Paracoccus kondratievae produces poly(3-hydroxybutyrate) under elevated temperature conditions.

As part of ongoing efforts to discover novel polyhydroxyalkanoate-producing bacterial species, we embarked on characterizing the thermotolerant species, Paracoccus kondratievae, for biopolymer synthesis. Using traditional chemical and thermal characterization techniques, we found that P. kondratievae accumulates poly(3-hydroxybutyrate) (PHB), reaching up to 46.8% of the cell's dry weight after a 24-h incubation at 42°C. Although P. kondratievae is phylogenetically related to the prototypical polyhydroxyalkanoate producer, Paracoccus denitrificans, we observed significant differences in the PHB production dynamics between these two Paracoccus species. Notably, P. kondratievae can grow and produce PHB at elevated temperatures ranging from 42 to 47°C. Furthermore, P. kondratievae reaches its peak PHB content during the early stationary growth phase, specifically after 24 h of growth in a flask culture. This is then followed by a decline in the later stages of the stationary growth phase. The depolymerization observed in this growth phase is facilitated by the abundant presence of the PhaZ depolymerase enzyme associated with PHB granules. We observed the highest PHB levels when the cells were cultivated in a medium with glycerol as the sole carbon source and a carbon-to-nitrogen ratio of 10. Finally, we found that PHB production is induced as an osmotic stress response, similar to other polyhydroxyalkanoate-producing species.

Sea-Ice Bacteria Halomonas sp. Strain 363 and Paracoccus sp. Strain 392 Produce Multiple Types of Poly-3-Hydroxyalkaonoic Acid (PHA) Storage Polymers at Low Temperature.

Poly-3-hydroxyalkanoic acids (PHAs) are bacterial storage polymers commonly used in bioplastic production. Halophilic bacteria are industrially interesting organisms, as their salinity tolerance and psychrophilic nature lowers sterility requirements and subsequent production costs. We investigated PHA synthesis in two bacterial strains, Halomonas sp. 363 and Paracoccus sp. 392, isolated from Southern Ocean sea ice and elucidated the related PHA biopolymer accumulation and composition with various approaches, such as transcriptomics, microscopy, and chromatography. We show that both bacterial strains produce PHAs at 4°C when the availability of nitrogen and/or oxygen limited growth. The genome of Halomonas sp. 363 carries three phaC synthase genes and transcribes genes along three PHA pathways (I to III), whereas Paracoccus sp. 392 carries only one phaC gene and transcribes genes along one pathway (I). Thus, Halomonas sp. 363 has a versatile repertoire of phaC genes and pathways enabling production of both short- and medium-chain-length PHA products. IMPORTANCE Plastic pollution is one of the most topical threats to the health of the oceans and seas. One recognized way to alleviate the problem is to use degradable bioplastic materials in high-risk applications. PHA is a promising bioplastic material as it is nontoxic and fully produced and degraded by bacteria. Sea ice is an interesting environment for prospecting novel PHA-producing organisms, since traits advantageous to lower production costs, such as tolerance for high salinities and low temperatures, are common. We show that two sea-ice bacteria, Halomonas sp. 363 and Paracoccus sp. 392, are able to produce various types of PHA from inexpensive carbon sources. Halomonas sp. 363 is an especially interesting PHA-producing organism, since it has three different synthesis pathways to produce both short- and medium-chain-length PHAs.

In vivo creation of plasmid pCRT01 and its use for the construction of carotenoid-producing Paracoccus spp. strains that grow efficiently on industrial wastes.

BACKGROUND: Carotenoids are natural tetraterpene pigments widely utilized in the food, pharmaceutical and cosmetic industries. Currently, chemical synthesis of these compounds outperforms their production in Escherichia coli or yeast due to the limited efficiency of the latter. The use of natural microbial carotenoid producers, such as bacteria of the genus Paracoccus (Alphaproteobacteria), may help to optimize this process. In order to couple the ability to synthesize these pigments with the metabolic versatility of this genus, we explored the possibility of introducing carotenoid synthesis genes into strains capable of efficient growth on simple low-cost media. RESULTS: We constructed two carotenoid-producing strains of Paracoccus carrying a new plasmid, pCRT01, which contains the carotenoid synthesis gene locus crt from Paracoccus marcusii OS22. The plasmid was created in vivo via illegitimate recombination between crt-carrying vector pABW1 and a natural "paracoccal" plasmid pAMI2. Consequently, the obtained fusion replicon is stably maintained in the bacterial population without the need for antibiotic selection. The introduction of pCRT01 into fast-growing "colorless" strains of Paracoccus aminophilus and Paracoccus kondratievae converted them into efficient producers of a range of both carotenes and xanthophylls. The exact profile of the produced pigments was dependent on the strain genetic background. To reduce the cost of carotenoid production in this system, we tested the growth and pigment synthesis efficiency of the two strains on various simple media, including raw industrial effluent (coal-fired power plant flue gas desulfurization wastewater) supplemented with molasses, an industrial by-product rich in sucrose. CONCLUSIONS: We demonstrated a new approach for the construction of carotenoid-producing bacterial strains which relies on a single plasmid-mediated transfer of a pigment synthesis gene locus between Paracoccus strains. This strategy facilitates screening for producer strains in terms of synthesis efficiency, pigment profile and ability to grow on low-cost industrial waste-based media, which should increase the cost-effectiveness of microbial production of carotenoids.

Evaluation of denitrification performance and bacterial community of a sequencing batch reactor under intermittent aeration.

Effects of operational parameters (initial nitrite concentration, initial nitrate concentration, carbon source, and COD/N ratio) on denitrification performance was evaluated using a sequencing batch reactor (SBR) under intermittent aeration. Complete denitrification was observed without N2O accumulation when the initial nitrite concentration was 100-500 mg-N·L-1. When the initial nitrate concentration was 75-300 mg-N·L-1, 95-96% of NO3--N was completely reduced to N2 gas. Acetate was the most effective sole carbon source for the complete denitrification of the SBR under intermittent aeration, and 99% of NO3--N was reduced to N2 gas. The optimum COD/N ratio was 8-12 for the complete denitrification, while NO2- accumulation was observed at low COD/N ratios of 1 and 2. In this study, N2O accumulation was not observed during the denitrification process regardless of operational condition. Paracoccus (15-68%), a representative aerobic denitrifying bacterium, was dominant in the SBR during the denitrification process, and the intermittent aeration condition could affect the abundance of Paracoccus in this study.

Biodegradation of aged polycyclic aromatic hydrocarbons in agricultural soil by Paracoccus sp. LXC combined with humic acid and spent mushroom substrate.

Paracoccus sp. LXC combined with humic acid (HA) and spent mushroom substrate (SMS) obtained from Auricularia auricular and Sarcomyxa edulis was tested for the remediation of agricultural soil contaminated with aged polycyclic aromatic hydrocarbons (PAHs). The biomass and diversity of bacteria and fungi and the soil enzyme activity were analyzed. PAH removal and dissipation kinetics were examined. The highest degradation rate of PAHs was 56.5% when soil was amended with Paracoccus sp. LXC combined with HA and unsterilized SMS from A. auricular. The half-life of PAHs decreased from 2323.3 days in natural attenuation to 66.6-277.2 days in amended treatments. Soil treated with Paracoccus sp. LXC combined with HA and SMS from A. auricular acquired high contents of organic matter and nutrients. HA and SMS aided the growth of PAH-degrading bacteria and promoted the diversity of bacteria but not of fungi. The degradation rate of PAHs was mainly correlated positively with soil laccase activity. Low- and middle-molecular-weight PAHs were significantly removed by Paracoccus sp. LXC, HA and SMS. High-molecular-weight PAHs were removed by SMS but not by Paracoccus sp. LXC. Biodegradation by Paracoccus sp. LXC combined with HA and SMS is a promising choice for remediating aged PAH-contaminated agricultural soils.

Isolation and characterization of a lindane degrading bacteria Paracoccus sp. NITDBR1 and evaluation of its plant growth promoting traits

Lindane contamination in different environmental compartments is still posing a serious threat to our environment and effective measures need to be taken for the detoxification of lindane. Soil bacteria isolated from agricultural fields are known to possess certain plant growth promoting traits like the production of phytohormones, production of ammonia, nitrogen fixation and solubilization of phosphorus, etc. In the present study, an indigenous bacterial strain Paracoccus sp. NITDBR1 have been isolated from an agricultural field in Manipur, India which could grow on 100 mg L−1 lindane as the sole source of carbon and could degrade up to 90% of lindane in mineral salt media under liquid culture conditions in 8 days. The strain NITDBR1 was able to form biofilm in lindane media and the addition of substrate like glucose and sucrose enhanced the biofilm formation by 1.3 and 1.17-fold respectively in 3 days. The strain NITDBR1 could produce glycolipid and glycoprotein based biosurfactants. It was also found to possess plant growth promoting traits like nitrogen fixation and indole-3-acetic acid production to assist crop production. The phytotoxicity studies carried out on mustard seeds revealed that the degradation products formed after treatment with NITDBR1 could lower the toxicity of lindane for root elongation by 1.3-fold. Therefore, strain NITDBR1 could be useful for the bioremediation of soil contaminated with lindane with lesser damage to the environment, biofilm forming ability may help the bacteria survive under stressed environmental conditions, and biosurfactant production will help in increasing the bioavailability of contaminants. The plant growth promoting traits can be beneficial for agriculture. With such soil friendly activities coupled with pesticide degradation, this strain can be used for environmental as well as agricultural applications

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Isolation and characterization of a lindane degrading bacteria Paracoccus sp. NITDBR1 and evaluation of its plant growth promoting traits.

Lindane contamination in different environmental compartments is still posing a serious threat to our environment and effective measures need to be taken for the detoxification of lindane. Soil bacteria isolated from agricultural fields are known to possess certain plant growth promoting traits like the production of phytohormones, production of ammonia, nitrogen fixation and solubilization of phosphorus, etc. In the present study, an indigenous bacterial strain Paracoccus sp. NITDBR1 have been isolated from an agricultural field in Manipur, India which could grow on 100 mg L-1 lindane as the sole source of carbon and could degrade up to 90% of lindane in mineral salt media under liquid culture conditions in 8 days. The strain NITDBR1 was able to form biofilm in lindane media and the addition of substrate like glucose and sucrose enhanced the biofilm formation by 1.3 and 1.17-fold respectively in 3 days. The strain NITDBR1 could produce glycolipid and glycoprotein based biosurfactants. It was also found to possess plant growth promoting traits like nitrogen fixation and indole-3-acetic acid production to assist crop production. The phytotoxicity studies carried out on mustard seeds revealed that the degradation products formed after treatment with NITDBR1 could lower the toxicity of lindane for root elongation by 1.3-fold. Therefore, strain NITDBR1 could be useful for the bioremediation of soil contaminated with lindane with lesser damage to the environment, biofilm forming ability may help the bacteria survive under stressed environmental conditions, and biosurfactant production will help in increasing the bioavailability of contaminants. The plant growth promoting traits can be beneficial for agriculture. With such soil friendly activities coupled with pesticide degradation, this strain can be used for environmental as well as agricultural applications.

Biodegradation of Pendimethalin by Paracoccus sp. P13.

In this study, a bacterial strain P13 capable of degrading pendimethalin was isolated from the soil of a fruit garden. Based on observed cellular morphology and physiology characteristics and a phylogenetic analysis of 16S rRNA gene sequences, strain P13 was identified as a member of the genus Paracoccus. Strain P13 grew on pendimethalin as the sole carbon source, and could degrade 100 mg/L pendimethalin within 2 days and 200 mg/L pendimethalin within 5 days. Pendimethalin degradation was proposed to be initiated by oxidation ring cleavage to yield 1,3-dinitro-2-(pentan-3-ylamino)butane-1,4-diol, an alkane organic compound that was identified by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), which then underwent a series of enzymatic reactions to produce CO2 and H2O. The optimal pH and temperature for pendimethalin degradation by strain P13 were 7.0 and 30 °C, respectively. This study identified the bacterial strain Paracoccus sp. P13, which degraded pendimethalin with a relatively high efficiency, and presents a previously unreported microbial pendimethalin degradation pathway.

Towards smart biocide-free anti-biofilm strategies: Click-based synthesis of cinnamide analogues as anti-biofilm compounds against marine bacteria.

A set of triazole-based analogues of N-coumaroyltyramine was designed to discover potential leads that may help in the control of bacterial biofilms. the most potent compounds act as inhibitors of biofilm development with EC50 closed to ampicillin (EC50 = 11 µM) without toxic effect on bacterial growth even at high concentrations(100 µM).

Biodiversity of aerobic methylobacteria associated with the phyllosphere of the southern Moscow Oblast.

During the summer period (15­25°C), 34 strains of methylotrophic bacteria associated with different species of herbs, shrub, and trees in Pushchino (Moscow oblast, Russia) were isolated on the medium with methanol. Predominance of pink-colored Methylobacterium strains in the phyllosphere of many plants was confirmed by microscopy, enumeration of the colonies from grass leaves, and sequencing of the 16S rRNA genes. Colorless and yellow-pigmented methylotrophs belonged to the genera Methylophilus, Methylobacillus, Hansschlegelia, Methylopila, Xanthobacter, and Paracoccus. All isolates were able to synthesize plant hormones auxins from L-tryptophan (5−50 µg/mL) and are probably plant symbionts.

Comparative genomics of Paracoccus sp. SM22M-07 isolated from coral mucus: insights into bacteria-host interactions.

One of the main goals of coral microbiology is to understand the ways in which coral-bacteria associations are established and maintained. This work describes the sequencing of the genome of Paracoccus sp. SM22M-07 isolated from the mucus of the endemic Brazilian coral species Mussismilia hispida. Comparative analysis was used to identify unique genomic features of SM22M-07 that might be involved in its adaptation to the marine ecosystem and the nutrient-rich environment provided by coral mucus, as well as in the establishment and strengthening of the interaction with the host. These features included genes related to the type IV protein secretion system, erythritol catabolism, and succinoglycan biosynthesis. We experimentally confirmed the production of succinoglycan by Paracoccus sp. SM22M-07 and we hypothesize that it may be involved in the association of the bacterium with coral surfaces.

Screening of bromotyramine analogues as antifouling compounds against marine bacteria.

Rapid and efficient synthesis of 23 analogues inspired by bromotyramine derivatives, marine natural products, by means of CuSO4-catalysed [3+2] alkyne-azide cycloaddition is described. The final target was then assayed for anti-biofilm activity against three Gram-negative marine bacteria, Pseudoalteromonas ulvae (TC14), Pseudoalteromonas lipolytica (TC8) and Paracoccus sp. (4M6). Most of the synthesised bromotyramine/triazole derivatives are more active than the parent natural products Moloka'iamine (A) and 3,5-dibromo-4-methoxy-ß-phenethylamine (B) against biofilm formation by the three bacterial strains. Some of these compounds were shown to act as non-toxic inhibitors of biofilm development with EC50 < 200 µM without any effect on bacterial growth even at high concentrations (200 µM).

A rapid, sensitive, simple plate assay for detection of microbial alginate lyase activity.

Screening of microorganisms capable of producing alginate lyase enzyme is commonly carried out by investigating their abilities to grow on alginate-containing solid media plates and occurrence of a clearance zone after flooding the plates with agents such as 10% (w/v) cetyl pyridinium chloride (CPC), which can form complexes with alginate. Although the CPC method is good, advantageous, and routinely used, the agar in the media interferes with the action of CPC, which makes judgment about clearance zones very difficult. In addition, this method takes a minimum of 30 min to obtain the zone of hydrolysis after flooding and the hydrolyzed area is not sharply discernible. An improved plate assay is reported herein for the detection of extracellular alginate lyase production by microorganisms. In this method, alginate-containing agar plates are flooded with Gram's iodine instead of CPC. Gram's iodine forms a bluish black complex with alginate but not with hydrolyzed alginate, giving sharp, distinct zones around the alginate lyase producing microbial colonies within 2-3 min. Gram's iodine method was found to be more effective than the CPC method in terms of visualization and measurement of zone size. The alginate-lyase-activity area indicated using the Gram's iodine method was found to be larger than that indicated by the CPC method. Both methods (CPC and Gram's iodine) showed the largest alginate lyase activity area for Saccharophagus degradans (ATCC 43961) followed by Microbulbifer mangrovi (KCTC 23483), Bacillus cereus (KF801505) and Paracoccus sp. LL1 (KP288668) grown on minimal sea salt medium. The rate of growth and metabolite production in alginate-containing minimal sea salt liquid medium, followed trends similar to that of the zone activity areas for the four bacteria under study. These results suggested that the assay developed in this study of Gram's iodine could be useful to predict the potential of microorganisms to produce alginate lyase. The method also worked well for screening and identification of alginate lyase producers and non-producers from environmental samples on common laboratory media. They did this by clearly showing the presence or absence of clearance zones around the microbial colonies grown. This new method is rapid, efficient, and could easily be performed for screening a large number of microbial cultures. This is the first report on the use of Gram's iodine for the detection of alginate lyase production by microorganisms using plate assay.

Chronic impact of sulfamethoxazole on acetate utilization kinetics and population dynamics of fast growing microbial culture.

The study evaluated the chronic impact of sulfamethoxazole on metabolic activities of fast growing microbial culture. It focused on changes induced on utilization kinetics of acetate and composition of the microbial community. The experiments involved a fill and draw reactor, fed with acetate and continuous sulfamethoxazole dosing of 50 mg/L. The evaluation relied on model evaluation of the oxygen uptake rate profiles, with parallel assessment of microbial community structure by 454-pyrosequencing. Continuous sulfamethoxazole dosing inflicted a retardation effect on acetate utilization in a way commonly interpreted as competitive inhibition, blocked substrate storage and accelerated endogenous respiration. A fraction of acetate was utilized at a much lower rate with partial biodegradation of sulfamethoxazole. Results of pyrosequencing with a replacement mechanism within a richer more diversified microbial culture, through inactivation of vulnerable fractions in favor of species resistant to antibiotic, which made them capable of surviving and competing even with a slower metabolic response.

Influence of zero-valent iron nanoparticles on nitrate removal by Paracoccus sp.

Nitrate contamination in drinking water is a major threat to public health. This study investigated the efficiency of denitrification of aqueous solutions in the co-presence of synthesized nanoscale zero-valent iron (nZVI; diameter: 20-80 nm) and a previously isolated Paracoccus sp. strain YF1. Various influencing factors were studied, such as oxygen, pH, temperature, and anaerobic corrosion products (Fe(2+), Fe(3+) and Fe3O4). With slight toxicity to the strain, nZVI promoted denitrification efficiency by providing additional electron sources under aerobic conditions. For example, 50 mg L(-1) nZVI increased the nitrate removal efficiency from 66.9% to 85.2%. However, a high concentration of nZVI could lead to increased production of Fe(2+), a toxic ion which could compromise the removal efficiency. Kinetic studies suggest that denitrification by both free cells, and nZVI-amended cells fitted well to the zero-order model. Temperature and pH are the major factors affecting nitrate removal and cell growth, with or without the presence of nZVI. In this study, nitrate removal and cell growth increased in the pH range of 6.5-8.0, and temperature range of 25-35 °C. These conditions favor the growth of the strain, which dominated denitrification in all scenarios involved. As for anaerobic corrosion products, compared with Fe(2+) and Fe(3+), Fe3O4 promoted denitrification by serving as an electron donor. Finally, scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) confirmed attachments of nZVI on the surface of the cell, and the formation of iron oxides. This study indicated that, as an electron donor source with minimal cellular toxicity, nZVI could be used to promote denitrification efficiency under biotic conditions.

Impact of iron-based nanoparticles on microbial denitrification by Paracoccus sp. strain YF1.

This study investigates the effects of Fe and Fe/Ni nanoparticles on biological denitrification when using Paracoccus sp. strain YF1. Results show that adding Fe and Fe/Ni nanoparticles to the cells decreased their growth and denitrification rate. Compared to that of free cells (control 89.47%), a decrease (64.33%) in the presence of 1000 mg/L Fe/Ni nanoparticles was observed, while a small decline in the denitrification rate (76.36%) was obtained when the concentration of Fe nanoparticles was 1000 mg/L. These were further confirmed by adding Fe(2+), Fe(3+), Fe3O4, Fe(2+)/Ni(2+) and Fe(3+)/Ni(2+) individually to the free cell system. Furthermore, Fe and Fe/Ni nanoparticles influenced the nitrate removal and bacterial growth under different pH and temperature conditions. SEM, XRD and EDS demonstrated that iron oxides formed as a result of nanoparticles corrosion in biological medium. Finally the presence of nanoparticles around some bacteria was observed.

Paracoccus zhejiangensis sp. nov., isolated from activated sludge in wastewater-treatment system.

A bacterial strain, designated J6(T), was isolated from activated sludge, collected from a chemical wastewater treatment system in Zhejiang Province of China. The cells stained Gram-negative, were aerobic, pale-yellow, and non-motile short rods. Phylogenetic analysis of the 16S rRNA gene sequence indicated that the closest relative of this organism was Paracoccus aminophilus KACC 12262(T) = JCM 7686(T) (97.4 % sequence similarity). Strain J6(T) grew at 10-37 °C (optimum 30 °C), at pH 6.0-8.0 (optimum pH 7.0) and with 0-5 % NaCl (optimum 3 %, w/v). The predominant cellular fatty acid found was summed feature 8(C18:1 ω7c and/or C18:1 ω6c; 82.8 %). The major respiratory quinone-detected was Q-10 and the DNA G+C content was 61.9 mol %. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine and several unknown polar lipids. Strain J6(T) showed low DNA-DNA relatedness values with P. aminophilus KACC 12262(T) (28 ± 3 %). The phylogenetic analysis, DNA-DNA hybridization, whole-cell fatty acid composition as well as biochemical characteristics allowed clear differentiation of the isolate from the other type strains of already described Paracoccus species. It is evident from the genotypic, phenotypic and chemotaxonomic analyses that strain J6(T) should be classified as a novel species of the genus Paracoccus, for which the name P. zhejiangensis sp. nov. is proposed. The type strain is J6(T) (KACC 16703(T) = CCTCC AB 2012031(T)).

Role of transition metals in UV-B-induced damage to bacteria.

The purpose of this study was to explore the possible link between metals and UV-B-induced damage in bacteria. The effect of growth in the presence of enhanced concentrations of different transition metals (Co, Cu, Fe, Mn and Zn) on the UV-B sensitivity of a set of bacterial isolates was explored in terms of survival, activity and oxidative stress biomarkers (ROS generation, damage to DNA, lipid and proteins and activity of antioxidant enzymes). Metal amendment, particularly Fe, Cu and Mn, enhanced bacterial inactivation during irradiation by up to 35.8%. Amendment with Fe increased ROS generation during irradiation by 1.2-13.3%, DNA damage by 10.8-37.4% and lipid oxidative damage by 9.6-68.7%. Lipid damage during irradiation also increased after incubation with Cu and Co by up to 66.8% and 56.5% respectively. Mn amendment decreased protein carbonylation during irradiation by up to 44.2%. These results suggest a role of Fe, Co, Cu and Mn in UV-B-induced bacterial inactivation and the importance of metal homeostasis to limit the detrimental effects of ROS generated during irradiation.

[Bioaugmented removal of pyridine and the microbial community dynamic analysis].

The bioaugmented removal of pyridine was investigated through introducing immobilized Paracoccus sp. strain KT-5 capable of degrading pyridine into the lab-scale sequencing batch reactor (SBR) inoculated with activated sludge. The terminal restriction fragment length polymorphisms (T-RFLP) was used to analyzed the microbial community dynamics of two reactors during the whole operation process. The experimental results indicated that the introduction of immobilized strain KT-5 into the SBR could speed up the start-up of reactor, compared to the non-bioaugmented SBR. When the initial concentration of pyridine varied from 195.6 mg x L(-1) to 586.8 mg x L(-1), the bioaugmented effect was not significant; however, when the initial concentration of pyridine was 782.4-2934 mg x L(-1), the bioaugmentation role in pyridine degradation was obvious. The analysis of T-RFLP indicated that the introduced immobilized strain KT-5, as a dominant strain, always existed in both free and immobilized biomass of the bioaugmented SBR.

Functional consortium for denitrifying sulfide removal process.

Denitrifying sulfide removal (DSR) process simultaneously converts sulfide, nitrate, and chemical oxygen demand from industrial wastewaters to elemental sulfur, nitrogen gas, and carbon dioxide, respectively. This investigation utilizes a dilution-to-extinction approach at 10(-2) to 10(-6) dilutions to elucidate the correlation between the composition of the microbial community and the DSR performance. In the original suspension and in 10(-2) dilution, the strains Stenotrophomonas sp., Thauera sp., and Azoarcus sp. are the heterotrophic denitrifiers and the strains Paracoccus sp. and Pseudomonas sp. are the sulfide-oxidizing denitrifers. The 10(-4) dilution is identified as the functional consortium for the present DSR system, which comprises two functional strains, Stenotrophomonas sp. strain Paracoccus sp. At 10(-6) dilution, all DSR performance was lost. The functions of the constituent cells in the DSR granules were discussed based on data obtained using the dilution-to-extinction approach.