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.     

Analytical precision and repeatability of respiratory quinones for quantitative study of microbial community structure in environmental samples

Microbial community structure is one of the important factors controlling the pollutant-degrading capacity of ecosystems. The analysis of microbial quinones has gained increased recognition as a simple and useful approach for studying microbial structure in environmental samples. The analytical precision of quinone characterization using high performance liquid chromatograph (HPLC) with a UV-detector was studied in this study. Activated sludge was used as a typical mixed culture. The coefficient of variation of quinone content was lower than 6%, and that of microbial diversity calculated from the composition of quinones was as low as 3%. Statistical analyses on the analytical precision of quinones demonstrated that the critical value of dissimilarity between two quinone profiles of activated sludge, which is used to make a judgement whether the two quinone profiles are different or not, is 0.1 for the analytical method used in this study. The values of minimum biomass required for quinone analysis to have a reliable analytical result of microbial quinones were 2 mg-dry-cell for activated sludge.     

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.     

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。     

Distribution of Isoprenoid Quinones in Halophilic Bacteria

Menaquinones were the sole isoprenoid quinones found in 28 of the 34 halophilic organisms examined. Unsaturated and dihydrogenated menaquinones with eight isoprene units were found in some alkalophilic halophiles and representatives of the genera Halobacterium and Halococcus. Brevibacterium halotolerans and Micrococcus halobius possessed major amounts of unsaturated menaquinones with seven and eight isoprene units, respectively. Actinopolyspora halophila possessed complex mixtures of partially hydrogenated menaquinones with tetrahydrogenated menaquinones with nine isoprene units predominating. Vibrio costicola possessed both menaquinones and ubiquinones with eight isoprene units. Ectothiorhodospira halophila, Flavobacterium halmephilum, Paracoccus halodenitrificans and Pseudomonas beijerinkii contained ubiquinones as their sole respiratory quinones. Ectothiorhodospira halophila possessed major amounts of ubiquinones with eight isoprene units whereas ubiquinones with nine isoprene units predominated in Flavobacterium halmephilum, Paracoccus halodenitrificans and Pseudomonas beijerinkii.     

Isoprenoid quinone composition of representatives of the genus Campylobacter

The isoprenoid quinone composition of 17 strains representing nine species or sub-species of the genus Campylobacter was investigated. All strains produced similar respiratory quinone patterns consisting of unsaturated menaquinones with six isoprene units and a novel unidentified quinone. Mass spectral analysis indicate the unknown compound has six isoprene units and a formula C₄₂H₅₈O₂. The present study indicates respiratory quinones may be useful generic markers for Campylobacter.     

Quinone profiles in lake sediments: Implications for microbial diversity and community structures

Microbial quinone compositions of sediment mud samples from five different lakes in Japan were studied by spectrochromatography and mass spectrometry. The total quinone content of these samples ranged from 1.97 to 18.0 nmol/g dry weight of sediment, of which a combined fraction of ubiquinones and menaquinones accounted for 42 to 74%. The remaining fraction (26 to 58%) consisted of the photosynthetic quinones, plastoquinones and phylloquinone. The sediment samples produced PQ-9 or Q-8 as the most abundant quinone type regardless of their geographic locations and depths. These results indicate that oxygenic phototrophic microorganisms and Q-8-containing proteobacteria constituted major parts of microbial populations in the lake sediment. In the surface water of the same sampling sites, plastoquinones and phylloquinone occurred in much higher proportions. These findings suggested that the high abundance of oxygenic phototrophs in the sediment muds resulted from their constant movement or sedimentation from the surface water. Numerical analyses of the quinone profiles showed that the microbial communities of the sediment were diverse and different in different lakes but similar to each other in the diversity of bioenergetic modes. Three physiological groups of microbes showing ubiquinone-mediated aerobic respiration, oxygenic photosynthesis, and menaquinone-associated respiration were suggested to inhabit the lake sediments in balance.     

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.     

Changes in the microbial community structure during thermophilic composting of manure as detected by the quinone profile method

Quinone profiles and physico-chemical properties were measured to characterize the microbial community structure during a 14-day thermophilic composting of cattle manure mixed with rice straw as a bulking agent. The change in total quinone content (TQ) and the divergence of quinone (DQ) indicated that the microbial biomass reached a peak followed by a decrease, whereas the microbial community diversity increased continuously during the composting process. The high mole fraction of ubiquinones in the raw materials, and at the beginning of the composting period suggested that fungi and/or Proteobacteria were present. The predominance of MK-7 from days 3 to 7 suggested that Bacillus spp. were the main microbial species. An increase in partially saturated and long-chain menaquinones during the latter composting period indicated that the proliferation of various species of Actinobacteria was occurring. The microbial community structure, as expressed by TQ and DQ, corresponded well to physico-chemical properties such as the C/N ratio, pH, O₂ consumption and compost mass reduction.     

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.     

Isoprenoid Quinone Composition as a Guide to the Classification of Sporolactobacillus and Possibly Related Bacteria

Menaquinones with seven isoprene units were the major isoprenoid quinones detected in the chloroform-methanol extracts of representative strains of the genera Bacillus and Sporolactobacillus. Neither menaquinones nor ubiquinones were detected in similar extracts of strains of the genus Lactobacillus.     

A note on the isoprenoid quinone content of Bordetella avium and related species

The isoprenoid quinone content of isolates of Bordetella avium (four strains), Alcaligenes faecalis (one strain), Bordetella bronchiseptica (one strain) and a Bordetella avium-like organism (four strains) was determined by reverse-phase high-performance liquid chromatography. All the isolates contained ubiquinones with eight isoprene units as the major component. No menaquinones were detected.     

NADH: quinone oxidoreductase as a site of Na+-dependent activation in the respiratory chain of marine Vibrio alginolyticus.

The site of Na+-dependent activation in the respiratory chain of the marine bacterium, Vibrio alginolyticus, was investigated. The respiratory chain system contained ubiquinones (Q), menaquinones (MK), cytochromes b(560), c(553), d(630), and o(560). The membrane-bound and partially purified NADH dehydrogenase was stimulated 2- to 3-fold by the addition of 0.2 M Na+ or K+ and no specific requirement for Na+ was observed in this reaction step. The cytochrome oxidase showed no requirement for monovalent cations. The respiratory activity (NADH oxidase) of the membrane was lost on removal of the quinones, and the reincorporation of authentic Q-10 or MK-4 restored the activity. The rate of MK-4 reduction by NADH (menaquinone reductase) as measured using MK-4 incorporated membrane was activated by Na+, but only slightly by K+. The apparent Ka for Na+ was 78 mM for both menaguinone reductase and NADH oxidase. The requirement for Na+ of menaquinone reductase was greatly reduced in the presence of 0.2 M K+. Ubiquinone reductase as measured by using Q-10 incorporated membrane was also activated more effectively by Na+ than by K+. These results strongly suggested that the site of Na+-dependent activation in the respiratory chain of marine V. alginolyticus was at the step of NADH; quinone oxidoreductase.     

A note on the isoprenoid quinone content of Bordetella avium and related species

The isoprenoid quinone content of isolates of Bordetella avium (four strains), Alcaligenes faecalis (one strain), Bordetella bronchiseptica (one strain) and a Bordetella avium -like organism (four strains) was determined by reverse-phase high-performance liquid chromatography. All the isolates contained ubiquinones with eight isoprene units as the major component. No menaquinones were detected.     

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.     

Lipid composition of novel Shewanella species isolated from far Eastern seas

A comparative study of the lipid composition of 26 strains (including type strains) of marine Gammaproteobacteria belonging to the genera Shewanella, Alteromonas, Pseudoalteromonas, Marinobacterium, Microbulbifer, and Marinobacter was carried out. The bacteria exhibited genus-specific profiles of ubiquinones, phospholipids, and fatty acids, which can serve as reliable chemotaxonomic markers for tentative identification of new isolates. The studied species of the genus Shewanella were distinguished by the presence of two types of isoprenoid quinones, namely, ubiquinones Q-7 and Q-8 and menaquinones MK-7 and MMK-7; five phospholipids typical of this genus, namely, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), lyso-PE, and acyl-PG; and the fatty acids 15:0, 16:0, 16:1 (n-7), 17:1 (n-8), i-13:0, and i-15:0. The high level of branched fatty acids (38-45%) and the presence of eicosapentaenoic acid (4%) may serve as criteria for the identification of this genus. Unlike Shewanella spp., bacteria of the other genera contained a single type of isoprenoid quinone: Q-8 (Alteromonas, Pseudoalteromonas, Marinobacterium, and Microbulbifer) or Q-9 (Marinobacter). The phospholipid compositions of these bacteria were restricted to three components: two major phospholipids (PE and PG) and a minor phospholipid, bisphosphatidic acid (Alteromonas and Pseudoalteromonas) or DPG (Marinobacterium, Microbulbifer, and Marinobacter). The bacteria exhibited genus-specific profiles of fatty acids.     

<Emphasis Type="Italic">Shewanella upenei</Emphasis> sp. nov., a lipolytic bacterium isolated from bensasi goatfish <Emphasis Type="Italic">Upeneus bensasi</Emphasis>

A Gram-staining-negative, motile, non-spore-forming and rod-shaped bacterial strain, 20-23R^T, was isolated from intestine of bensasi goatfish, Upeneus bensasi, and its taxonomic position was investigated by using a polyphasic study. Phylogenetic analyses based on 16S rRNA gene sequences revealed that strain 20-23R^T belonged to the genus Shewanella. Strain 20-23R^T exhibited 16S rRNA gene sequence similarity values of 99.5, 99.2, and 97.5% to Shewanella algae ATCC 51192^T, Shewanella haliotis DW01^T, and Shewanella chilikensis JC5^T, respectively. Strain 20-23R^T exhibited 93.1–96.0% 16S rRNA gene sequence similarity to the other Shewanella species. It also exhibited 98.3–98.4% gyrB sequence similarity to the type strains of S. algae and S. haliotis. Strain 20-23R^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-8 and Q-7. The fatty acid profiles of strain 20–23R^T, S. algae KCTC 22552^T and S. haliotis KCTC 12896^T were similar; major components were iso-C_15:0, C_16:0, C_16:1 ω7c and/or iso-C_15:0 2-OH and C_17:1 ω8c. The DNA G+C content of strain 20-23R^T was 53.9 mol%. Differential phenotypic properties and genetic distinctiveness of strain 20–23R^T, together with the phylogenetic distinctiveness, revealed that this strain is distinguishable from recognized Shewanella species. On the basis of the data presented, strain 20-23R^T represents a novel species of the genus Shewanella, for which the name Shewanella upenei sp. nov. is proposed. The type strain is 20–23R^T (=KCTC 22806^T =CCUG 58400^T).     

Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater

A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the γ-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4–10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.     

Investigations on the influence of headgroup substitution and isoprene side-chain length in the function of primary and secondary quinones of bacterial reaction centers

The contributions of headgroup and side-chain in the binding and function of the primary (QA) and secondary (QB) quinones of isolated reaction centers (RCs) from Rhodobacter sphaeroides were investigated. Various ubiquinones and structurally similar quinones were reconstituted into RCs depleted of one (1Q-RCs) or both (0Q-RCs) quinones. The influence of partition coefficients on the apparent binding affinities was minimized by expressing dissociation constants in terms of the mole fraction of quinone partitioned into the detergent. It was then apparent that the size of the isoprenyl side-chain was of little consequence in determining the binding affinity or the functional competence of either QA or QB, although an alkyl chain of equivalent size was a poor substitute. The degree of substitution of the headgroup, however, was a sensitive determinant of binding. For both quinone sites, the trisubstituted plastoquinones bond more weakly than the fully substituted ubiquinones. Similarly, for binding to the QA site, duroquinone (tetramethylbenzoquinone) bound much more strongly than trimethylbenzoquinone. The affinity of the QA site for ubiquinones was about 20-times stronger than the QB site, but the QB site is probably not more specific than the QA site. However, QB function depends on a suitable redox free-energy drop from QA as well as binding, and of all the quinones tested only the ubiquinones simultaneously supported full QA and QB activity. Even plastoquinone-A, which fills both roles in Photosystem II, was unable to do so in bacterial RCs, although it did bind. The unique ability of ubiquinones to both bind and provide the appropriate redox span is discussed. The temperature dependence of binding of the isoprenyl ubiquinones at the QA site changed markedly with chain length. For Q-10-Q-7, the binding enthalpy was positive and net binding was entirely driven by entropic factors. For the shorter-chain ubiquinones, Q-6-Q-1, both entropy and enthalpy of binding were favorable. This strong entropy-enthalpy compensation is suggested to arise from antagonistic interactions (anticooperativity) between headgroup and tail binding. For QB function by hydrophobic quinones, the temperature dependence of the micelle properties prevented easy access to thermodynamic parameters. However, for water-soluble Q-0, binding to the QB site was determined to be enthalpically driven.(ABSTRACT TRUNCATED AT 400 WORDS)     

Starter culture of <Emphasis Type="Italic">Pseudomonas veronii</Emphasis> strain B for aerobic granulation

Aggregation of bacterial cells is used in formation of microbial granules. Aerobically grown microbial granules can be used as the bio-agents in the treatment of wastewater. However, there are problems with start up of microbial granulation and biosafety of this process. Aim of this research was selection and testing of safe microbial strain with high cell aggregation ability to shorten period of microbial granules formation. Five bacterial strains with cell aggregation index higher than 50% have been isolated from the granules. Strain of Pseudomonas veronii species was considered as most probably safe starter culture for granulation because other strains belonged to the species known as human pathogens. The microbial granules were formed after 3 days of cultivation in case when P. veronii strain B was applied to start-up aerobic granulation process using model wastewater. The granules were produced from activated sludge after 9 days of cultivation. Microbial aggregates produced from starter culture of P. veronii strain B were more compact (sludge volume index was 70 ml/g) than those produced from activated sludge (sludge volume index was 106 ml/g). It is a first proof that application of selected safe starter pure culture with high cell aggregation ability can accelerate and enhance formation of microbial granules.