Aerobic and anaerobic growth of rifampin-resistant denitrifying bacteria in soil

The growth and survival of several rifampin-resistant isolates of denitrifying bacteria were examined under anaerobic (denitrifying) and aerobic conditions. Two isolates added to nonsterile Bruno soil at densities of between 10(4) and 10(6) CFU g dry soil-1 exhibited an initial period of growth followed by a gradual decline in numbers. After 28 days, both isolates maintained viable populations of between 10(4) and 10(5) CFU g dry soil-1 under both denitrifying and aerobic conditions. One of the isolates consistently grew better under denitrifying conditions, and the other isolate consistently grew better under aerobic conditions. The relative pattern of denitrifying versus aerobic growth for each organism was not affected by the addition of glucose. The growth yields of the two isolates varied with soil type, but the relative pattern of denitrifying versus aerobic growth was consistent in three soils with greatly different properties. Five of nine isolates introduced into Bruno soil at low population densities (approximately 10(5) CFU g dry soil-1) exhibited better growth after 2 days under denitrifying conditions. It was not possible to predict the prevalence of the denitrifying or aerobic mode of growth in nonsterile soil from the growth characteristics of the isolates in pure cultures or sterile soil.     

Use of Tn5 Mutants To Assess the Role of the Dissimilatory Nitrite Reductase in the Competitive Abilities of Two Pseudomonas Strains in Soil

We examined the influence of soil aeration state and plant root presence on the comparative survival of wild-type bacteria and isogenic Tn5 (Nir(sup-)) mutants lacking the ability to synthesize nitrite reductase. Two denitrifying Pseudomonas strains with different nitrite reductase types were used. Enumeration of bacteria in sterile and nonsterile soils was based on differential antibiotic resistance. The validity of the bacterial models studied (i.e., equal growth of wild-type and mutant bacteria under aerobic conditions and significantly better growth of wild-type bacteria under denitrifying conditions) was verified in pure-culture studies. In sterile soil, both strains survived better under aerobic than under anaerobic conditions. The lower efficiency of denitrification than O(inf2) respiration in supporting bacterial growth explained this result, and the physical heterogeneity of soil did not strongly modify the results obtained in pure-culture studies. In nonsterile soil, one of the Pseudomonas strains survived better under anaerobic conditions while the other competed equally with the indigenous soil microflora under aerobic and anaerobic conditions. However, when the Nir(sup-)-to-total inoculant ratios (wild type plus Nir(sup-) mutant) were analyzed, it appeared that the presence of nitrite reductase conferred on both Pseudomonas strains a competitive advantage for anaerobic environment or rhizosphere colonization. This is the first attempt to demonstrate with isogenic nondenitrifying mutants that denitrification can contribute to the persistence and distribution of bacteria in fluctuating soil environments.     

Competition between Two Isolates of Denitrifying Bacteria Added to Soil

We examined the competitive relationship between two isolates of denitrifying bacteria, both of which grow well under aerobic conditions but differ in their ability to grow under denitrifying conditions. The growth and persistence of the two isolates, added to sterile soil or added to soil previously colonized by the other isolate, were monitored under aerobic and denitrifying (anaerobic) conditions. When isolates were added together to sterile soil, the isolate added at the higher density reduced the growth of the isolate added at the lower density. The magnitude of the growth reduction varied depending on the competitive abilities of the individual isolates and the aeration state of the soil. Prior colonization of soil with one of the isolates conferred a competitive advantage on the colonized isolate but did not lead to the disappearance of the challenging isolate. Fluctuations in aeration state caused large changes in the population density of one isolate and altered the competitive relationship between the two isolates. The competitive effectiveness of each isolate varied with cell density, the degree of prior colonization of the soil by the other isolate, and the aeration state of the soil.     

Isolation and characterization of a new denitrifying spirillum capable of anaerobic degradation of phenol

Two kinds of phenol-degrading denitrifying bacteria, Azoarcus sp. strain CC-11 and spiral bacterial strain CC-26, were isolated from the same enrichment culture after 1 and 3 years of incubation, respectively. Both strains required ferrous ions for growth, but strain CC-26 grew better than strain CC-11 grew under iron-limited conditions, which may have resulted in the observed change in the phenol-degrading bacteria during the enrichment process. Strain CC-26 grew on phenol, benzoate, and other aromatic compounds under denitrifying conditions. Phylogenetic analysis of 16S ribosomal DNA sequences revealed that this strain is most closely related to a Magnetospirillum sp., a member of the alpha subclass of the class Proteobacteria, and is the first strain of a denitrifying aromatic compound-degrading bacterium belonging to this group. Unlike previously described Magnetospirillum strains, however, this strain did not exhibit magnetotaxis. It grew on phenol only under denitrifying conditions. Other substrates, such as acetate, supported aerobic growth, and the strain exhibited microaerophilic features.     

Enumeration of Tn5 mutant bacteria in soil by using a most- probable-number-DNA hybridization procedure and antibiotic resistance

Investigations were made into the utility of DNA hybridization in conjunction with a microdilution most-probable-number procedure for the enumeration of Rhizobium spp. and Pseudomonas putida in soil. Isolates of Rhizobium spp. and P. putida carrying the transposon Tn5 were added to sterile and nonsterile Burbank sandy loam soil and enumerated over time. Soil populations of rhizobia were enumerated by colony hybridization, most-probable-number-DNA hybridization procedure, plate counts, plant infectivity most probable number, and fluorescent antibody counts. Population values compared well for all methods at 5 and 30 days after the addition of cells, although the fluorescent antibody method tended to overestimate the viable population. In nonsterile soil, most-probable-number-DNA hybridization procedure enumerated as few as 10 P. putida Tn5 cells g of soil-1 and 100 R. leguminosarum bv. phaseoli Tn5 cells g of soil-1 and should have utility for following the fate of genetically engineered microorganisms released to the environment. Among the Kmr isolates containing Tn5, approximately 5% gave a dark, more intense autoradiograph when probed with 32P-labeled pGS9 DNA, which facilitated their detection in soil. Hybridization with a pCU101 probe (pGS9 without Tn5) indicated that donor plasmid sequences were being maintained in the bacterial chromosome. Transposon-associated antibiotic resistance was also utilized as a phenotypic marker. Tn5 vector-integrate mutants were successfully enumerated at low populations (10 to 100 cells g of soil-1) in soil by both phenotypic (Kmr) and genotypic (DNA probe) analysis. However, determination of the stability of Tn5 or Tn5 and vector sequences in the bacteria is necessary.     

Characterization of streptomyces lydicus WYEC108 as a potential biocontrol agent against fungal root and seed rots.

The actinomycete Streptomyces lydicus WYEC108 showed strong in vitro antagonism against various fungal plant pathogens in plate assays by producing extracellular antifungal metabolites. When Pythium ultimum or Rhizoctonia solani was grown in liquid medium with S. lydicus WYEC108, inhibition of growth of the fungi was observed. When WYEC108 spores or mycelia were used to coat pea seeds, the seeds were protected from invasion by P. ultimum in an oospore-enriched soil. While 100% of uncoated control seeds were infected by P. ultimum within 48 h after planting, less than 40% of coated seeds were infected. When the coated seeds were planted in soil 24 h prior to introduction of the pathogen, 96 h later, less than 30% of the germinating seeds were infected. Plant growth chamber studies were also carried out to test for plant growth effects and for suppression by S. lydicus WYEC108 of Pythium seed rot and root rot. When WYEC108 was applied as a spore-peat moss-sand formulation (10(8) CFU/g) to P. ultimum-infested sterile or nonsterile soil planted with pea and cotton seeds, significant increases in average plant stand, plant length, and plant weight were observed in both cases compared with untreated control plants grown in similar soils. WYEC108 hyphae colonized and were able to migrate downward with the root as it elongated. Over a period of 30 days, the population of WYEC108 colonized emerging roots of germinating seeds and remained stable (10(5) CFU/g) in the rhizosphere, whereas the nonrhizosphere population of WYEC108 declined at least 100-fold (from 10(5) to 10(3) or fewer CFU/g). The stability of the WYEC108 population incubated at 25 degrees C in the formulation, in sterile soil, and in nonsterile soil was also evaluated. In all three environments, the population of WYEC108 maintained its size for 90 days or more. When pea, cotton, and sweet corn seeds were placed into sterile and nonsterile soils containing 10(6) or more CFU of WYEC108 per g, it colonized the emerging roots. After a 1-week growing period, WYEC108 populations of 10(5) CFU/g (wet weight) of root were found on pea roots in the amended sterile soil environment versus 10(4) CFU/g in amended nonsterile soil. To further study the in vitro interaction between the streptomycete and P. ultimum, mycelia of WYEC108 were mixed with oospores of P. ultimum in agar, which was then used as a film to coat slide coverslips.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enumeration of Tn5 mutant bacteria in soil by using a most- probable-number-DNA hybridization procedure and antibiotic resistance.

Investigations were made into the utility of DNA hybridization in conjunction with a microdilution most-probable-number procedure for the enumeration of Rhizobium spp. and Pseudomonas putida in soil. Isolates of Rhizobium spp. and P. putida carrying the transposon Tn5 were added to sterile and nonsterile Burbank sandy loam soil and enumerated over time. Soil populations of rhizobia were enumerated by colony hybridization, most-probable-number-DNA hybridization procedure, plate counts, plant infectivity most probable number, and fluorescent antibody counts. Population values compared well for all methods at 5 and 30 days after the addition of cells, although the fluorescent antibody method tended to overestimate the viable population. In nonsterile soil, most-probable-number-DNA hybridization procedure enumerated as few as 10 P. putida Tn5 cells g of soil-1 and 100 R. leguminosarum bv. phaseoli Tn5 cells g of soil-1 and should have utility for following the fate of genetically engineered microorganisms released to the environment. Among the Kmr isolates containing Tn5, approximately 5% gave a dark, more intense autoradiograph when probed with 32P-labeled pGS9 DNA, which facilitated their detection in soil. Hybridization with a pCU101 probe (pGS9 without Tn5) indicated that donor plasmid sequences were being maintained in the bacterial chromosome. Transposon-associated antibiotic resistance was also utilized as a phenotypic marker. Tn5 vector-integrate mutants were successfully enumerated at low populations (10 to 100 cells g of soil-1) in soil by both phenotypic (Kmr) and genotypic (DNA probe) analysis. However, determination of the stability of Tn5 or Tn5 and vector sequences in the bacteria is necessary.     

Microbiological Comparison of Surface Soil and Unsaturated Subsurface Soil from a Semiarid High Desert

Thirty-two chemoheterotrophic bacteria were isolated from unsaturated subsurface soil samples obtained from ca. 70 m below land surface in a high desert in southeastern Idaho. Most isolates were gram positive (84%) and strict aerobes (79%). Acridine orange direct counts of microbes in one subsurface sample showed lower numbers than similar counts performed on surface soils from the same location (ca. 5 × 10⁵ versus 2 × 10⁶ cells per g [dry weight] of soil), but higher numbers than those from plate counts performed on the subsurface material. Another sample taken from the same depth at another location showed no evidence of colonies under identical conditions. Soil analyses indicated that subsurface sediments versus surface soils were slightly alkaline (pH 7.9 versus 7.4), had a higher water content (25.7 versus 6.3%), and had lower organic carbon concentrations (0.05 to 0.17 versus 0.25% of soil dry weight). Analyses of biologically relevant gases from the unsaturated subsurface indicated an aerobic environment. As in other unsaturated soil environments, either a high proportion of bacteria in these subsurface sediments are not viable or they are incapable of growth on conventional media under aerobic conditions. The presence and numbers of bacteria in these deep sediments may be influenced by colonization opportunities afforded by periodic percolation of surface water through fractures in overlying strata.     

Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by<Emphasis Type="Italic"> Pseudomonas stutzeri</Emphasis> strain JJ

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.     

Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production(1)

Bacteria capable of denitrification are spread among phylogenetically diverse groups. In the present investigation, molecular methods (amplified ribosomal DNA restriction analysis (ARDRA) and partial 16S rDNA gene sequencing) were used to determine the genetic diversity of culturable denitrifying soil bacteria. The purpose of this work was to study the microbial density and diversity of denitrifying communities isolated from two luvisols and a rendosol. The denitrifying bacterial density was significantly higher in the two luvisols (3x10(6) and 4x10(6) bacteria g(-1) dry soil) than in the rendosol (4x10(5) bacteria g(-1) dry soil). Denitrifying isolates from soils were grouped according to the similarity of their restriction patterns into 26 ARDRA types. Interestingly ARDRA analysis suggests that some denitrifying isolates are specific to a soil type while others seem to be geographically widespread. The number of individual isolates found in each ARDRA type appeared to be highly variable between the two sampling dates but some denitrifying types were capable of persisting in soil. The tree obtained from the partial sequences revealed five major branches exhibiting highest identity to the following genera: (i) Burkholderia-Ralstonia, (ii) Pseudomonas, (iii) Xanthomonas-Frateuria, (iv) Bacillus and (v) Streptomyces. Our 16S rDNA-based analysis clearly reveals broad diversity exceeding that previously described in the literature.     

Influence of aerobic microorganisms upon virus survival in soil

Survival of human poliovirus type 1 in a sandy loam soil appeared to be deleteriously influenced by aerobic microorganisms. This effect was determined by comparing the survival of virus in soil under four different possible combinations of aerobic versus anaerobic (H2-CO2) atmosphere and sterile versus nonsterile condition. Storage of samples was done in humid chambers to prevent soil desiccation. The effect attributed to aerobic microorganisms was measurable and statistically significant at all three incubation temperatures used in the study (1, 23, and 37 degrees C), with the increase in inactivation rate attributable to aerobic microorganisms generally being two to threefold. No comparable effect was observed to occur for anaerobic microorganisms under the sets of conditions employed in the study.     

Hydrocarbon bioremediation potential of an unimpacted Kuwaiti oil-field environment

Seasonal variations in the hydrocarbon-degrading potential of soil samples from an unimpacted site in the Kuwaiti Burgan oil field environment were studied under mesophilic conditions. Hydrocarbon-degrading microorganisms occurred but varied all-year-round, and their numbers ranged from 1.3 x 10(7) to 9.3 x 10(7) CFU g(-1) dry soil, while hydrocarbon-degrading fungi ranged from 3.0 x 10(4) - 3.8 x 10(5) CFU g(-1) dry soil, depending on the sampling period. These hydrocarbon-degraders also comprised variable but generally high proportions of the total aerobic heterotrophic organisms (2 to > 98%) for bacteria and lower levels (7-9%) for fungi. The crude oil-degrading capacity of the oil-degrading populations (bacteria and fungi) ranged from 80-95% of the hexane-extractable fractions. Differential inhibition studies carried out on soil samples showed that bacteria were the greater contributors to hydrocarbon degradation (79-92%) than fungi. Pure hydrocarbon substrates, hexadecane and phenanthrene, were degraded to near completion after a 28-day incubation by both the bacterial and fungal portions of the soil flora.     

Effect of Parental Growth on Dynamics of Conjugative Plasmid Transfer in the Pea Spermosphere

Plasmid transfer rates for the conjugative plasmid R388::Tn1721 from Pseudomonas cepacia (donor) to Pseudomonas fluorescens (recipient) on agar media, in broth, and in microcosms containing sterile or nonsterile soil, in the presence or absence of germinating pea seeds, were determined. Donors, recipients, and transconjugants were enumerated on selective media after 1 day on agar or in broth culture and over a 7-day period in soil or pea spermosphere microcosms. Donor and recipient growth rates and plasmid transfer rate constants [(gamma), where (gamma) = transconjugants (middot) (donors (middot) recipients)(sup-1) (middot) h(sup-1)] were calculated for three initial parental densities (10(sup4), 10(sup6), or 10(sup8) CFU/g or ml) in each system. For all initial density levels, values of (gamma) in agar and broth matings were higher than those in soil or in the pea spermosphere-rhizosphere microcosms. Values of (gamma) were not influenced by the pea spermosphere or by sterile or nonsterile conditions of the soil. However, (gamma) values in microcosm experiments were inversely related to initial parental density and were directly related to donor growth rates. Values of (gamma) averaged 4 x 10(sup-10), 4 x 10(sup-12), and 3 x 10(sup-14) when initial donor and recipient cell densities were 10(sup4), 10(sup6), and 10(sup8) CFU/g, respectively. These results suggest that the plasmid transfer rate constant is independent of parental cell density only when parental growth is not limited. In a resource-limited environment, intra- or interspecific competition may reduce the transfer rate by limiting parental growth.     

Degradation of n-hexadecane and its metabolites by Pseudomonas aeruginosa under microaerobic and anaerobic denitrifying conditions

A strategy for sequential hydrocarbon bioremediation is proposed. The initial O(2)-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosa fermentation. The effects of dissolved oxygen (DO) concentration on n-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O(2)-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO(2)(-)-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO(2)(-)-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO(3)(-)-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N(2)). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.     

Growth,photosynthesis, nitrogen fixation and carbohydrate production by a unicellular cyanobacterium,Synechococcus sp. (Cyanophyta)

A polymer-producing strain of unicellular cyanobacteria, Synechococcus sp., was isolated from a coastal lagoon in Florida. This strain, designated BG0011, excreted a highly viscous polysaccharide. Maximum observed growth rates for BG0011 were 2.5 div. day^-2. BG0011 also exhibited nitrogen fixation (nitrogenase) activity under aerobic conditions and grew at near maximum rates in medium lacking reduced nitrogen. Growth and carbohydrate production were enhanced by carbon dioxide enrichment. Rheological study of the extracellular polysaccharide revealed a viscosity versus shear rate curve similar in shape to that of xanthan gum. Maximum observed rate of carbohydrate production was 1 g dry weight liter^-1 month^-1.     

Detection and characterization of plasmid pJP4 transfer to indigenous soil bacteria

Prior to gene transfer experiments performed with nonsterile soil, plasmid pJP4 was introduced into a donor microorganism, Escherichia coli ATCC 15224, by plate mating with Ralstonia eutropha JMP134. Genes on this plasmid encode mercury resistance and partial 2, 4-dichlorophenoxyacetic acid (2,4-D) degradation. The E. coli donor lacks the chromosomal genes necessary for mineralization of 2,4-D, and this fact allows presumptive transconjugants obtained in gene transfer studies to be selected by plating on media containing 2,4-D as the carbon source. Use of this donor counterselection approach enabled detection of plasmid pJP4 transfer to indigenous populations in soils and under conditions where it had previously not been detected. In Madera Canyon soil, the sizes of the populations of presumptive indigenous transconjugants were 10(7) and 10(8) transconjugants g of dry soil(-1) for samples supplemented with 500 and 1,000 microg of 2,4-D g of dry soil(-1), respectively. Enterobacterial repetitive intergenic consensus PCR analysis of transconjugants resulted in diverse molecular fingerprints. Biolog analysis showed that all of the transconjugants were members of the genus Burkholderia or the genus Pseudomonas. No mercury-resistant, 2, 4-D-degrading microorganisms containing large plasmids or the tfdB gene were found in 2,4-D-amended uninoculated control microcosms. Thus, all of the 2,4-D-degrading isolates that contained a plasmid whose size was similar to the size of pJP4, contained the tfdB gene, and exhibited mercury resistance were considered transconjugants. In addition, slightly enhanced rates of 2,4-D degradation were observed at distinct times in soil that supported transconjugant populations compared to controls in which no gene transfer was detected.     

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.     

Microbial development in distillers wet grains produced during fuel ethanol production from corn (Zea mays)

Distillers grains are coproduced with ethanol and carbon dioxide during the production of fuel ethanol from the dry milling and fermentation of corn grain, yet there is little basic microbiological information on these materials. We undertook a replicated field study of the microbiology of distillers wet grains (DWG) over a 9 day period following their production at an industrial fuel ethanol plant. Freshly produced DWG had a pH of about 4.4, a moisture content of about 53.5% (wet mass basis), and 4 x 10(5) total yeast cells/g dry mass, of which about 0.1% were viable. Total bacterial cells were initially below detection limits (ca. 10(6) cells/g dry mass) and then were estimated to be approximately 5 x 10(7) cells/g dry mass during the first 4 days following production. Culturable aerobic heterotrophic organisms (fungi plus bacteria) ranged between 10(4) and 10(5) CFU/g dry mass during the initial 4 day period, and lactic acid bacteria increased from 36 to 10(3) CFU/g dry mass over this same period. At 9 days, total viable bacteria and yeasts and (or) molds topped 10(8) CFU/g dry mass and lactic acid bacteria approached 10(6) CFU/g dry mass. Community phospholipid fatty acid analysis indicated a stable microbial community over the first 4 days of storage. Thirteen morphologically distinct isolates were recovered, of which 10 were yeasts and molds from 6 different genera, 2 were strains of the lactic-acid-producing Pediococcus pentosaceus and only one was an aerobic heterotrophic bacteria, Micrococcus luteus. The microbiology of DWG is fundamental to the assessment of spoilage, deleterious effects (e.g., toxins), or beneficial effects (e.g., probiotics) in its use as feed or in alternative applications.     

Diaphorobacter nitroreducens gen nov,sp nov,a poly(3-hydroxybutyrate)-degrading denitrifying bacterium isolated from activated sludge

Three denitrifying strains of bacteria capable of degrading poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were isolated from activated sludge and characterized. All of the isolates had almost identical phenotypic characteristics. They were motile gram-negative rods with single polar flagella and grew well with simple organic compounds, as well as with PHB and PHBV, as carbon and energy sources under both aerobic and anaerobic denitrifying conditions. However, none of the sugars tested supported their growth. The cellular fatty acid profiles showed the presence of C16:1omega7cis and C16:0 as the major components and of 3-OH-C10:0 as the sole component of hydroxy fatty acids. Ubiquinone-8 was detected as the major respiratory quinone. A 16S rDNA sequence-based phylogenetic analysis showed that all the isolates belonged to the family Comamonadaceae, a major group of beta-Proteobacteria, but formed no monophyletic cluster with any previously known species of this family. The closest relative to our strains was an unidentified bacterium strain LW1 (=DSM 13225) (99.9% similarity), reported previously as a 1-chloro-4-nitrobenzene degrading bacterium. DNA-DNA hybridization levels among the new isolates were more than 60%, whereas those between our isolates and strain DSM 13225 were less than 50%. The G+C content of genomic DNA of the new strains was 64 to 65 mol%. Based on these results, we concluded that the PHBV-degrading denitrifying isolates should be classified as a new genus and a new species, for which we propose the name Diaphorobacter nitroreducens. The type strain is strain NA10B (=JCM 11421=CIP 107294). We also propose to classify strain DSM 13225 as a genospecies of Diaphorobacter.