微生物醌指纹法在环境微生物群体组成研究中的应用

微生物醌是能量代谢过程的电子传递体,不同的微生物含有不同种类和分子结构的醌。因此,环境样品中微生物醌的组成,即醌指纹可在一定程度上反映微生物的群体结构。本简要介绍了微生物醌的分析方法,并利用醌指纹对活性污泥中的微生物群体组成进行了解析。     

Application of modified supercritical carbon dioxide extraction to microbial quinone analysis

Supercritical carbon dioxide (scCO₂) was applied to extract microbial quinones from activated sludge. Identification and analysis was then performed using high-performance liquid chromatography (HPLC) equipped with ultraviolet–visible (UV–Vis) detector and photodiode array detector (PDA). Extracted microbial quinones were trapped and separated as menaquinones (MK) and ubiquinones (Q) species using two Sep-Pak Plus Silica cartridges joined in series. Four ubiquinones and 12 menaquinones species were identified in 0.1 g dried activated sludge based on retention time and spectrum analysis. Among the tested various polar solvents, methanol showed to be the best modifier, based on the highest total quinone content extracted and the lowest dissimilarity index. The diversity index of quinone and the number of quinone species using methanol-modified scCO₂ were similar to that of the conventional method (organic solvent extraction).     

Analysis of the differences in microbial community structures between suspended and sessile microorganisms in rivers based on quinone profile

In this study, a quinone profiling method was applied to clarify the differences in community structure between suspended and sessile microorganisms in rivers. The compositions of microbial quinone of 6 sites for 4 rivers were analyzed. Ubiquinone (UQ)-8, UQ-10, menaquinone (MK)-7, and plastoquinone (PQ)-9 were observed in all samples of suspended and sessile microorganisms for the sites investigated. The dominant quinone species in suspended microorganisms was ubiquinone, and that in sessile microorganism was photosynthetic quinones (namely PQ-9 and vitamin K1). This indicated that aerobic bacteria were abundant in the suspended microorganisms, and photosynthetic microorganisms such as micro-algae and cyanobacteria dominated in the sessile microorganisms. The quinone concentration in the river waters tested, which reflects the concentration of suspended microorganisms, ranged from 0.045 to 1.813 nmol/L. The microbial diversities of suspended and sessile microorganisms calculated based on the composition of all quinones were in the range from 3.4 to 7.5, which was lower than those for activated sludge and soils. Moreover, the diversity of heterotrophic bacteria for sessile microorganisms in the rivers was higher than that for the suspended microorganisms.     

Quinone profiling of bacterial communities in natural and synthetic sewage activated sludge for enhanced phosphate removal

Respiratory quinones were used as biomarkers to study bacterial community structures in activated sludge reactors used for enhanced biological phosphate removal (EBPR). We compared the quinone profiles of EBPR sludges and standard sludges, of natural sewage and synthetic sewage, and of plant scale and laboratory scale systems. Ubiquinone (Q) and menaquinone (MK) components were detected in all sludges tested at molar MK/Q ratios of 0.455 to 0.981. The differences in MK/Q ratios were much larger when we compared different wastewater sludges (i.e., raw sewage and synthetic sewage) than when we compared sludges from the EBPR and standard processes or plant scale and laboratory scale systems. In all sludges tested a Q with eight isoprene units (Q-8) was the most abundant quinone. In the MK fraction, either tetrahydrogenated MK-8 or MK-7 was the predominant type, and there was also a significant proportion of MK-6 to MK-8 in most cases. A numerical cluster analysis of the profiles showed that the sludges tested fell into two major clusters; one included all raw sewage sludges, and the other consisted of all synthetic sewage sludges, independent of the operational mode and scale of the reactors and the phosphate accumulation. These data suggested that Q-8-containing species belonging to the class Proteobacteria (i.e., species belonging to the beta subclass) were the major constituents of the bacterial populations in the EBPR sludge, as well as in standard activated sludge. Members of the class Actinobacteria (gram-positive bacteria with high DNA G+C contents) were the second most abundant group in both types of sludge. The bacterial community structures in activated sludge processes may be affected more by the nature of the influent wastewater than by the introduction of an anaerobic stage into the process or by the scale of the reactors.     

Phospholipid-Derived Fatty Acids and Quinones as Markers for Bacterial Biomass and Community Structure in Marine Sediments

Phospholipid-derived fatty acids (PLFA) and respiratory quinones (RQ) are microbial compounds that have been utilized as biomarkers to quantify bacterial biomass and to characterize microbial community structure in sediments, waters, and soils. While PLFAs have been widely used as quantitative bacterial biomarkers in marine sediments, applications of quinone analysis in marine sediments are very limited. In this study, we investigated the relation between both groups of bacterial biomarkers in a broad range of marine sediments from the intertidal zone to the deep sea. We found a good log-log correlation between concentrations of bacterial PLFA and RQ over several orders of magnitude. This relationship is probably due to metabolic variation in quinone concentrations in bacterial cells in different environments, whereas PLFA concentrations are relatively stable under different conditions. We also found a good agreement in the community structure classifications based on the bacterial PLFAs and RQs. These results strengthen the application of both compounds as quantitative bacterial biomarkers. Moreover, the bacterial PLFA- and RQ profiles revealed a comparable dissimilarity pattern of the sampled sediments, but with a higher level of dissimilarity for the RQs. This means that the quinone method has a higher resolution for resolving differences in bacterial community composition. Combining PLFA and quinone analysis as a complementary method is a good strategy to yield higher resolving power in bacterial community structure.     

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Characterization of the bacterial population structure in an anaerobic-aerobic activated sludge system on the basis of respiratory quinone profiles.

Bacterial respiratory quinones were used as biomarkers for studying the bacterial population structure, especially the content of Acinetobacter species, in a laboratory-scale anaerobic-aerobic activated sludge system and in the standard aerobic system. All tested sludges contained both ubiquinone and menaquinone, with a molar ratio of about 1:0.5. High-performance liquid chromatography showed that ubiquinone with eight isoprene units (Q-8) was present as the predominant ubiquinone, Q-10 was the second most common type, and Q-9 and other homologs were minor components in the anaerobic-aerobic sludge and the standard aerobic sludge. Bacteriological examination indicated that, in both sludge systems, Q-8-containing bacteria constituted a large proportion of the aerobic heterotrophic bacterial flora, but only a few strains with Q-9 were found. These findings demonstrate that the population of Acinetobacter species, which contain Q-9 as the major quinone, is negligible in those environments. The present results suggest that the introduction of anaerobic conditions into the aerobic batch process has little influence on the bacterial community structure.     

Effects of UV pretreatment on microbial community structure and metabolic characteristics in a subsequent biofilter treating gaseous chlorobenzene

To provide insight into effects of UV pretreatment on microorganisms in subsequent biofilters, the changes of microbial community structure and metabolic characteristics of biofilters with (UV–BF) and without (BF) UV pretreatment were studied. The respiratory quinone and BIOLOG methods were used to analyze microbial community structure and metabolic characteristics, respectively. The results indicated the quinone profiles, the species of dominant quinone and its molar fraction of the biofilm in both biofilters showed different behaviors. Ubiquinones-8 and menaquinone-9(H₂) was the dominant quinones in BF and UV–BF processes, respectively. The dissimilarity index of two biofilters markedly increased to nearly 60 after turning on the UV lamp. The microbial samples from UV–BF process showed higher metabolic activities of 0.040 cm⁻¹ h⁻¹ than 0.028 cm⁻¹ h⁻¹ in BF process. Moreover, the microorganisms in both biofilters demonstrated distinct metabolic characteristics. Further, the performance of biofilters showed good correlation with microbial community structure and metabolic characteristics.     

Changes in Quinone Profiles of Hot Spring Microbial Mats with a Thermal Gradient

The respiratory and photosynthetic quinones of microbial mats which occurred in Japanese sulfide-containing neutral-pH hot springs at different temperatures were analyzed by spectrochromatography and mass spectrometry. All of the microbial mats that developed at high temperatures (temperatures above 68°C) were so-called sulfur-turf bacterial mats and produced methionaquinones (MTKs) as the major quinones. A 78°C hot spring sediment had a similar quinone profile. Chloroflexus-mixed mats occurred at temperatures of 61 to 65°C and contained menaquinone 10 (MK-10) as the major component together with significant amounts of either MTKs or plastoquinone 9 (PQ-9). The sunlight-exposed biomats growing at temperatures of 45 to 56°C were all cyanobacterial mats, in which the photosynthetic quinones (PQ-9 and phylloquinone) predominated and MK-10 was the next most abundant component in most cases. Ubiquinones (UQs) were not found or were detected in only small amounts in the biomats growing at temperatures of 50°C and above, whereas the majority of the quinones of a purple photosynthetic mat growing at 34°C were UQs. A numerical analysis of the quinone profiles was performed by using the following three parameters: dissimilarity index (D), microbial divergence index (MD_q), and bioenergetic divergence index (BD_q). A D matrix tree analysis showed that the hot spring mats consisting of the sulfur-turf bacteria, Chloroflexus spp., cyanobacteria, and purple phototrophic bacteria formed distinct clusters. Analyses of MD_q and BD_q values indicated that the microbial diversity of hot spring mats decreased as the temperature of the environment increased. The changes in quinone profiles and physiological types of microbial mats in hot springs with thermal gradients are discussed from evolutionary viewpoints.     

rRNA-targeted寡核苷酸探针识别活性污泥微生物群落结构与功能研究进展

活性污泥中微生物群落内部关系非常复杂 ,及时对活性污泥中优势菌群和群落内部关系进行监测是污水处理中采取正确措施的关键。历史研究表明传统培养方法经常导致活性污泥优势菌群检测的失败 ,而r RNA- targeted寡核苷酸探针作为一种快速原位监测活性污泥微生物群落结构和功能的新工具被引入 ,使我们对参与污水净化的微生物群落结构和优势菌群能有较全面的了解。就该方法在识别除磷污泥、脱氮污泥、污泥泡沫和膨胀污泥中微生物群落结构和功能的典型应用进行综述 ,分析了该方法存在的优点和缺点 ,并对目前已建立且应用于活性污泥微生物检测的 r RNA- targeted寡核苷酸探针进行了详细总结     

Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.     

Intact Phospholipid and Quinone Biomarkers to Assess Microbial Diversity and Redox State in Microbial Mats

Microbial mats are stratified microbial communities composed by highly inter-related populations and therefore are frequently chosen as model systems to study diversity and ecophysiological strategies. The present study describes an integrated approach to analyze microbial quinones and intact polar lipids (IPLs) in microbial mats within layers as thin as 500 μm by liquid chromatography–tandem mass spectrometry. Quinone profiles revealed important depth-related differences in community composition in two mat systems. The higher abundance of ubiquinones, compared to menaquinones, reflected the clear predominance of microorganisms belonging to aerobic α-, β-, and γ-Proteobacteria in Ebro delta estuarine mats. Hypersaline photosynthetic Camargue mats (France) showed a predominance of menaquinone-9 at the top of the mat, which is consistent with an important contribution of facultative aerobic or anaerobic bacteria in its photic zone. Quinone indices also indicated a higher diversity of non-phototrophs and a more anaerobic character in the hypersaline mats. Besides, the dissimilarity index suggested that the samples were greatly influenced by a depth-related redox state gradient. In the analysis of IPLs, there was a predominance of phosphatidylglycerols and sulfoquinovosyldiacylglycerols, the latter being an abundant biomarker of Cyanobacteria. This combined approach based on quinone and IPL analysis has proven to be a useful method to establish differences in the microbial diversity and redox state of highly structure microbial mat systems at a fine-scale level.     

Analysis of microbial community structure in a biofilm on membrane surface in the submerged membrane bioreactor treating domestic wastewater on the basis of respiratory quinone profiles

The objective of this study was to investigate the microbial community structure of the biofouling film formed on hollow-fiber membrane surfaces in the submerged membrane bioreactor (SMBR) with a nitrification-denitrification process. In this experiment, aeration was conducted intermittently (60 min off, 90 min on) cyclic anoxic and oxic conditions in the SMBR. The dominant quinone types of biofilm on the membrane surface in an intermittently aerated SMBR were ubiquinone (UQs)-8, -10, followed by menaquinones (MKs)-8(H4), -8(H2) and -7, but those of suspended microorganisms were UQ-8, UQ-10 followed by MKs-8, -9(H4) and -6. The change in quinone profiles of biofilm on the membrane surface suggested that UQ-9, MK-7, MK-8(H2) and MK-8(H4) contributed to microbiological fouling in the intermittently aerated SMBR treating domestic wastewater. The microbial diversities of suspended microorganisms and biofilm, calculated based on the composition of all quinones, were 9.5 and 10.9, respectively.     

Characterization of quinone tautomerase activity in the hemolymph of Sarcophaga bullata larvae

The hemolymph of Sarcophaga bullata larvae was activated with either zymosan or proteolytic enzymes such as chymotrypsin or subtilisin and assayed for phenoloxidase activity by two different assays. While oxygen uptake studies readily attested to the wide specificty of activated phenoloxidase, visible spectral studies failed to confirm the accumulation of quinone products in the case of 4-alkyl substituted catechols such as N-acetyldopamine and N-β-alanyldopamine. Sepharose 6B column chromatography of the activated hemolymph resolved phenoloxidase activity into two fractions, designated as A and B. Peak A possessed typical o-diphenoloxidase (o-diphenol, oxygen oxidoreductase EC 1.10.3.1) activity, while peak B oxidized physiologically important catecholamine derivatives such as N-acetyldopamine, N-acetylnorepinephrine, and N-β-alanyldopamine into N-acetylnorepinephrine, N-acetylarterenone, and N-β-alanylnorepinephrine, respectively, and converted 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol into 3,4-dihydroxymandelic acid, 3,4-dihydroxybenzaldehyde, and 2-hydroxy-3′,4′-dihydroxyacetophenone, respectively. These transformations are consistent with the conversion of phenoloxidase-generated quinones to quinone methides and subsequent non-enzymatic transformations of quinone methides. Accordingly, Peak B contained both o-diphenoloxidase activity and quinone tautomerase activity. Sepharose 6B column chromatography of unactivated hemolymph resulted in the separation of quinone tautomerase from prophenoloxidase. The tautomerase rapidly converted both chemically made and mushroom tyrosinase-generated quinones to quinone methides. Thus the failure to observe the accumulation of quinones with N-acyl derivatives of dopamine and related compounds in the whole hemolymph is due to the rapid conversion of these long lived toxic quinones to short lived quinone methides. The latter, being unstable, undergo rapid non-enzymatic transformations to form side-chain-oxygenated products that are non-toxic. The possible roles of quinone isomerase and its reaction products—quinone methides—as essential components of sclerotization of cuticle and defense reaction of Sarcophaga bullata are discussed.     

厌氧消化污泥微生物组研究方法及应用

污泥厌氧消化是在消化污泥微生物组的协调下将剩余污泥中有机物转化为甲烷的微生物过程。与传统厌氧消化过程不同,污泥厌氧消化系统的进料底物为含有大量微生物细胞及胞外多聚物等复杂大分子有机物的剩余污泥。因此,厌氧消化污泥微生物组的种群组成、功能及种群间互作关系等异常复杂,使厌氧消化污泥微生物组分析成为难点问题。但近年来高通量测序技术及生物信息学分析方法的快速发展为消化污泥微生物组研究提供了契机,并迅速推动了该研究领域的发展。本文从4个方面梳理、总结厌氧消化污泥微生物组的研究及应用现状:剩余活性污泥结构、组成及其厌氧消化;基于16SrRNA基因序列测序的微生物组研究;基于宏基因组及宏转录组分析的微生物组研究;厌氧消化污泥微生物组研究案例分析。最后我们提出了厌氧消化污泥微生物组研究亟待解决的关键科学问题。     

Development of ecotoxicity assay based on inhibition of respiring activity in microbial community using XTT reduction

The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC(50)) value of 3,5-dichlorophenol (3,5-DCP). The EC(50) value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

Phylogenetic Specificity and Reproducibility and New Method for Analysis of Terminal Restriction Fragment Profiles of 16S rRNA Genes from Bacterial Communities

Terminal restriction fragment (TRF) analysis of 16S rRNA genes is an increasingly popular method for rapid comparison of microbial communities, but analysis of the data is still in a developmental stage. We assessed the phylogenetic resolution and reproducibility of TRF profiles in order to evaluate the limitations of the method, and we developed an essential analysis technique to improve the interpretation of TRF data. The theoretical phylogenetic resolution of TRF profiles was determined based on the specificity of TRFs predicted from 3,908 16S rRNA gene sequences. With sequences from the Proteobacteria or gram-positive division, as much as 73% of the TRFs were phylogenetically specific (representing strains from at most two genera). However, the fraction decreased when sequences from the two divisions were combined. The data show that phylogenetic inference will be most effective if TRF profiles represent only a single bacterial division or smaller group. The analytical precision of the TRF method was assessed by comparing nine replicate profiles of a single soil DNA sample. Despite meticulous care in producing the replicates, numerous small, irreproducible peaks were observed. As many as 85% of the 169 distinct TRFs found among the profiles were irreproducible (i.e., not present in all nine replicates). Substantial variation also occurred in the height of synonymous peaks. To make comparisons of microbial communities more reliable, we developed an analytical procedure that reduces variation and extracts a reproducible subset of data from replicate TRF profiles. The procedure can also be used with other DNA fingerprinting techniques for microbial communities or microbial genomes.     

Changes in respiratory quinone profiles of enhanced biological phosphorus removal activated sludge under different influent phosphorus/carbon ratio conditions

Changes in the microbial community of an enhanced biological phosphorus removal (EBPR) activated sludge system under different influent phosphorus/carbon (P/C) ratio conditions were investigated through evaluation of population respiratory quinone profiles. A total of 13 types of respiratory quinone homologs consisting of 3 types of ubiquinones (UQ) and 10 types of menaquinones (MK) were identified in this study. The dominant quinones were UQ-8 and MK-7 throughout the operational period. A higher P/C ratio (0.1) in the influent stimulated an increase in the mole fractions of UQ-8, MK-7, MK-8(H₄), MK-9(H₄) and MK-8(H₈), suggesting that actinobacterial polyphosphate-accumulating organisms (PAO) containing partially hydrogenated MK, mainly MK-8(H₄), were contributing to EBPR. However, when the P/C ratio gradually decreased from 0.1 to 0.01, the mole fractions of UQ-8 increased from 0.46 to 0.58, while MK-7, MK-8(H₂), MK-8(H₄), MK-9(H₄), MK-8(H₈) and MK-9(H₆) markedly decreased. These changes in the respiratory quinone profiles suggest that glycogen-accumulating organisms corresponding to some Gammaproteobacteria had become dominant populations with a decrease in actinobacterial PAO. On the other hand, increasing abruptly the P/C ratio to 0.1 further caused an increase in the mole fraction of UQ-8, indicating that Rhodocyclus-related organisms were important PAO.