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PubMed2001

Production of PHA from starchy wastewater via organic acids.

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.

Alcaligenes↗
PubMed2000

Denitrification with methane as electron donor in oxygen-limited bioreactors.

The microbial population from a reactor using methane as electron donor for denitrification under microaerophilic conditions was analyzed. High numbers of aerobic methanotrophic bacteria (3 10(7) cells/ml) and high numbers of acetate-utilizing denitrifying bacteria (2 10(7) cells/ml) were detected, but only very low numbers of methanol-degrading denitrifying bacteria (4 10(4) cells/ml) were counted. Two abundant acetate-degrading denitrifiers were isolated which, based on 16S rRNA analysis, were closely related to Mesorhizobium plurifarium (98.4% sequence similarity) and a Stenotrophomonas sp. (99.1% sequence similarity). A methanol-degrading denitrifying bacterium isolated from the bioreactor morphologically resembled Hyphomicrobium sp. and was moderately related to H. vulgare (93.5% sequence similarity). The initial characterization of the most abundant methanotrophic bacterium indicated that it belongs to class II of the methanotrophs. "In vivo" 13C-NMR with concentrated cell suspensions showed that this methanotroph produced acetate under oxygen limitation. The microbial composition of reactor material together with the NMR experiments suggest that in the reactor methanotrophs excrete acetate, which serves as the direct electron donor for denitrification.

Anaerobiosis↗
PubMed2005

Bioremediation of petroleum hydrocarbons in contaminated soils: comparison of biosolids addition, carbon supplementation, and monitored natural attenuation.

Two methods of biostimulation were compared in a laboratory incubation study with monitored natural attenuation (MNA) for total petroleum hydrocarbon (TPH) degradation in diesel-contaminated Tarpley clay soil with low carbon content. One method utilized rapid-release inorganic fertilizers rich in N and P, and the other used sterilized, slow-release biosolids, which added C in addition to N and P. After 8 weeks of incubation, both biostimulation methods degraded approximately 96% of TPH compared to MNA, which degraded 93.8%. However, in the first week of incubation, biosolids-amended soils showed a linear two orders of magnitude increase in microbial population compared to MNA, whereas, in the fertilizer-amended soils, only a one order of magnitude increase was noted. In the following weeks, microbial population in the fertilizer-amended soils dropped appreciably, suggesting a toxic effect owing to fertilizer-induced acidity and/or NH(3) overdosing. Results suggest that biosolids addition is a more effective soil amendment method for biostimulation than the commonly practiced inorganic fertilizer application, because of the abilities of biosolids to supplement carbon. No statistically significant difference was observed between the biostimulation methods and MNA, suggesting that MNA can be a viable remediation strategy in certain soils with high native microbial population.

Bacteria↗
PubMed2003

Advantages of using subsurface flow constructed wetlands for wastewater treatment in space applications: ground-based Mars Base prototype.

Research and design of subsurface flow wetland wastewater treatment systems for a ground-based experimental prototype Mars Base facility has been carried out, using a subsurface flow approach. These systems have distinct advantages in planetary exploration scenarios: they are odorless, relatively low-labor and low-energy, assist in purification of water and recycling of atmospheric CO2, and will support some food crops. An area of 6-8 m2 may be sufficient for integration of wetland wastewater treatment with a prototype Mars Base supporting 4-5 people. Discharge water from the wetland system will be used as irrigation water for the agricultural crop area, thus ensuring complete recycling and utilization of nutrients. Since the primary requirements for wetland treatment systems are warm temperatures and lighting, such bioregenerative systems may be integrated into early Mars base habitats, since waste heat from the lights may be used for temperature maintenance in the human living environment. "Wastewater gardens (TM)" can be modified for space habitats to lower space and mass requirements. Many of its construction requirements can eventually be met with use of in-situ materials, such as gravel from the Mars surface. Because the technology requires little machinery and no chemicals, and relies more on natural ecological mechanisms (microbial and plant metabolism), maintenance requirements are minimized, and systems can be expected to have long operating lifetimes. Research needs include suitability of Martian soil and gravel for wetland systems, system sealing and liner options in a Mars Base, and wetland water quality efficiency under varying temperature and light regimes.

Ecological Systems, Closed↗
PubMed2026

Discovery of Glycosylated β-Amino Acid-Containing Macrolactams from Nonomuraea sp. 0L2P via Genome Mining.

β-Amino acid-containing macrolactams (β-AACMs) are a class of bioactive natural products characterized by nitrogen-containing starter units within polyketide-derived macrocycles. Here, we report four previously undescribed macrolactams, gruelactams A-D (1-4), from Nonomuraea sp. 0L2P, discovered through an integrated approach combining genome mining, 15N-labeling, and antibacterial screening. Their planar structures were elucidated by comprehensive spectroscopic analyses, including 1D and 2D NMR and HRESI-MS, and their configurations were partially assigned based on ROESY data and bioinformatic analysis. Genome sequencing and antiSMASH analysis identified a putative type I polyketide synthase (PKS) biosynthetic gene cluster, enabling the proposal of a biosynthetic pathway. Bioactivity assays showed that gruelactam D (4) exhibits antibacterial activity against Bacillus cereus and Staphylococcus aureus, with MIC values of 8 and 16 μg/mL, respectively. These findings expand the chemical diversity of β-AACMs and demonstrate the utility of genome-guided approaches for discovering bioactive natural products from rare actinomycetes.

Anti-Bacterial Agents↗
PubMed2004

Phenotypic and genotypic analysis of rhizobia isolated from pasture legumes native of Sardinia and Asinara Island.

Thirty-five rhizobial strains were isolated from nodules of Lotus edulis, L. ornithopodioides, L. cytisoides, Hedysarum coronarium, Ornithopus compressus and Scorpiurus muricatus growing in Sardinia and Asinara Island. Basic characteristics applied to identification of rhizobia such as symbiotic properties, antibiotic- and salt-resistance, temperate-sensitivities, utilization of different sources of carbon and nitrogen were studied. The results from the 74 metabolic tests were used for cluster analysis of the new rhizobial isolates and 28 reference strains, belonging to previously classified and unclassified fast-, intermediate- and slow-growing rhizobia. All strains examined were divided into two large groups at a linkage distance of 0.58. None of the reference strains clustered with the new rhizobial isolates, which formed five subgroups almost respective of their plant origin. RFLP analysis of PCR-amplified 16S-23S rDNA IGS showed that the levels of similarity between rhizobial isolates from Ornithopus, Hedysarum and Scorpiurus, and the type strains of Rhizobium leguminosarum, Mesorhizobium loti, M. ciceri, M. mediterraneum, Sinorhizobium meliloti and Bradyrhizobium japonicum were not more than 30%. Thus, it can be assumed that these groups of new rhizobial isolates are not closely related to the validly described rhizobial species.

DNA, Bacterial↗
PubMed1999

Genetic and physiologic characterization of urease of Actinomyces naeslundii.

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of Actinomyces naeslundii, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the ureC gene of A. naeslundii WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the A. naeslundii urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the ureABCEFGD genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5' to the start codon of ureA. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the ureC gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by A. naeslundii via a urease-dependent pathway and that ureolysis can protect A. naeslundii against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to A. naeslundii critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.

Actinomyces↗
PubMed1991

Ruminal digestion and microbial utilization of diets varying in type of carbohydrate and protein.

Three ruminally and duodenally cannulated, lactating Holstein cows were used in a 3 x 3 Latin square experiment to study the effects of differing levels of nonstructural carbohydrate and degradable intake protein on ruminal digestibility and microbial protein production. Three diets were formulated to contain 1) 38 and 13.2%, 2) 31 and 11.8%, and 3) 24 and 9% nonstructural carbohydrate and degradable intake protein as percentages of the DM, respectively. Dry matter intakes were similar for all diets (21.9, 21.1, and 18.3 kg/d for diets 1, 2, and 3, respectively). Likewise, microbial efficiency, as estimated from purine analysis, was unaffected by diet and averaged 24 g of microbial N/kg of OM digested for all treatments. Ruminal digestion of OM averaged 66.6, 65.1, and 55.7% for diets 1, 2, and 3, respectively, resulting in lower microbial N flow per day for diet 3 (317, 333, and 202 g, respectively). Digestion of nonstructural carbohydrate and CP followed similar trends as did OM digestion, whereas NDF digestion remained similar across all diets. These results indicate that nonstructural carbohydrate greater than 24% and ruminally degradable protein greater than 9% of DM will enhance microbial protein flow from the rumen.

Animal Feed↗
PubMed1990

Phosphonate utilization by bacterial cultures and enrichments from environmental samples.

A selection of axenic microbial strains and a variety of environmental samples were investigated with respect to the utilization of a series of natural and xenobiotic phosphonates as the sole phosphorus source for growth. Phosphonate degradation was observed only with bacteria and not with eucaryotic microorganisms. All representatives of the phosphonates examined supported bacterial growth, with the exception of methylphosphonate diethylester. Yet, distinctly different phosphonate utilization patterns were noted between phosphonate-positive strains. C-P bond cleavage by a photosynthetic bacterium is reported for the first time; growing photoheterotrophically, Rhodobacter capsulatus ATCC 23782 was able to utilize 2-aminoethylphosphonate and alkylphosphonates. Bacteria with the potential to utilize at least one of the phosphonate moieties from the xenobiotic phosphonates Dequest 2010, Dequest 2041, and Dequest 2060 were detected in all environments, with only two exceptions for Dequest 2010. Phosphonate P utilization to an extent of 94 and 97%, for Dequest 2010 and Dequest 2041, respectively, provided evidence that a complete breakdown of these compounds with respect to the C-P bond cleavage can be achieved by some bacteria. The results suggest that phosphonate-utilizing bacteria are ubiquitous, and that selected strains can degrade phosphonates that are more complex than those described previously.

Bacteria↗
PubMed1992

Effect of formic acid or formaldehyde treatment of alfalfa silage on nutrient utilization by dairy cows.

Third-cutting alfalfa with 37% DM was ensiled untreated or treated with either 2.8 g of formic acid/100 g of DM or .31 g of formaldehyde/100 g of DM and fed to lactating dairy cows in two experiments. Silage treated with formic acid had the lowest pH and concentrations of NPN, NH3, and total free AA. Both treatments decreased rumen in vitro protein degradability but did not affect in vitro rumen plus pepsin digestibility. In trial 1, part 1, 22 Holstein cows received a standard diet for 18 d postpartum and then were fed for 6 wk one of three diets containing 98% alfalfa silage DM. Although DMI was comparable, yields of milk, SCM, fat, protein, lactose, and SNF were higher when treated silages were fed. Plasma concentrations of branched-chain, essential, and total AA increased when formic acid-treated silage was fed. Rumen pH and concentrations of NH3 and VFA were similar for all diets. Rumen escape protein, estimated using 15N as a microbial protein marker, was increased more by formic acid than by formaldehyde treatment. In trial 1, part 2, supplementation with 4.8% fish meal increased concentration of milk protein and yields of milk, protein, lactose, and SNF. Milk urea concentration was higher on the untreated silage diet. Total tract apparent DM and N digestibilities were not affected by silage treatment, although fish meal decreased apparent DM digestibility. In trial 2, 80:20 alfalfa silage:ground corn diets were fed to 12 midlactation cows in a 3 x 3 Latin square study. Milk production was unaffected, but milk protein concentration and DMI were higher when treated silages were fed. Feeding treated silages increased plasma concentrations of branched-chain AA, essential AA, and total AA. Formaldehyde and especially formic acid treatment effectively improved utilization of nutrients in alfalfa silage by lactating dairy cows.

Amino Acids↗
PubMed1995

Food intake, nutrient utilization and water turnover in the lesser mouse-deer (Tragulus javanicus) given lundai (Sapium baccatum).

Voluntary food intake, digestibility and water turnover were determined in adult Malaysian lesser mouse-deer (Tragulus javanicus) given unlimited access to lundai foliage (Sapium baccatum). Daily dry matter (DM) intake was 42.4 g/kg metabolic live mass (M0.73) or 3.7% M. Digestible energy intake was 853 kJ/day (571 kJ metabolisable energy per M0.73), calculated to be used with 79% efficiency. Apparent digestibility (%) of organic matter was 83.8, crude fibre 63.7, acid detergent fibre 60.5, neutral detergent fibre 72.1 and crude protein 65.0. Urinary excretion of the purine derivative, allantoin, was 0.05 mg/g digestible DM intake suggesting rumen microbial yield efficiency may be lower than in other ruminant species. Total water intake was 182 ml/M0.82. The body-water content of the fed mouse-deer, from tritiated water dilution, was 77% M, consistent with a very lean carcass. Turnover of body water was 17% per day. The mouse-deer produced relatively dry, well-defined faecal pellets.

Allantoin↗
PubMed2003

Utilization of the buffering capacity of corn steep liquor in bacterial cellulose production by Acetobacter xylinum.

Acetobacter xylinum BPR2001 produces water-insoluble bacterial cellulose (BC). Using a pH sensor for the accurate control of pH, which is one of the most critical factors for efficient BC production, is difficult especially in a baffled shake-flask and an airlift reactor. The buffering capacity of corn steep liquor (CSL) was estimated by measuring beta (buffering capacity) values in advance and was used to maintain the pH within the optimal range during the production of BC. When CSL was added to either a shake-flask, a stirred-tank reactor or an airlift reactor, BC production was almost the same as that in cultivations where pH was controlled manually or by a pH sensor.

Bioreactors↗
PubMed1987

Diel interactions of oxygenic photosynthesis and n(2) fixation (acetylene reduction) in a marine microbial mat community.

Diel variations in N(2) fixation (acetylene reduction), CO(2) fixation, and oxygen concentrations were measured, on three separate occasions, in a marine microbial mat located on Shackleford Banks, North Carolina. Nitrogenase activity (NA) was found to be inversely correlated with CO(2) fixation and, in two of the three diel periods studied, was higher at night than during the day. Oxygen concentrations within the top 3 mm of the mat ranged from 0 to 400 muM on a diel cycle; anaerobic conditions generally persisted below 4 mm. NA in the mat was profoundly affected by naturally occurring oxygen concentrations. Experimentally elevated oxygen concentrations resulted in a significant depression of NA, whereas the addition of the Photosystem II inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea decreased oxygen concentrations within the mat and resulted in a significant short-term enhancement of NA. Mat N(2)-fixing microorganisms include cyanobacteria and heterotrophic, photoautotrophic, and chemolithotrophic eubacteria. Measured (whole-mat) NA is probably due to a combination of the NA of each of these groups of organisms. The relative contributions of each group to whole-mat NA probably varied during diel and seasonal (successional) cycles. Reduced compounds derived from photosynthetic CO(2) fixation appeared to be an important source of energy for NA during the day, whereas heterotrophic or chemolithotrophic utilization of reduced compounds appeared to be an important source of energy for NA at night, under reduced ambient oxygen concentrations. Previous estimates of N(2) fixation calculated on the basis of daytime measurements may have seriously underestimated diel and seasonal nitrogen inputs in mat systems.

Journal Article↗
PubMed1993

Identification of the sources of energy for nitrogen fixation and physiological characterization of nitrogen-fixing members of a marine microbial mat community.

Experimental manipulations of a microbial mat community were performed to determine sources of energy and reductant used for nitrogen fixation and to physiologically characterize the responsible diazotrophs. The dominant photolithotrophic members of this community were nonheterocystous cyanobacteria, but other potential nitrogen-fixing microorganisms were also present. Pronounced diel variability in rates of acetylene reduction was observed, with nighttime rates a factor of three to four higher than daytime rates. Acetylene reduction measured at night was dependent upon the occurrence of oxygenic photosynthesis the preceding day; mats incubated in the dark during the daytime reduced acetylene at rates comparable to those of light-incubated mats but were not able to reduce acetylene at the normally high rates the following night. The addition of various exogenous carbon compounds to these dark-incubated mats did not elicit nighttime acetylene reduction. Nighttime acetylene reduction apparently proceeds under anoxic conditions in these mats; the highest rates of acetylene reduction occur late at night. Additions of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of oxygenic photosynthesis) to mats resulted in a pronounced stimulation of acetylene reduction during the day, but acetylene reduction the next night proceeded at greatly reduced rates (relative to untreated mats). This daytime stimulation, under the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-induced anoxic conditions in the experimentally treated mats, was light dependent. These results suggest that nitrogen fixation in these mats may be attributed to the activities of nonheterocystous cyanobacteria utilizing storage products of oxygenic photosynthesis under anoxic conditions at night.

Journal Article↗
PubMed1978

The biology of the gonococcus.

Gonorrhea has been known since antiquity. Today, this disease is the most commonly reported infectious disease in the U.S. The natural environment of the etiological agent, Neisseria gonorrhoeae, is man. In this host, the organism usually parasitizes mucosal surfaces populated by columnar epithelial cells. Under certain conditions, the gonococcus may disseminate or spread to adjacent organs. The gonococcus is well adapted to its environment and is a successful parasite. Until recently, gonococci were uniformly sensitive to penicilin. However, a plasmid encoding beta-lactamase has been identified in some isolates. Most strains exhibit specific requirements for various amino acids, vitamins, purines, and pyrimidines. Only glucose, pyruvate, and lactate are utilized as sources of energy. Glucose is dissimilated by a combination of the Entner-Doudoroff and pentose phosphate pathways. A tricarboxylic acid cycle is also present and active under certain conditions. Structurally, the cell envelope of the gonococcus resembles that of a typical Gram-negative bacterium. Gonococci are highly autolytic, especially in older cultures or after depletion of the energy source. Autolysis is not due solely to peptidoglycan hydrolysis, but appears to involve a destabilization of the outer membrane as well. Cell surface components such as pili, lipopolysaccharide, outer membrane proteins, and a capsule are associated with the virulence and pathogenicity of this organism.

Amino Acids↗
PubMed1983

The nutritive value of rumen micro-organisms in ruminants. 2. The apparent digestibility and net utilization of microbial N for growing lambs.

Four experiments were conducted with eighteen lambs sustained entirely by intragastric nutrition at gross energy inputs varying from 430 to 860 kJ/kg live weight0.75 (W0.75). Isolated rumen micro-organisms (RMO) were infused into the abomasum in quantities varying from 0 to 2 g digestible N/kg W0.75 to assess the increase in N balance as a result of increasing RMO input when N was limiting. The over-all utilization of N from RMO (RMO-N) could be described by the equation y = 0.543 X -0.457, residual SD = 0.037, where y is the N balance and X is the abomasal input of RMO-N, both expressed in g/kg W0.75. Thus the coefficient of efficiency of utilization of infused RMO-N was 0.543 (SE 0.008). The coefficient of efficiency of utilization of RMO-N truly digested (i.e. the biological value) was 0.659 (SE 0.015). The RMO-N input (mean with SE) at N equilibrium was 0.843 (0.009) g/kg W0.75. The true digestibility of RMO-N was 0.813 (0.004). The urinary N excretion when no N was infused was 0.329 (0.008) g/kg W0.75 and the N excreted via the faeces with zero N input was 0.036 (0.009) g/kg W0.75.

Amino Acids↗
PubMed1983

Direct selection of Saccharomyces cerevisiae resistant to the antibiotic G418 following transformation with a DNA vector carrying the kanamycin-resistance gene of Tn903.

We have developed a new procedure for selecting yeast transformants without the need for complementing auxotrophic markers. The procedure is based on resistance to antibiotic G418 imparted to transformants by recombinant DNA vectors. We constructed several Escherichia coli-yeast shuttle vectors containing the kanamycin (G418)-resistance gene of Tn903, plus several yeast genes making dual selections possible. The efficiency for selecting G418-resistant transformants was dependent upon several factors including the composition of the growth medium and the time at which G418 selective pressure was administered. Media which contained levels of salts found in yeast nitrogen base rendered cells partially to completely resistant to G418 and could not be used for selecting G418-resistant transformants. On the other hand, untransformed cells remained sensitive to G418 when grown on YEPD medium thus allowing selection of G418-resistant transformants. A lag phase of 12 to 18 h, following growth at 30 degrees C, was required prior to administration of G418 to achieve maximal transformation frequency. Transformation frequencies ranged from 100 to 700 per micrograms of DNA and varied with the vector and strain used. The kanamycin gene imparted resistance to G418 in either the episomally or chromosomally integrated state. The gene was highly stable in the integrated state, even without selective pressure. The utility of the procedure was demonstrated by selecting transformants of four different strains of Saccharomyces cerevisiae and by cloning autonomous replication sequences (ARS) from the yeast Kluyveromyces lactis. We believe that this or related procedures could be used to develop transformation systems for many eukaryotic and prokaryotic cells for which no transformation procedure is available.

DNA, Recombinant↗
PubMed2004

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization.

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH(4)(+) as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (alpha-Proteobacteria), 23%; gamma-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. alpha- and gamma-Proteobacteria dominated the utilization of [(14)C]acetic acid and (14)C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-D-[1-(14)C]glucosamine (NAG). The GNSB accounted for 9% of the (14)C-amino acid-consuming bacteria and 27% of the [(14)C]NAG-consuming bacteria but did not utilize [(14)C]acetic acid. Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.

Autoradiography↗