Biodegradation of melamine and its hydroxy derivatives by a bacterial consortium containing a novel Nocardioides species

Melamine has recently been recognized as a food contaminant with adverse human health effects. Melamine contamination in some crops arises from soil and water pollution from various causes. To remove melamine from the polluted environment, a novel bacterium, Nocardioides sp. strain ATD6, capable of degrading melamine was enriched and isolated from a paddy soil sample. The enrichment culture was performed by the soil-charcoal perfusion method in the presence of triazine-degrading bacteria previously obtained. Strain ATD6 degraded melamine and accumulated cyanuric acid and ammonium, via the intermediates ammeline and ammelide. No gene known to encode for triazine-degrading enzymes was detected in strain ATD6. A mixed culture of strain ATD6 and a simazine-degrading Methyloversatilis sp. strain CDB21 completely degraded melamine, but the degradation rate of cyanuric acid was slow. The degradation of melamine and its catabolites by the mixed culture was greatly enhanced by including Bradyrhizobium japonicum strain CSB1 in the inoculum and adding ethanol to the culture medium. The melamine-degrading consortium consisting of strains ATD6, CDB21, and CSB1 appears to be potentially safer than other known melamine-degrading bacteria for the bioremediation of farmland and other contaminated sites, as no known pathogens were included in the consortium.     

Gene Sequence and Properties of an s-Triazine Ring-Cleavage Enzyme from Pseudomonas sp. Strain NRRLB-12227

Pesticides based on the s-triazine ring structure are widely used in cultivation of food crops. Cleavage of the s-triazine ring is an important step in the mineralization of s-triazine compounds and hence in their complete removal from the environment. Cyanuric acid amidohydrolase cleaves cyanuric acid (2,4,6-trihydroxy-s-triazine), which yields carbon dioxide and biuret; the biuret is subject to further metabolism, which yields CO₂ and ammonia. The trzD gene encoding cyanuric acid amidohydrolase was cloned into pMMB277 from Pseudomonas sp. strain NRRLB-12227, a strain that is capable of utilizing s-triazines as nitrogen sources. Hydrolysis of cyanuric acid was detected in crude extracts of Escherichia coli containing the cloned gene by monitoring the disappearance of cyanuric acid and the appearance of biuret by high-performance liquid chromatography (HPLC). DEAE and hydrophobic interaction HPLC were used to purify cyanuric acid amidohydrolase to homogeneity, and a spectrophotometric assay for the purified enzyme was developed. The purified enzyme had an apparent K_m of 0.05 mM for cyanuric acid at pH 8.0. The enzyme did not cleave any other s-triazine or hydroxypyrimidine compound, although barbituric acid (2,4,6-trihydroxypyrimidine) was found to be a strong competitive inhibitor. Neither the nucleotide sequence of trzD nor the amino acid sequence of the gene product exhibited a significant level of similarity to any known gene or protein.     

Mineralization of s-triazine herbicides by a newly isolated Nocardioides species strain DN36

A novel s-triazine-mineralizing bacterium—Nocardioides sp. strain DN36—was isolated from paddy field soil treated with ring-U-¹⁴C-labeled simetryn ([¹⁴C]simetryn) in a model paddy ecosystem (microcosm). In a tenfold-diluted R2A medium, strain DN36 liberated ¹⁴CO₂ from not only [¹⁴C]simetryn but also three ring-U-¹⁴C-labeled s-triazines: atrazine, simazine, and propazine. We found that DN36 mineralized ring-U-¹⁴C–cyanuric acid added as an initial substrate, indicating that the bacterium mineralized s-triazine herbicides via a common metabolite, namely, cyanuric acid. Strain DN36 harbored a set of genes encoding previously reported s-triazine-degrading enzymes (TrzN-AtzB-AtzC), and it also transformed ametryn, prometryn, dimethametryn, atraton, simeton, and prometon. The findings suggest that strain DN36 can mineralize a diverse range of s-triazine herbicides. To our knowledge, strain DN36 is the first Nocardioides strain that can individually mineralize s-triazine herbicides via the ring cleavage of cyanuric acid. Further, DN36 could not grow on cyanuric acid, and the degradation seemed to occur cometabolically.     

Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates

The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was examined under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography analysis, followed by liquid chromatography–tandem mass spectrometry identification of metabolites. Organisms in WRF microcosms degraded 180 μM RDX within 4 h. Nitroso-intermediates hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were present as early as 0.25 h and were detected throughout the 24-h incubation period, representing one reductive pathway of ring cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also produced, which were unstable and resulted in rapid ring cleavage to a common metabolite consistent with an m/z of 149. These represent two additional reductive pathways for RDX degradation in ovine WRF, which have not been previously reported. The 24 ruminal isolates degraded RDX with varying efficiencies (0–96 %) over 120 h. Of the most efficient degraders identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, while Anaerovibrio lipolyticus, Prevotella ruminicola, and Streptococcus bovis IFO utilized RDX as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, delineated the metabolic pathway for its biodegradation.     

Purification and Characterization of an Inducible s-Triazine Hydrolase from Rhodococcus corallinus NRRL B-15444R

The widespread use and relative persistence of s-triazine compounds such as atrazine and simazine have led to increasing concern about environmental contamination by these compounds. Few microbial isolates capable of transforming substituted s-triazines have been identified. Rhodococcus corallinus NRRL B-15444 has previously been shown to possess a hydrolase activity that is responsible for the dechlorination of the triazine compounds deethylsimazine (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine) (CEAT) and deethylatrazine (6-chloro-N-isopropyl-1,3,5-triazine-2,4-diamine) (CIAT). The enzyme responsible for this activity was purified and shown to be composed of four identical subunits of 54,000 Da. Kinetic experiments revealed that the purified enzyme is also capable of deaminating the structurally related s-triazine compounds melamine (2,4,6-triamino-1,3,5-triazine) (AAAT) and CAAT (2-chloro-4,6-diamino-1,3,5-triazine), as well as the pyrimidine compounds 2,4,6-triaminopyrimidine (AAAP) and 4-chloro-2,6-diaminopyrimidine (CAAP). The triazine herbicides atrazine and simazine inhibit the hydrolytic activities of the enzyme but are not substrates. Induction experiments demonstrate that triazine hydrolytic activity is inducible and that this activity rises approximately 20-fold during induction.     

Enzymatic Degradation of Chlorodiamino-s-Triazine

2-Chloro-4,6-diamino-s-triazine (CAAT) is a metabolite of atrazine biodegradation in soils. Atrazine chlorohydrolase (AtzA) catalyzes the dechlorination of atrazine but is unreactive with CAAT. In this study, melamine deaminase (TriA), which is 98% identical to AtzA, catalyzed deamination of CAAT to produce 2-chloro-4-amino-6-hydroxy-s-triazine (CAOT). CAOT underwent dechlorination via hydroxyatrazine ethylaminohydrolase (AtzB) to yield ammelide. This represents a newly discovered dechlorination reaction for AtzB. Ammelide was subsequently hydrolyzed by N-isopropylammelide isopropylaminohydrolase to produce cyanuric acid, a compound metabolized by a variety of soil bacteria.     

Catabolism of terbuthylazine by mixed bacterial culture originating from s-triazine-contaminated soil

The s-triazine herbicide terbuthylazine (TERB) has been used as the main substitute of atrazine in many EU countries for more than 10 years. However, the ecological consequences of this substitution are still not fully understood. Since the fate of triazine herbicides is primarily dependent on microbial degradation, in this paper, we investigated the ability of a mixed bacterial culture, M3-T, originating from s-triazine-contaminated soil, to degrade TERB in liquid culture and soil microcosms. The M3-T culture grown in mineral medium with TERB as the N source and citrate as the C source degraded 50 mg L^?1 of TERB within 3 days of incubation. The culture was capable of degrading TERB as the sole C and N source, though at slower degradation kinetics. A thorough LC-MS analysis of the biodegradation media showed the formation of hydroxyterbuthylazine (TERB-OH) and N-t-butylammelide (TBA) as major metabolites, and desethylterbuthylazine (DET), hydroxydesethylterbuthylazine (DET-OH) and cyanuric acid (CA) as minor metabolites in the TERB degradation pathway. TBA was identified as a bottleneck in the catabolic pathway leading to its transient accumulation in culture media. The supplementation of glucose as the exogenous C source had no effect on TBA degradation, whereas citrate inhibited its disappearance. The addition of M3-T to sterile soil artificially contaminated with TERB at 3 mg kg^?1 of soil resulted in an accelerated TERB degradation with t _1/2 value being about 40 times shorter than that achieved by the native microbial community. Catabolic versatility of M3-T culture makes it a promising seed culture for accelerating biotransformation processes in s-triazine-contaminated environment.     

Roseivivax atlanticus sp. nov., isolated from surface seawater of the Atlantic Ocean

A taxonomic study was carried out on strain 22II-S10s^T, which was isolated from the surface seawater of the Atlantic Ocean. The bacterium was found to be Gram-negative, oxidase and catalase positive, rod shaped and motile by subpolar flagella. The isolate was capable of gelatine hydrolysis but unable to reduce nitrate to nitrite or degrade Tween 80 or aesculin. Growth was observed at salinities of 0.5–18 % (optimum, 2–12 %), at pH of 3–10 (optimum, 7) and at temperatures of 10–41 °C (optimum 28 °C). Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 22II-S10s^T belongs to the genus Roseivivax, with highest sequence similarity to Roseivivax halodurans JCM 10272^T (97.2 %), followed by Roseivivax isoporae LMG 25204^T (97.0 %); other species of genus Roseivivax shared 95.2–96.7 % sequence similarity. The DNA–DNA hybridization estimate values between strain 22II-S10s^T and the two type strains (R. halodurans JCM 10272^T and R. isoporae LMG 25204^T) were 22.00 and 21.40 %. The principal fatty acids were identified as Summed Feature 8 (C_18:1 ω7c/ω6c) (67.4 %), C_18:0 (7.2 %), C_19:0 cyclo ω8c (7.1 %), C_18:1 ω7c 11-methyl (6.8 %) and C_16:0 (5.9 %). The respiratory quinone was determined to be Q-10 (100 %). Phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an aminolipid, a glycolipid and three phospholipids were present. The G+C content of the chromosomal DNA was determined to be 67.5 mol%. The combined genotypic and phenotypic data show that strain 22II-S10s^T represents a novel species within the genus Roseivivax, for which the name Roseivivax atlanticus sp. nov. is proposed, with the type strain 22II-S10s^T (= MCCC 1A09150^T = LMG 27156^T).     

Utilization of chlorinated s-triazines by a new strain of Klebsiella pneumoniae

A bacterium utilizing 2-chloro-4,6-diamino-s-triazine (CAAT) as sole nitrogen source was isolated under a N₂-free atmosphere and identified as Klebsiella pneumoniae. Concomitant to CAAT degradation the protein content increased and chloride was released into the medium. Under air and a N₂-atmosphere no reduction of CAAT degradation resulted, though this strain is able to fix molecular nitrogen, but the decomposition accelerated under anaerobic conditions. The degradation rate increased continuously with increasing CAAT concentration. A continuous CAAT degradation without CAAT accumulation was possible up to a influx rate of 4.8 mol·l^–1 h^–1 (dilution rate = 0.007 h^–1). K. pneumoniae A2 was also able to utilize deethylsimazine (CEAT) and deethylatrazine (CIAT) as nitrogen source. Both under aerobic and anaerobic conditions CEAT could be degraded faster than CIAT. The degradation sequence of mixed s-triazines was cyanuric acid < CAAT < CEAT < CIAT, which was reflected by the degradation times of single compounds. Complete degradation was assumed for all investigated s-triazine derivatives.     

Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in<Emphasis Type="Italic"> Clostridium bifermentans</Emphasis> HAW-1

A fast hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading [28.1 mol h^–1 g (dry weight) cells^–1; biomass, 0.16 g (dry weight) cells^–1] and strictly anaerobic bacterial strain, HAW-1, was isolated and identified as Clostridium bifermentans using a 16S-rRNA-based method. Based on initial rates, strain HAW-1 transformed RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) with yields of 56, 7.3 and 0.2%, respectively. Complete removal of RDX and its nitroso metabolites produced (%, of total C or N) methanol (MeOH, 23%), formaldehyde (HCHO, 7.4%), carbon dioxide (CO₂, 3.0%) and nitrous oxide (N₂O, 29.5%) as end products. Under the same conditions, strain HAW-1 transformed MNX separately at a rate of 16.9 mol h^–1 g (dry weight) cells^–1 and produced DNX (25%) and TNX (0.4%) as transient products. Final MNX transformation products were (%, of total C or N) MeOH (21%), HCHO (2.9%), and N₂O (17%). Likewise strain HAW-1 degraded TNX at a rate of 7.5 mol h^–1 g (dry weight) cells^–1 to MeOH and HCHO. Furthermore, removal of both RDX and MNX produced nitrite (NO₂^–) as a transient product, but the nitrite release rate from MNX was quicker than from RDX. Thus, the predominant pathway for RDX degradation is based on initial reduction to MNX followed by denitration and decomposition. The continued sequential reduction to DNX and TNX is only a minor route.     

Use of an algD Promoter-Driven Expression System for the Degradation of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Pseudomonas sp. HK-6

Pseudomonas sp. HK-6 is able to utilize hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a sole nitrogen source. The HK-6 strain was stimulated to produce an exopolymer, mainly alginate, as a stress response when grown in LB broth containing RDX, synthesizing ~230 μg/mL after 48 h. The algA mRNA levels in HK-6 increased by 7–8-fold after 2–6 h of exposure to 0.1 mM RDX, as measured by RT-qPCR. HK-6 was able to degrade ~25 % of 0.1 mM RDX after 20 days and 60 % after 50 days, whereas the pnrB null mutant only degraded less than 1 % after 50 days. The introduction of an algD promoter–pnrB gene fusion into the pnrB mutant fully restored RDX-degradation capability. To facilitate a study of PnrB action on RDX, a His6-PnrB fusion protein was heterologously expressed in E. coli BL21 cells, and the enzymatic activity on RDX was assayed by measuring the decrease in absorbance at 340 nm due to NADH oxidation. At the fixed condition of 0.1 mM RDX, 0.2 mM NADH, and 1 μg His6-PnrB, the absorbance at 340 nM gradually decreased and reached to its minimum value after 30 min. However, calculating the V _max and K _m values of PnrB for RDX was challenging due to extremely low solubility of RDX in water. The results clearly indicate the potential use of the algD promoter in studies of some genes in Pseudomonas species.     

Thermostable Cyanuric Acid Hydrolase from Moorella thermoacetica ATCC 39073

Cyanuric acid, a metabolic intermediate in the degradation of many s-triazine compounds, is further metabolized by cyanuric acid hydrolase. Cyanuric acid also accumulates in swimming pools due to the breakdown of the sanitizing agents di- and trichloroisocyanuric acid. Structurally stable cyanuric acid hydrolases are being considered for usage in pool water remediation. In this study, cyanuric acid hydrolase from the thermophile Moorella thermoacetica ATCC 39073 was cloned, expressed in Escherichia coli, and purified to homogeneity. The recombinant enzyme was found to have a broader temperature range and greater stability, at both elevated and low temperatures, than previously described cyanuric acid hydrolases. The enzyme had a narrow substrate specificity, acting only on cyanuric acid and N-methylisocyanuric acid. The M. thermoacetica enzyme did not require metals or other discernible cofactors for activity. Cyanuric acid hydrolase from M. thermoacetica is the most promising enzyme to use for cyanuric acid remediation applications.s-Triazine compounds have diverse applications as herbicides, resins, and disinfectants. The s-triazine herbicides, such as atrazine, help to promote high-yield, sustainable agriculture. Melamine, or triamino-s-triazine, is a high-volume industrial chemical. Melamine-based polymers have outstanding thermosetting properties, ideal for their use in kitchen utensils and plates, as high-pressure laminates such as Formica, and as whiteboards. Di- and tri-chloroisocyanuric acids find widespread application as disinfectants, algicides, and bactericides. The chlorinated isocyanuric acids are used in wastewater treatment, in the textile industry as bleaching compounds, and in preventing and curing diseases in husbandry and fisheries. A major use of these compounds is for swimming pool chlorination. They have outstanding performance for maintaining a high, stable chlorine content by dissolving slowly in water, allowing a continuous metered dosing of chlorine.Degradation of these and other s-triazine compounds results in the production of cyanuric acid (Fig. ​(Fig.1).1). Cyanuric acid has come under increased scrutiny because of its potential involvement in comediating toxicity resulting from the ingestion of melamine (10). Recently, melamine has been found in adulterated pet food and baby formula. Melamine and its metabolite cyanuric acid cocrystallize at low concentrations and are implicated in acute renal failure in cats that have consumed adulterated food products (10). Cyanuric acid degradation is also of interest from the perspective of environmental remediation. The use of di- or trichloroisocyanuric acid in pool water results in spontaneous chemical dechlorination that disinfects the water but also produces, as a by-product, large amounts of cyanuric acid. High levels of cyanuric acid perturb the equilibrium, thus preventing dechlorination by additional chlorinated isocyanuric acid, such that disinfection is not achieved. As a result, swimming pools must be emptied and refilled, using water and causing discharge issues. It would be desirable to remediate pool water in situ, conserving water, saving money, and extending pool water use. In this context, there is a need to better understand cyanuric acid degradation and to identify highly stable biological catalysts to use for this purpose.Open in a separate windowFIG. 1.Atrazine, ametryn, trichloroisocyanuric acid, and melamine are all metabolized via cyanuric acid that is transformed to biuret by the action of cyanuric acid hydrolases.Microbial enzymatic degradation of cyanuric acid has been studied previously (3, 4, 8, 18). Two distinct but homologous enzymes, AtzD from Pseudomonas sp. strain ADP (8) and TrzD from Pseudomonas sp. strain NRRLB-12227 (now called Acidovorax avenae subsp. citrulli) (11), have been studied in detail. These enzymes, known as cyanuric acid hydrolases, catalyze the conversion of cyanuric acid to biuret (Fig. ​(Fig.1).1). Biuret is not considered toxic to humans and degrades more readily than cyanuric acid.Barbiturase is the only protein known to be homologous to cyanuric acid hydrolase that has a defined and different physiological function. Barbiturase catalyzes the conversion of barbituric acid to ureidomalonic acid in organisms that catabolize pyrimidines by the oxidative pathway. Barbiturase is unstable at 4°C in the absence of ethylene glycol and dithiothreitol (DTT). Furthermore, activity is completely lost when the protein is maintained at 55°C for 30 min (20). AtzD and TrzD are relatively stable at 4°C, but they lose activity when frozen (our unpublished data). Moreover, the thermostability properties of AtzD and TrzD are not well studied, but these enzymes are derived from mesophilic bacteria. In this context, we initiated a search to identify a stable cyanuric acid hydrolase. Enzymes that are more stable in response to temperature changes are more stable in response to many environmental factors. Thus, a thermostable enzyme would be most applicable to pool water and other remediation efforts.We employed bioinformatic techniques that identified a cyanuric acid hydrolase homolog in Moorella thermoacetica ATCC 39073, an anaerobic, acetogenic bacterium that is able to grow at 65°C. The gene was cloned into E. coli, the protein was expressed at high levels, the recombinant E. coli strain degraded cyanuric acid, and the enzyme was obtained in homogeneous form by a convenient one-step purification. The enzyme''s function as a cyanuric acid hydrolase was confirmed, and it was shown to be significantly more stable than other known members of the cyanuric acid protein family.     

Novel sulfonamide incorporating piperazine,aminoalcohol and 1,3,5-triazine structural motifs with carbonic anhydrase I,II and IX inhibitory action

A new series of s-triazine derivatives incorporating sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and piperazine or aminoalcohol structural motifs is reported. Molecular docking was exploited to select compounds from virtual combinatorial library for synthesis and subsequent biological evaluation. The compounds were prepared by using step by step nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The compounds were tested as inhibitors of physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II and tumor-associated hCA IX. These compounds show appreciable inhibition. hCA I was inhibited with K_Is in the range of 8.5–2679.1 nM, hCA II with K_Is in the range of 4.8–380.5 nM and hCA IX with K_Is in the range of 0.4–307.7 nM. As other similar derivatives, some of the compounds showed good or excellent selectivity ratios for inhibiting hCA IX over hCA II, of 3.5–18.5. 4-[({4-Chloro-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)methyl] benzene sulfonamide demonstrated subnanomolar affinity for hCA IX (0.4 nM) and selectivity (18.50) over the cytosolic isoforms. This series of compounds may be of interest for the development of new, unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX.     

Fed-Batch Cultivation of Arthrospira platensis Using Carbon Dioxide from Alcoholic Fermentation and Urea as Carbon and Nitrogen Sources

To reduce CO₂ emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m^?2 s^?1?≤?I?≤?240 μmol m^?2 s^?1). The type of carbon source (pure CO₂ or CO₂ from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y _X/N ), and the maximum cell concentration (X _m ) and productivity (P _X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X _m ?=?2,960?±?35 g m^?3, P _X ?=?425?±?5.9 g m^?3 day^?1 and Y _X/N ?=?15?±?0.2 g g^?1) was obtained at I?=?120 μmol m^?2 s^?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.     

Inhibition of atrazine degradation by cyanazine and exogenous nitrogen in bacterial isolate M91-3

A variety of s-triazine herbicides and nitrogen fertilizers frequently occur as co-contaminants at pesticide manufacturing and distribution facilities. The degradation of atrazine and cyanazine by the bacterial isolate M91-3 was investigated in washed-cell suspensions and crude cellular extracts. Cyanazine competitively inhibited atrazine degradation. The maximum atrazine degradation rate (V _max) was 41 times higher and the half-saturation constant for the inhibitor (K _i) was 1.3 times higher in the crude cellular extract than in the washed-cell suspension, suggesting that cellular uptake influenced degradation of the s-triazines. Cultures that had received prior exposure to atrazine and simazine exhibited comparable atrazine degradation rates, while cells exposed to cyanazine, propazine, ametryne, cyanuric acid, 2-hydroxyatrazine, biuret, and urea exhibited a lack of atrazine-degradative activity. Growth in the presence of exogenous inorganic nitrogen inhibited subsequent atrazine-degradative activity in washed-cell suspensions, suggesting that regulation of s-triazine and nitrogen metabolism are linked in this bacterial isolate. These findings have significant implications for the environmental fate of s-triazines in agricultural settings since these herbicides are frequently applied to soils receiving N fertilizers. Furthermore, these results suggest that bioremediation of s-triazine-contaminated sites (common at pesticide distribution facilities in the cornbelt) may be inhibited by the presence of N fertilizers that occur as co-contaminants. Received: 3 March 1998 / Received revision: 24 September 1998 / Accepted: 11 October 1998     

Marinobacter persicus sp. nov., a moderately halophilic bacterium from a saline lake in Iran

A Gram-negative, non-endospore-forming, rod shaped, strictly aerobic, moderately halophilic bacterium, designated strain M9B^T, was isolated from the hypersaline lake Aran-Bidgol in Iran. Cells of strain M9B^T were found to be motile and produce colonies with an orange-yellow pigment. Growth was determined to occur between 5 and 20 % (w/v) NaCl and the isolate grew optimally at 7.5–10 % (v/w) NaCl. The optimum pH and temperature for growth of the strain were determined to be pH 7.0 and 35 °C, respectively, while it was able to grow over pH and temperature ranges of 6–8 and 25–45 °C, respectively. Phylogenetic analysis based on the comparison of 16S rRNA gene sequences revealed that strain M9B^T is a member of the genus Marinobacter. The closest relative to this strain was found to be Marinobacter hydrocarbonoclasticus MBIC 1303^T with a similarity level of 97.7 %. DNA–DNA hybridization between the novel isolate and this phylogenetically related species was 13 ± 2 %. The major cellular fatty acids of the isolate were identified as C_16:0, C_19:1 ω6c, C_18:1 ω9c and C_16:1 ω9c. The polar lipid pattern of strain M9B^T was determined to consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and three phospholipids. Ubiquinone 9 (Q-9) was the only lipoquinone detected. The G+C content of the genomic DNA of this strain was determined to be 58.6 mol%. Phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness data suggest that this strain represents a novel species of the genus Marinobacter, for which the name Marinobacter persicus sp. nov. is proposed. The type strain of Marinobacter persicus is strain M9B^T (=IBRC-M 10445^T = CCM 7970^T = CECT 7991^T = KCTC 23561^T).     

Oceanospirillum nioense sp. nov., a marine bacterium isolated from sediment sample of Palk bay, India

A novel Gram-negative, spiral shaped, motile bacterium, designated strain NIO-S6^T, was isolated from a sediment sample collected from Off-shore Rameswaram, Tamilnadu, India. Strain NIO-S6^T was found to be positive for oxidase, DNase and lysine decarboxylase activities and negative for catalase, gelatinase, lipase, ornithine decarboxylase, nitrate reductase, aesculinase, amylase and urease activities. The fatty acids were determined to be dominated by C_10:0 3OH, C_16:0, C_16:1 and C_18:1. Strain NIO-S6^T contains Q-8 as the major respiratory quinone. The DNA G+C content of the strain NIO-S6^T was determined to be 49.5 ± 0.6 mol %. Phylogenetic analysis based on 16S rRNA gene sequence of strain NIO-S6^T indicated Oceanospirillum linum and Oceanospirillum maris of the family Oceanospirillaceae (phylum Proteobacteria) are the closest related species with sequence similarities of 98.4 and 97.8 % respectively. Other members of the family showed sequence similarities <96.4 %. However, DNA–DNA hybridization with Oceanospirillum linum LMG 5214^T and Oceanospirillum maris LMG 5213^T showed a relatedness of 31.5 and 46.9 % with respect to strain NIO-S6^T. Based on the phenotypic characteristics and on phylogenetic inference, strain NIO-S6^T is proposed as a novel species of the genus Oceanospirillum as Oceanospirillum nioense sp. nov. and the type strain is NIO-S6^T (=MTCC 11154^T = KCTC 32008^T).     

Nonhongiella spirulinensis gen. nov., sp. nov., a bacterium isolated from a cultivation pond of Spirulina platensis in Sanya, China

A Gram-negative, aerobic, motile rod strain, designated Ma-20^T, was isolated from a pool of marine Spirulina platensis cultivation, Sanya, China, and was subjected to a polyphasic taxonomy study. Strain Ma-20^T can grow in the presence of 0.5–11 % (w/v) NaCl, 10–43 °C and pH 6–10, and grew optimally at 30 °C, pH 7.5–9.0 in natural seawater medium. The polar lipids were composed of phosphatidylethanolamine, three unidentified phospholipids and three unidentified polar lipids. The respiratory quinone was ubiquinone 8 (Q-8) and the major fatty acids were C_18:1ω6c/C_18:1ω7c (summed feature 8, 32.84 %), C_16:1ω6c/C_16:1ω7c (summed feature 3, 30.76 %), C_16:0 (13.54 %), C_12:03-OH (4.63 %), and C_12:0 (4.09 %). The DNA G+C content of strain Ma-20^T was 58 mol %. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain Ma-20^T belonging to Gammaproteobacteria, it shared 88.46–91.55 and 89.21–91.26 % 16S rRNA gene sequence similarity to the type strains in genus Hahella and Marinobacter, respectively. In addition to the large 16S rRNA gene sequence difference, Ma-20^T can also be distinguished from the reference type strains Hahella ganghwensis FR1050^T and Marinobacter hydrocarbonoclasticus sp. 17^T by several phenotypic characteristics and chemotaxonomic properties. On the basis of phenotypic, chemotaxonomic and phylogenetic properties, strain Ma-20^T is suggested to represent a novel species of a new genus in Gammaproteobacteria, for which the name Nonhongiella spirulinensis gen. nov., sp. nov. is proposed. The type strain is Ma-20^T (=KCTC 32221^T=LMG 27470^T).     

Hymenobacter qilianensis sp. nov., isolated from a subsurface sandstone sediment in the permafrost region of Qilian Mountains,China and emended description of the genus Hymenobacter

A red–pink, Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain DK6-37 was isolated from the permafrost region of Qilian Mountains in northwest of China. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that this isolate represents a novel member of the genus Hymenobacter, with low sequence similarities (<97 %) to recognized Hymenobacter species. Optimum growth was observed at 28 °C, pH 7.0 and 0 % NaCl. The strain was found to contain MK-7 as the predominant menaquinone. The polar lipids were identified as phosphatidylethanolanmine, two unknown aminophospholipids, one unknown aminolipid and three unknown polar lipids. The major fatty acids were identified as summed feature 3 (C_16:1 ω7c/C_16:1 ω6c as defined by MIDI), summed feature 4 (anteiso-C_17:1 B/iso-C_17:1 I), C_16:1 ω5c, iso-C_17:0 3-OH, iso-C_15:0 and C_18:0. The DNA G + C content was determined to be 67.4 mol %. On the basis of the polyphasic evidence presented, it is proposed that strain DK6-37 represents a novel species of the genus Hymenobacter, for which the name Hymenobacter qilianensis sp. nov. is proposed. The type strain is DK6-37^T (= CGMCC 1.12720^T = JCM 19763^T).