Assimilatory Sulfur Metabolism in Marine Microorganisms: Considerations for the Application of Sulfate Incorporation into Protein as a Measurement of Natural Population Protein Synthesis

The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [³⁵S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of ³⁵S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [³⁵S]sulfate incorporation was compared with [¹⁴C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with ³⁵S. Redistribution of ¹⁴C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [³⁵S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.     

Biofilm Dynamics and Kinetics during High-Rate Sulfate Reduction under Anaerobic Conditions

The sulfate kinetics in an anaerobic, sulfate-reducing biofilm were investigated with an annular biofilm reactor. Biofilm growth, sulfide production, and kinetic constants (K_m and V_max) for the bacterial sulfate uptake within the biofilm were determined. These parameters were used to model the biofilm kinetics, and the experimental results were in good agreement with the model predictions. Typical zero-order volume rate constants for sulfate reduction in a biofilm without substrate limitation ranged from 56 to 93 μmol of SO²₄-cm⁻³ h⁻¹ at 20°C. The temperature dependence (Q₁₀) of sulfate reduction was equivalent to 3.4 at between 9 and 20°C. The measured rates of sulfate reduction could explain the relatively high sulfide levels found in sewers and wastewater treatment systems. Furthermore, it has been shown that sulfate reduction in biofilms just a few hundred micrometers thick is limited by sulfate diffusion into biofilm at concentrations below 0.5 mM. This observation might, in some cases, be an explanation for the relatively poor capacity of the sulfate-reducing bacteria to compete with the methanogenic bacteria in anaerobic wastewater treatment in submerged filters.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain identification of phospholipids as calcium-independent activators.

Phosphatidyl inositol and lysophosphatidyl choline have been identified as activators of a partially purified brain cyclic nucleotide phosphodiesterase previously shown to be regulated in vitro by Ca²⁺ and a Ca²⁺-binding protein. Microgram quantities of either phospholipid produced a linear, immediate and reversible activation of the enzyme in the absence of Ca²⁺ and the Ca²⁺-dependent regulator (CDR). Fatty acids were also found to activate the phosphodiesterase to varying degrees, with oleic acid being the most effective. Phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine and lysophosphatidyl ethanolamine were not effective as activators. Only sodium dodecyl sulfate, of a variety of nonionic, cationic, and anionic detergents tested, activated the phosphodiesterase. Sodium dodecyl sulfate produced a modest degree of activation over a narrow concentration range, followed by enzyme denaturation at higher concentrations.The interaction of the phosphodiesterase with the phospholipid activators has been compared to its interaction with the Ca²⁺·CDR complex. Both Ca²⁺·CDR and lysophosphatidyl choline decreased the thermal stability of the enzyme to a similar extent. The apparent K_m of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 30 μm with guanosine-3′,5′-monophosphate (cGMP) as substrate and 1 mm with adenosine-3′,5′-monophosphate (cAMP) as substrate. With increasing lysophosphatidyl choline concentration, the apparent K_m for each nucleotide remained unchanged while the V increased. The apparent K_d for Mg²⁺ of the lysophosphatidyl choline-dependent phosphodiesterase activity was approximately 3 μm and was unaffected by lysophosphatidyl choline concentration. Activation of the phosphodiesterase by lysophosphatidyl choline was characterized by a high degree of positive cooperativity, exhibiting a Hill coefficient of 3.8. Fluphenazine was a competitive inhibitor of both Ca²⁺·CDR and lysophosphatidyl choline activation of the enzyme.     

3-D-QSAR and docking studies on the neuronal choline transporter

The high affinity neuronal choline transporter (CHT1) is responsible for the uptake of choline into the pre-synaptic terminal of cholinergic neurons. Considering our past experience with modeling the blood–brain barrier choline transporter (BBBCHT) as drug delivery vector to the CNS, we investigated the 3-D-quantitative structure–activity relationship of the neuronal choline transporter. Comparative molecular field analysis (CoMFA) and comparative similarity index analysis (CoMSIA) yielded cross-validated models with a q² of 0.5, and a non-cross validated r² of 0.8. The electrostatic results of the 3-D-QSAR models are corroborated with a docking study into the bacterial choline transporter. Using this electrostatic map, we propose a putative binding site in a homology model of the CHT1. Knowledge gained from this study is useful to better understand the CHT1 as well as can be used in medicinal chemistry programs targeting this transporter.     

Cloning, Expression, and Purification of Choline Dehydrogenase from the Moderate Halophile Halomonas elongata

Choline dehydrogenase (EC 1.1.99.1) catalyzes the four-electron oxidation of choline to glycine-betaine via a betaine-aldehyde intermediate. Such a reaction is of considerable interest for biotechnological applications in that transgenic plants engineered with bacterial glycine-betaine-synthesizing enzymes have been shown to have enhanced tolerance towards various environmental stresses, such as hypersalinity, freezing, and high temperatures. To date, choline dehydrogenase has been poorly characterized in its biochemical and kinetic properties, mainly because its purification has been hampered by instability of the enzyme in vitro. In the present report, we cloned and expressed in Escherichia coli the betA gene from the moderate halophile Halomonas elongata which codes for a hypothetical choline dehydrogenase. The recombinant enzyme was purified to more than 70% homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by treatment with 30 to 50% saturation of ammonium sulfate followed by column chromatography using DEAE-Sepharose. The purified enzyme showed similar substrate specificities with either choline or betaine-aldehyde as the substrate, as indicated by the apparent V/K values (where V is the maximal velocity and K is the Michaelis constant) of 0.9 and 0.6 μmol of O₂ min⁻¹ mg⁻¹ mM⁻¹ at pH 7 and 25°C, respectively. With 1 mM phenazine methosulfate as the primary electron acceptor, the apparent V_max values for choline and betaine-aldehyde were 10.9 and 5.7 μmol of O₂ min⁻¹ mg⁻¹, respectively. These V_max values decreased four- to sevenfold when molecular oxygen was used as the electron acceptor. Altogether, the kinetic data are consistent with the conclusion that H. elongata betA codes for a choline dehydrogenase that can also act as an oxidase when electron acceptors other than molecular oxygen are not available.     

Structural analysis by mass spectrometry and NMR spectroscopy of the glycolipid sulfate from Halobacterium salinarium and a note on its possible function

The major glycolipid sulfate of the extreme halophile Halobacterium salinarium was isolated and characterised mainly by mass spectrometry and NMR spectroscopy. The mass spectrum of the permethylated, desulfated and trimethylsilylated derivative showed the molecule to be a trihexosyl glycerol C₂₀-diether with the sulfate group on the terminal hexose. A 3-position of the sulfate was indicated by the mass spectrum obtained after acetylation and trimethylsilylation (solvolysis of sulfate and replacement by a trimethylsilyl group). The NMR spectrum of the desulfated permethylated glycolipid gave conclusive evidence for the presence of one β and two α anomeric protons. With the knowledge of degradation data it was possible to assign the β signal to galactose (terminal hexone), and the α signals to glucose and mannose. These data together make it likely that the glycolipid sulfate is identical in structure with the glycolipid from Halobacterium cutirubrum characterised previously (M. Kates and P.W. Deroo, J. Lipid Res., 14 (1973) 438).On the basis of a suggested function of cerebroside sulfate of animal origin (identical polar end with the bacterial glycolipid: β-galactopyranose-3-sulfate) and the present knowledge of ion transport in Halobacteria, it is proposed that the bacterial glycolipid may function as a selective K⁺ receptor for the K⁺ transport from a high-Na⁺ and low-K⁺ outside medium.     

Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S⁰ under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S₈) and inorganic polysulfides (S_n²⁻). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that S_n²⁻ species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS⁻ as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S⁰ deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.     

Biofilm growth kinetics of a monomethylamine producing Alphaproteobacteria strain isolated from an anaerobic reactor

Industrial fishing effluents are characterized by high loads of protein and sulfate that stimulate the activity of proteolytic and sulfate reducing bacteria during anaerobic digestion. Their metabolic products (NH₃ and H₂S respectively) have a well-known detrimental effect on the activity of methanogens.Since methylamine is a carbon source used by methylaminotrophic methane producing archaea (mMPA) but not by sulfate reducing bacteria (SRB), enriched mMPA anaerobic biofilms have been developed on ceramics. We propose that methylated amines could be produced in the biofilm by using betaine, a known precursor of methylamine, as a carbon and energy source.We isolated an anaerobic betainotrophic methylaminogenic bacterial strain (bMB) from an anaerobic bioreactor, using betaine as the only carbon and energy source. This strain was identified by a standard biochemical test (API 20NE), cloning, and 16S rDNA sequencing.bMB biofilm structure and biofilm growth kinetic parameters were determined by means of scanning electron microscopy (SEM), and the Gompertz growth model, respectively. Monomethylamine production was determined by infrared spectroscopy and by high pressure liquid chromatography.The isolated bMB strain was determined as Stappia stellulata (Proteobacteria phylum). It was able to form biofilm on ceramics and its kinetic growth parameters resulted in: maximum biofilm bacterial count (A) of 6.25 × 10⁸ UFC/cm² and maximum specific growth rate (μ_m) of 0.022 1/h. Production of monomethylamine was about 4.027 atogram/cell/day (at/cell/day) after 15 days of incubation in biofilms.This study confirms the adhesion capacity of this bMB strain on ceramic supports, assuring that monomethylamine production in biofilms could be enriched with mMPA that use monomethylamine.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Radioisotope Assay for the Quantification of Sulfate-Reducing Bacteria in Sediment and Water

A radioisotope enrichment culture method was developed to estimate the physiologically active component of a population of sulfate-reducing bacteria in environmental water and sediment samples. Aliquots of water or sediment were added to 50-ml serum bottles filled with ³⁵S-sulfate broth incubated for approximately 30 h. After incubation, the disintegration rate per milliliter of spent medium was measured, and the percentage of loss of activity resulting from bacterial sulfate reduction was determined. This loss of sulfate from the medium was then translated to a specific number of Desulfovibrio desulfuricans cells that would reduce an equivalent amount of sulfate in the same incubation time. This comparison was done using a series of growth curves of D. desulfuricans covering a range of inoculum densities between 10² and 10⁷ cells. The radioassay was used to follow the effects of a pulp mill on a small anoxic river in Florida. The activity of the sulfate-reducing bacteria in the river was greatly suppressed when the mill was closed for annual maintenance. The initiation of waste treatment resulted in improved water quality in 1 week, but the river sediments required a month to show a 10-fold reduction in the population of sulfate-reducing bacteria.     

Bacterial Population Dynamics in Dairy Waste during Aerobic and Anaerobic Treatment and Subsequent Storage

The objective of this study was to model a typical dairy waste stream, monitor the chemical and bacterial population dynamics that occur during aerobic or anaerobic treatment and subsequent storage in a simulated lagoon, and compare them to those of waste held without treatment in a simulated lagoon. Both aerobic and anaerobic treatment methods followed by storage effectively reduced the levels of total solids (59 to 68%), biological oxygen demand (85 to 90%), and sulfate (56 to 65%), as well as aerobic (83 to 95%), anaerobic (80 to 90%), and coliform (>99%) bacteria. However, only aerobic treatment reduced the levels of ammonia, and anaerobic treatment was more effective at reducing total sulfur and sulfate. The bacterial population structure of waste before and after treatment was monitored using 16S rRNA gene sequence libraries. Both treatments had unique effects on the bacterial population structure of waste. Aerobic treatment resulted in the greatest change in the type of bacteria present, with the levels of eight out of nine phyla being significantly altered. The most notable differences were the >16-fold increase in the phylum Proteobacteria and the approximately 8-fold decrease in the phylum Firmicutes. Anaerobic treatment resulted in fewer alterations, but significant decreases in the phyla Actinobacteria and Bacteroidetes, and increases in the phyla Planctomycetes, Spirochetes, and TM7 were observed.     

Substrate Uptake by Uncultured Bacteria from the Genus Achromatium Determined by Microautoradiography

Microautoradiography was used to investigate substrate uptake by natural communities of uncultured bacteria from the genus Achromatium. Studies of the uptake of ¹⁴C-labelled substrates demonstrated that Achromatium cells from freshwater sediments were able to assimilate ¹⁴C from bicarbonate, acetate, and protein hydrolysate; however, ¹⁴C-labelled glucose was not assimilated. The pattern of substrate uptake by Achromatium spp. was therefore similar to those of a number of other freshwater and marine sulfur-oxidizing bacteria. Different patterns of radiolabelled bicarbonate uptake were noted for Achromatium communities from different geographical locations and indicated that one community (Rydal Water) possessed autotrophic potential, while the other (Hell Kettles) did not. Furthermore, the patterns of organic substrate uptake within a single population suggested that physiological diversity existed in natural communities of Achromatium. These observations are consistent with and may relate to the phylogenetic diversity observed in Achromatium communities. Incubation of Achromatium-bearing sediment cores from Rydal Water with ³⁵S-labelled sulfate in the presence and absence of sodium molybdate demonstrated that this bacterial population was capable of oxidizing sulfide to intracellular elemental sulfur. This finding supported the role of Achromatium in the oxidative component of a tightly coupled sulfur cycle in Rydal Water sediment. The oxidation of sulfide to sulfur and ultimately to sulfate by Achromatium cells from Rydal Water sediment is consistent with an ability to conserve energy from sulfide oxidation.     

Competition and coexistence of sulfate-reducing bacteria,acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol⁻¹, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol⁻¹, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.     

Microbial Ecophysiology of Whey Biomethanation: Characterization of Bacterial Trophic Populations and Prevalent Species in Continuous Culture

The organization and species composition of bacterial trophic groups associated with lactose biomethanation were investigated in a whey-processing chemostat by enumeration, isolation, and general characterization studies. The bacteria were spatially organized as free-living forms and as self-immobilized forms appearing in flocs. Three dominant bacterial trophic group populations were present (in most probable number per milliliter) whose species numbers varied with the substrate consumed: hydrolytic, 10¹⁰; acetogenic, 10⁷ to 10¹⁰; and methanogenic, 10⁶ to 10⁹. The three prevalent species utilizing lactose were identified as Leuconostoc mesenteroides, Klebsiella oxytoca, and Clostridium butyricum. Clostridium propionicum and Desulfovibrio vulgaris were the dominant lactate-consuming, hydrogen-producing acetogenic bacteria, while D. vulgaris was the only significant ethanol-degrading species. Methanosarcina barkeri and Methanothrix soehngenii were identified as the dominant acetate-utilizing methanogens, and Methanobacterium formicicum was the prevalent hydrogen-utilizing methanogen. A microbial food chain is proposed for lactose biomethanation that comprises multiple species in three different groups, with the major hydrogen-producing acetogen being a sulfate-reducing species, D. vulgaris, which functioned in the absence of significant levels of environmental sulfate.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.     

Dynamics of Bacterial Sulfate Reduction in a Eutrophic Lake

Bacterial sulfate reduction in the surface sediment and the water column of Lake Mendota, Madison, Wis., was studied by using radioactive sulfate (³⁵SO₄²⁻). High rates of sulfate reduction were observed at the sediment surface, where the sulfate pool (0.2 mM SO₄²⁻) had a turnover time of 10 to 24 h. Daily sulfate reduction rates in Lake Mendota sediment varied from 50 to 600 nmol of SO₄²⁻ cm⁻³, depending on temperature and sampling date. Rates of sulfate reduction in the water column were 10³ times lower than that for the surface sediment and, on an areal basis, accounted for less than 18% of the total sulfate reduction in the hypolimnion during summer stratification. Rates of bacterial sulfate reduction in the sediment were not sulfate limited at sulfate concentrations greater than 0.1 mM in short-term experiments. Although sulfate reduction seemed to be sulfate limited below 0.1 mM, Michaelis-Menten kinetics were not observed. The optimum temperature (36 to 37°C) for sulfate reduction in the sediment was considerably higher than in situ temperatures (1 to 13°C). The response of sulfate reduction to the addition of various electron donors metabolized by sulfate-reducing bacteria in pure culture was investigated. The degree of stimulation was in this order: H₂ > n-butanol > n-propanol > ethanol > glucose. Acetate and lactate caused no stimulation.     

Uptake of Choline and Its Conversion to Glycine Betaine by Bacteria in Estuarine Waters

The uptake and degradation of nanomolar levels of [methyl-¹⁴C]choline in estuarine water samples and in seawater filtrate cultures composed mainly of natural free-living bacteria was studied. Uptake of [¹⁴C]choline exhibited Michaelis-Menten kinetics, with K_t + S_n values of 1.7 to 2.9 nM in filtrate cultures and 1.7 to 4.1 nM in estuarine-water samples. V_max values ranged from 0.5 to 3.3 nM · h⁻¹. The uptake system for choline in natural microbial assemblages therefore displays very high affinity and appears able to scavenge this compound at the concentrations expected in seawater. Uptake of choline was inhibited by some natural structural analogs and p-chloromercuribenzoate, indicating that the transporter may be multifunctional and may involve a thiol binding site. When 11 nM [¹⁴C]choline was added to water samples, a significant fraction (>50%) of the methyl carbon was respired to CO₂ in incubations lasting 10 to 53 h. Cells taking up [¹⁴C]choline produced [¹⁴C]glycine betaine ([¹⁴C]GBT), and up to 80% of the radioactivity retained by cells was in the form of GBT, a well-known osmolyte. Alteration of the salinity in filtrate cultures affected the relative proportion of [¹⁴C]choline degraded or converted to [¹⁴C]GBT, without substantially affecting the total metabolism of choline. Increasing the salinity from 14 to 25 or 35 ppt caused more [¹⁴C]GBT to be produced from choline but less ¹⁴CO₂ to be produced than in the controls. Lowering the salinity to 7 ppt decreased [¹⁴C]GBT production and increased ¹⁴CO₂ production slightly. Intracellular accumulations of [¹⁴C]GBT in the salt-stressed cultures were osmotically significant (34 mM). Choline may be used as an energy substrate by estuarine bacteria and may also serve as a precursor of the osmoprotectant GBT, particularly as bacteria are mixed into higher-salinity waters.     

Effects of sulfate on lactate and C2-, C3- volatile fatty acid anaerobic degradation by a mixed microbial culture

The effects of sulfate on the anaerobic degradation of lactate, propionate, and acetate by a mixed bacterial culture from an anaerobic fermenter fed with wine distillery waste water were investigated. Without sulfate and with both sulfate and molybdate, lactate was rapidly consumed, and propionate and acetate were produced; whereas with sulfate alone, only acetate accumulated. Propionate oxidation was strongly accelerated by the presence of sulfate, but sulfate had no effect on acetate consumption even when methanogenesis was inhibited by chloroform. The methane production was not affected by the presence of sulfate. Counts of lactate- and propionate-oxidizing sulfate-reducing bacteria in the mixed culture gave 4.5×10⁸ and 1.5×10⁶ viable cells per ml, respectively. The number of lactate-oxidizing fermentative bacteria was 2.2×10⁷ viable cells per ml, showing that sulfate-reducing bacteria outcompete fermentative bacteria for lactate in the ecosystem studied. The number of acetoclastic methanogens was 3.5×10⁸ viable cells per ml, but only 2.5×10⁴ sulfate reducers were counted on acetate, showing that acetotrophic methanogens completely predominated over acetate-oxidizing sulfate-reducing bacteria. The contribution of acetate as electron donor for sulfate reduction in the ecosystem studied was found to be minor.     

Benthic decomposition of Zostera marina roots: a controlled laboratory experiment

The initial benthic decomposition of Zostera marina roots was studied in a controlled flow-through chamber experiment for 23 days. Sediment chambers without added roots served as controls. The inflowing and outflowing artificial seawater (ASW) was analyzed for O₂, ΣCO₂, urea-N, NH₄⁺ and NO₂⁻+NO₃⁻. Sediment profiles of Eh, particulate organic carbon (POC) and nitrogen, dissolved organic nitrogen (DON), dissolved free amino acids (DFAA), urea-N, NH₄⁺, DFAA and urea turnover rates, sulfate reduction and counts of total anaerobic heterotrophic bacteria and different functional groups were determined. Fluxes of O₂, ΣCO₂, urea-N and NH₄⁺ were stimulated during root decomposition compared to the unamended control. There were indications of stimulated bacterial growth based on counts of total anaerobic heterotrophic bacteria, anaerobic phosphatase utilizers, ammonifyers and sulfate reducers. Independent estimates of nitrogen and carbon incorporation into bacterial biomass during root decomposition indicate that a major fraction of the nitrogen for microbial growth was mobilized from the indigenous particulate organic nitrogen (PON) pool, whereas the energy source for bacterial growth was mainly obtained from the added eelgrass roots. Most of the nitrogen mineralized during root decomposition was incorporated into the bacterial biomass resulting in a low efflux of urea-N and inorganic nitrogen from the sediment to the water column.