affecting the existence of different populations to different levels,whilst H2 greatly changed the abundance of different methanogenic populations,and induced growth of new species.     

高钙废水颗粒污泥中古菌菌群结构变化的分析

为了解高浓度Ca~(2+)与颗粒污泥中古菌菌群结构的关系,利用高通量测序分析IC(Internal Circulation)反应器(以废纸造纸废水为处理对象)内不同高度处颗粒污泥中古菌菌群组成,并利用SEM(Scanning Electron Microscope)和EDS(Energy Dispersive X-ray Spectroscopy)分析颗粒污泥的形态结构和元素组成,同时借助红外光谱和XRD(X-ray diffraction)分析不同高度处颗粒污泥化学结构中各官能团的分布情况和无机成分.结果表明,随着反应器高度的增加,颗粒污泥中的无机成分CaCO_3也随着增加,而种泥中钙含量只有1.81%.种泥和ICR反应器内的颗粒污泥中古菌在门分类水平上主要包括Euryarchaeota和Bathyarchaeota,两者之和占90%以上.在属分类水平上,主要包括Methanosaeta、Methanoregula、Methanobacterium、Methanosarcina、Methanolinea、Methanospirillum等.种泥中古菌的物种多样性要明显高于IC反应器中,在IC反应器中不同高度处古菌生物多样性由高到低的顺序是5 m2 m7 m,5 m处的古菌物种更加丰富.随着反应器高度的增加,乙酸营养型甲烷菌的相对丰度呈现递增趋势,氢营养型甲烷菌则呈现递减趋势,而种泥中氢营养型甲烷菌相对丰度较大.这表明颗粒污泥在高钙废水的长期作用下,氢营养型甲烷菌的相对丰度减少,乙酸营养型甲烷菌的相对丰度增多,系统中古菌物种的相对丰度和多样性有所下降.CaCO_3沉淀使颗粒污泥的产甲烷活性降低,主要表现为其对氢营养型甲烷菌具有较大的抑制作用.     

pH、ORP制约的产酸相发酵类型及顶级群落

由于利用效果存在一定差异，不同产酸相发酵类型的末端产物作为产甲烷细菌的底物，影响整个二相厌氧生物处理系统的稳定、高效运行。pH、ORP作为产酸相系统中的非生物因子，在一定程度上影响产酸相发酵类型的形成。本实验采用连续流搅拌槽式厌氧反应器（CSTR）考察并分析了特定pH、ORP组成对产酸相发酵类型决定情况，同时以种群生态学理论为基础，确立了受pH、ORP制约的不同发酵类型的顶级群落及优势种群。     

利用FISH和DGGE对产甲烷颗粒污泥中微生物种群的研究

利用FISH和DGGE技术对厌氧反应器内处于不同运行阶段的4个产甲烷颗粒污泥进行研究,考察其中真细菌和古细菌的种群结构,并对其中的优势古细菌进行系统发育分析.FISH结果表明,颗粒污泥中真细菌含量明显高于古细菌,真细菌主要分布在颗粒污泥外层,古细菌则主要分布在内层;DGGE结果表明,随着反应器COD负荷的增加以及运行时间的延长,真细菌种群结构相对较稳定,而古细菌种群结构则发生了较明显变化,其中占优势的古细菌种类逐渐减少;将有代表性的7个古细菌条带切胶回收并测序,结果显示,反应器运行后期占优势的菌种主要包括甲烷微粒菌(Methanocorpusculum)、甲烷杆菌(Methanobacterium)和甲烷髦毛菌(Methanosaeta)等.     

微生物群落对氨胁迫响应的宏基因组学研究

为探析厌氧消化微生物群落对氨胁迫的响应,在串联批次实验中引入氨氮（TAN）胁迫,结合宏基因组学分析,研究了不同TAN浓度下,厌氧消化系统的过程参数、群落结构/组成及其功能基因/代谢途径的响应.结果显示,TAN为3000mg/L时,系统性能逐步优化;TAN为6000mg/L时,甲烷产率逐步下降,并伴随着丙酸、丁酸积累.从群落组成上看,两个氨氮梯度下,抗性和冗余性保障了水解酸化菌代谢底物的能力.产氢产乙酸和产甲烷方面,低氨氮组两类功能菌群丰度持续增加,但高氨氮组增加幅度低甚至出现丰度下降,指示该环节可能受到了氨胁迫.进一步分析两阶段的功能基因变化,发现产甲烷和互营乙酸氧化途径在氨胁迫下表现出抗性和冗余性,但高浓度TAN （6000mg/L）抑制了丁酸和丙酸代谢途径中关键基因ACADS和PCCA的表达.可见,氨抑制失稳的关键环节是产氢产乙酸阶段.