Quinone Profiles Research Articles

Changes in Microbial Community Structure in Soil Percolated with Pentachlorophenol with Reference to Respiratory Quinone Profile

農薬によりもたらされる微生物群集構造の変化を追跡する方法として呼吸鎖キノンのプロファイル分析が有効な方法であることを, ペンタクロロフェノール (PCP) 環流土壌をモデルに用いて示した. 20mg/lのPCP溶液の環流によって, 土壌微生物バイオマス量の指標である全キノン量は増加し, 200mg/lのPCP溶液の環流では減少した. キノン組成では, ユビキノン-10 (H2) (10個のイソプレノイド側鎖に2水素が付加されたユビキノン) が20mg/lのPCP溶液の環流で消失し, 糸状菌の減少を示した. 高濃度PCP溶液 (200mg/l) の環流では, 微生物群集構造がより大きく変化した. すなわち, メナキノン-7 (メナキノンに7個のイソプレノイド側鎖がついたもの) が消失し, ユビキノン-9が出現した. メナキノン-7は, Bacillus のような低GC含量のグラム陽性菌および Cytophaga-Flavobacterium 群に属するグラム陰性菌を示し, またユビキノン-9は Pseudomonas 等のグラム陰性菌を示している. 20mg/lのPCP溶液で29日間培養して経時変化を見ると, ユビキノン種が増加し, メナキノン種が減少した. これは, 希釈平板法でクリスタルバイオレット耐性菌 (グラム陰性菌の指標) が増加したこととよく対応した.

Long-term changes in microbial community structure in soils subjected to different fertilizing practices revealed by quinone profile analysis

Quinone profile analysis of stored air-dried soils gave an approximation of the long-term changes in the microbial community structure in four soils subjected to different types of fertilizer application from 1987 to 1997: unfertilized soil (NF-soil), soil amended with chemical fertilizers (CF-soil), soil amended with chemical fertilizers and 40 t ha-1 y-1 of farmyard manure (CF+ FYM-soil), and soil amended with 400 t ha-1 y-1 of farmyard manure (FYM-soil). The carbon content increased, and the soil pH remained higher in the soils receiving farmyard manure. Principal component analysis of the quinone profiles of the soils indicated that the microbial community structure showed a high similarity among the four soils before the onset of cultivation and changed to a different community structure specific to the respective fertilizing practices except for the NF-soil. The specific quinone profile became stable after two cropping seasons in the FYM-soil, after 10 cropping seasons in the CF+ FYM-soil and after 15 cropping seasons in the CF-soil, respectively. The quinone profile of the NF-soil did not become stable, and no specific profile was developed. The specific quinone profiles in the FYM- and CF+ FYM-soils were both characterized by large mole fractions of menaquinone with seven isoprenoid units (MK-7). Farmyard manure itself contained a large mole fraction of MK-7. It was suggested that the amount of MK-7 increased due to the application of farmyard manure. MK-7 indicates the presence of Gram-positive bacteria with low guanine plus cytosine contents such as Bacillus and Gram-negative bacteria of the Cytophaga-Flavobacterium complex. The specific quinone profile in the CF-soil was characterized by the presence of menaquinone with seven isoprenoid units dihydrated (MK-7(H2)and MK-7(H4). Although Brevibacterium and Kocuria contain MK-7(H2), no microorganisms are known to have MK- 7(H.) as major quinone. The common major quinones were MK-8, MK-10(H4) and a mixture of MK-8(H4) and MK-9, suggesting the predominance of Grampositive bacteria in all the soils. Mole fractions of Ubiquinone with 8 isoprenoid units (Q-8) and Q-10 increased at various times in all the soils, indicating the sporadic growth of Gram-negative bacteria.

Effects of biodegradable substrates and microbial concentration on the acclimation of microbes to acrylonitrile in aerobic submerged biofilter

The effects of coexistence-of biodegradable substrates and microbial concentration on the acclimation of microbes to acrylonitrile (AN) in an aerobic submerged biofilter were investigated on the basis of kinetic and ecological studies. The experimental results show that the acclimation of microbial film to AN was promoted by a higher microbial concentration in the biofilter and by the coexistence of glucose and peptone in the influent. It was clarified that the upper limit of AN loading to the biofilter for the ultimate degradation, i.e., complete mineralization, of the influent AN was about 2.0-2.2 kg/m3. d. In addition, a new microbial quinone profile method was applied for the analysis of the microbial community change in the biofilter. The change in quinone profiles of the microbial film during the acclimation to AN suggested that Brevibacterium sp., Pseudomonas aeruginosa and Corynebacterium sp. could contribute to the degradation of AN in the aerobic biofilter.

Morphology, In-Situ characterization with rRNA targetted probes and respiratory quinone profiles of enhanced biological phosphorus removal sludge

The microbial communities in activated sludge acclimated with either acetate or glucose as the major carbon source under phosphorus limited or rich conditions were investigated morphologically, phylogenetically and chemotaxonomically. The sludge with a minimized polyphosphate content was dominated by tetrad shaped bacteria, which were suspected to be ‘glycogen accumulating bacteria (GAOs) or G bacteria’ The sludge containing high polyphosphate was dominated by cluster forming coccus bacteria. Quinone analyses suggested that all the sludge tested contained various ubiquinones and menaquinones, of which the ubiquinones Q-8 and Q-10 were dominant. Analyses with rRNA targeted probes showed that beta sub class of Proteobacteria was most predominant in all sludges tested. Morphological, phylogenetic and chemotaxonomic investigation all indicated that both high and low P sludges are microbiologically diverse.

Analysis of respiratory quinones in soil for characterization of microbiota

Method for the analysis of respiratory quinones in soil was developed to characterize soil microbiota. The respiratory quinones were extracted with a mixture of chloroform and methanol using a Wahling blender or a sonicator and cleaned-up by a silicagel column cartridge. The quinone species were determined by reverse-phase high performance liquid chromatography. Spectra of peaks were measured with a photodiode array detector to examine the purity. More than 90% of extractable quinones in soil were recovered by three extractions. The recovery of quinones, added to soil as freeze-dried powder of microorganisms, was higher than 96%. This procedure led to higher representative results as follows. Fluctuations within 95% of cumulative frequency were 13% for the extracted amount of quinones and 20% for the dissimilarity, respectively. The quinone profiles of four soils were determined as follows: a soil from the aerobic layer of a paddy field, an ando soil and two yellow upland soils which had received chemical fertilizers with and without farmyard manure. The quinone profiles displayed clear differences in the microbial composition and in the microbial diversity among the soils. It was demonstrated that the analysis of the respiratory quinone profile was useful to characterize the microbial community structure in soil.

Effect of drying treatment on the respiratory quinone profile in soil

Recently, the analysis of the microbial community structure in soil has received a great deal of attention. Various analytical methods based on biomarkers have been developed: 16S-rDNA, total DNA, phospholipid fatty acids, ergosterols, muramic acids, etc. (Tunlid and White 1992; Carter and Lynch 1993). In a previous paper, we reported that respiratory quinones are useful biomarkers to characterize the microbial community structure in soil (Fujie et al. 1998). In these analyses, only fresh moist soils or frozen soils had been used as samples. However, the soil samples are generally stored in the dark after air drying in many research institutes and experimental stations. It was considered that the analysis of microbial communities in dried soil samples was not possible. In this paper, we observed that the drying of soils did not affect the proportions of quinone species in soil although the treatment decreased the amount of extracted quinones. These findings suggest that the analysis of the respiratory quinone profile of dried soils reflects the micro biota present in fresh moist soils before drying.

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.

Analysis of microbial acclimation to refractory chemicals in wastewater using respiratory quinone profiles

The effects of initial microbial concentration and the coexistence of biodegradable substrates on the acclimation of microbes to acrylonitrile (AN) was investigated in an aerobic submerged biofilter using respiratory quinone profiles. The acclimation of microbes to AN was accelerated by increased initial microbial concentrations in the biofilter and by the coexistence of glucose and peptone in the influent. The change in quinone profiles of microbial films during the course of acclimation suggested that Brevibacterium sp., Pseudomonas aeruginosa and Corynebacterium sp. contributed to the degradation of AN in the aerobic biofilter.

Population dynamics of chromate reducing bacteria in a bioreactor system developed for the treatment of chromate wastewater

The population dynamics of microbes was investigated in a bioreactor system for the treatment of chromate wastewater supplemented with chromate reducing bacteria Enterobacter cloacae strain HO-1. In this study, respiratory quinone profile and PCR techniques were used to analyse the microbial population. The change in microbial community structure and density was observed on the basis of the respiratory quinone profile since the predominant quinone of HO-1 is ubiquinone-8 (Q-8) and the amount of quinone is proportional to the cell mass. the quantity of microbes and the Q-8 fraction of the total quinones were increased after cultivation with aeration, while long-term operation brought about a decrease in the fraction of Q-8. In addition, the amount of HO-1 was determined by the PCR amplification rate under conditions where the amplification was exponential to the reaction cycles. The cell density of HO-1 and total microbes determined from these procedures compared well with the numbers of HO-1 cells counted selectively using agar plates containing chromate.

Analysis of microbial acclimation to refractory chemicals in wastewater using respiratory quinone profiles

The effects of initial microbial concentration and the coexistence of biodegradable substrates on the acclimation of microbes to acrylonitrile (AN) was investigated in an aerobic submerged biofilter using respiratory quinone profiles. The acclimation of microbes to AN was accelerated by increased initial microbial concentrations in the biofilter and by the coexistence of glucose and peptone in the influent. The change m quinone profiles of microbial films during the course of acclimation suggested that Brevibacterium sp., Pseiulomonas aeruginosa and Corynebacterium sp. contributed to the degradation of AN in the aerobic biofilter.

Effects of biodegradable substrates and microbial concentration on the acclimation of microbes to acrylonitrile in aerobic submerged biofilter

The effects of coexistence-of biodegradable substrates and microbial concentration on the acclimation of microbes to acrylonitrile (AN) in an aerobic submerged biofilter were investigated on the basis of kinetic and ecological studies. The experimental results show that the acclimation of microbial film to AN was promoted by a higher microbial concentration in the biofilter and by the coexistence of glucose and peptone in the influent. It was clarified that the upper limit of AN loading to the biofilter for the ultimate degradation, i.e., complete mineralization, of the influent AN was about 2.0–2.2 kg/m3. d. In addition, a new microbial quinone profile method was applied for the analysis of the microbial community change in the biofilter. The change in quinone profiles of the microbial film during the acclimation to AN suggested that Brevibacterium sp., Pseudomonas aeruginosa and Corynebacterium sp. could contribute to the degradation of AN in the aerobic biofilter.

A New Approach to Numerical Analyses of Microbial Quinone Profiles in the Environment.

The quinone profiles of microbial biomass samples from wastewater and natural aquatic environments were studied. A soil sample was also analyzed for comparison. Attempts were made to perform numerical analyses on these profiles by using three parameters, the microbial divergence index (MDq), the bioenergetic divergence index (BDq), and the dissimilarity index (D), and the algorithm of the neighbor-joining method was used to make clustering trees based on D value matrix data. For these purposes, a new personal computer program named “BioCLUST” was developed. The neighbor-joining dendrograms were useful for the grouping of different microbial communities based on D value matrix data. Also, the communities could be classified effectively by plotting MDq data against BDq values. The numerical analysis indicated that sewage activated sludges formed a tight cluster independent of the location of plants and scale of reactors. This was also the case for influent raw sewage. Sewage microbial mats and river water were located in a group with the raw sewage cluster as the sister group. The soil and lake showed their respective independent lines. The use of our new program should facilitate microbial community analyses based on quinone patterns.

Population dynamics of chromate reducing bacteria in a bioreactor system developed for the treatment of chromate wastewater

The population dynamics of microbes was investigated in a bioreactor system for the treatment of chromate wastewater supplemented with chromate reducing bacteria Enterobacter cloacae strain HO-1. In this study, respiratory quinone profile and PCR techniques were used to analyse the microbial population. The change in microbial community structure and density was observed on the basis of the respiratory quinone profile since the predominant quinone of HO-1 is ubiquinone-8 (Q-8) and the amount of quinone is proportional to the cell mass the quantity of microbes and the Q-8 fraction of the total quinones were increased after cultivation with aeration, while long-term operation brought about a decrease in the fraction of Q-8. In addition, the amount of HO-1 was determined by the PCR amplification rate under conditions where the amplification was exponential to the reaction cycles. The cell density of HO-1 and total microbes determined from these procedures compared well with the numbers of HO-1 cells counted selectively using agar plates containing chromate.

Morphology, in-situ characterization with rRNA targetted probes and respiratory quinone profiles of enhanced biological phosphorus removal sludge

The microbial communities in activated sludge acclimated with either acetate or glucose as the major carbon source under phosphorus limited or rich conditions were investigated morphologically, phylogenetically and chemotaxonomically. The sludge with a minimized polyphosphate content was dominated by tetrad shaped bacteria, which were suspected to be ‘glycogen accumulating bacteria (GAOs) or G bacteria’ The sludge containing high polyphosphate was dominated by cluster forming coccus bacteria. Quinone analyses suggested that all the sludge tested contained various ubiquinones and menaquinones, of which the ubiquinones Q-8 and Q-10 were dominant. Analyses with rRNA targeted probes showed that beta sub class of Proteobacteria was most predominant in all sludges tested. Morphological, phylogenetic and chemotaxonomic investigation all indicated that both high and low P sludges are microbiologically diverse.

Respiratory quinone profile as a tool for refractory chemical biodegradation study

The respiratory quinone profile was successfully used as a tool for identifying the change of bacterial phase in the microbial film of aerobic submerged biofilter treating refractory chemicals such as dimethylformamide (DMF). It was ascertained that the coexistence of easily biodegradable substances such as glucose and peptone hindered the acclimation of bacteria inhabiting the microbial film in biofilter to DMF. The higher initial microbial concentration and the feeding of DMF as a sole organic substance accelerated the acclimation of microbial film to DMF. Appreciable changes in quinone profile were detected through the acclimation to DMF, and the results showed that DMF degrading bacteria such as Methylobacterium and Mycobacterium had grown in the biofilter.

Restriction Pattern Analysis by High-performance Liquid Chromatography of PCR-amplified 16S rDNA Fragments from Scum-forming Bacteria in Activated Sludge.

High-performance liquid chromatography (HPLC) with an anion-exchange nonporous column was used to analyze restriction fragment length polymorphism (RFLP) of the 16S ribosomal DNA (rDNA) of scum-forming actinomycetes found in three different activated sludge plants. 16S rDNA fragments from washed scum samples were amplified by PCR and cloned into plasmid vectors. The 16S rDNA inserts in the plasmids were reamplified in vitro, cut with restriction endonucleases, and separated directly by HPLC. Compared to conventional agarose electrophoresis, the HPLC system used allowed better separation of fragments up to 500bp. The HPLC-RFLP patterns of the scum clones were compared with those of PCR-amplified 16S rDNAs from authentic strains and with computer-predicted restriction patterns of the available 16S rDNA data on the actinomycetes. 16S rDNA-RFLP comparisons indicated that the predominat scum-forming bacteria were Gordona amarae in two activated sludge plants and Rhodococcus sp. in the remaining one. These findings agreed well with results of lipoquinone profiling of the scum performed concurrently. The results of this study indicated that a combination of 16S rDNA-RFLP analysis and quinone profiling should provide a more powerful non-cultivating tool for the identification of scum-forming bacteria in activated sludge.

キノンプロファイルによる人工廃水活性汚泥および実処理下水汚泥の微生物群集構造の比較解析

We studied microbial population shifts in laboratory-scale synthetic-waste activated sludge from plant-scale sewage sludge on the basis of respiratory quinone profiles. The laboratory sludge reactors were seeded with the sewage sludge and operated on a fill-and-draw basis in a 24 h batch cycle with either raw domestic sewage or each of three different synthetic wastewaters containing acetate, glucose, skim milk, and/or peptone as a primary component. After two months of acclimation, the quinone compositions of the laboratory sludges were analyzed compared with the seed sludge. Numerical analysis of quinone profiles showed that there was no significant difference in population structures between the seed sludge and the laboratory sludge loaded with raw sewage, while significant population shifts occurred in the three synthetic-waste laboratory sludges. These results suggest that laboratory activated sludge loaded with synthetic wastewater can not be regarded as a model of plant-scale sewage sludge in their microbiological aspects.

Respiratory quinone profile as a tool for refractory chemical biodegradation study

The respiratory quinone profile was successfully used as a tool for identifying the change of bacterial phase in the microbial film of aerobic submerged biofilter treating refractory chemicals such as dimethylformamide (DMF). It was ascertained that the coexistence of easily biodegradable substances such as glucose and peptone hindered the acclimation of bacteria inhabiting the microbial film in biofilter to DMF. The higher initial microbial concentration and the feeding of DMF as a sole organic substance accelerated the acclimation of microbial film to DMF. Appreciable changes in quinone profile were detected through the acclimation to DMF, and the results showed that DMF degrading bacteria such as Methylobacterium and Mycobacterium had grown in the biofilter.

Studies of microbial acclimation to hard chemicals on the basis of respiratory quinone profiles and kinetic analyses

The population dynamics of microbes in the biological wastewater treatment processes such as a submerged biofilter was investigated to obtain basic information to determine the optimal operating conditions. The effects of coexistence of biodegradable substances such as glucose and peptone on the acclimation of microbes in the biofilm to hard chemicals such as acrylonitrile (AN), which is poorly biodegradable and a volatile substance, was investigated on the basis of the respiratory quinone profile. Kinetic study of the removal of AN in the course of acclimation of microbes was investigated using a laboratory-scale submerged biofilter as well. It was ascertained that the acclimation of the microbes to AN was accelerated by coexistence of biodegradable substances, and the microbial phase after acclimation differed from those with the coexistence of glucose and peptone. The quinone profiles in the acclimation showed that Brevibacterium sp. and Pseudomonas aeruginosa, of which the predominant quinone of the respiratory chain is menaquinone-8(H2) and ubiquinone-9, respectively, multiplied selectively in the acclimation course without and with the coexistence of glucose and peptone, respectively. It was also made clear that there were few kinds and number of protozoa and metazoa in the biofilter treating the wastewater containing AN.

Studies of microbial acclimation to hard chemicals on the basis of respiratory quinone profiles and kinetic analyses

The population dynamics of microbes in the biological wastewater treatment processes such as a submerged biofilter was investigated to obtain basic information to determine the optimal operating conditions. The effects of coexistence of biodegradable substances such as glucose and peptone on the acclimation of microbes in the biofilm to hard chemicals such as acrylonitrile (AN), which is poorly biodegradable and a volatile substance, was investigated on the basis of the respiratory quinone profile. Kinetic study of the removal of AN in the course of acclimation of microbes was investigated using a laboratory-scale submerged biofilter as well. It was ascertained that the acclimation of the microbes to AN was accelerated by coexistence of biodegradable substances, and the microbial phase after acclimation differed from those with the coexistence of glucose and peptone. The quinone profiles in the acclimation showed that Brevibacterium sp. and Pseudomonas aeruginosa, of which the predominant quinone of the respiratory chain is menaquinone-8(H2) and ubiquinone-9, respectively, multiplied selectively in the acclimation course without and with the coexistence of glucose and peptone, respectively. It was also made clear that there were few kinds and number of protozoa and metazoa in the biofilter treating the wastewater containing AN.