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Maintenance of soil functioning following erosion of microbial diversity.

The paradigm that soil microbial communities, being very diverse, have high functional redundancy levels, so that erosion of microbial diversity is less important for ecosystem functioning than erosion of plant or animal diversity, is often taken for granted. However, this has only been demonstrated for decomposition/respiration functions, performed by a large proportion of the total microbial community, but not for specialized microbial groups. Here, we determined the impact of a decrease in soil microbial diversity on soil ecosystem processes using a removal approach, in which less abundant species were removed preferentially. This was achieved by inoculation of sterile soil microcosms with serial dilutions of a suspension obtained from the same non-sterile soil and subsequent incubation, to enable recovery of community size. The sensitivity to diversity erosion was evaluated for three microbial functional groups with known contrasting taxonomic diversities (ammonia oxidizers < denitrifiers < heterotrophs). Diversity erosion within each functional group was characterized using molecular fingerprinting techniques: ribosomal intergenic spacer analysis (RISA) for the eubacterial community, denaturing gradient gel electrophoresis (DGGE) analysis of nirK genes for denitrifiers, and DGGE analysis of 16S rRNA genes for betaproteobacterial ammonia oxidizers. In addition, we simulated the impact of the removal approach by dilution on the number of soil bacterial species remaining in the inoculum using values of abundance distribution of bacterial species reported in the literature. The reduction of the diversity of the functional groups observed from genetic fingerprints did not impair the associated functioning of these groups, i.e. carbon mineralization, denitrification and nitrification. This was remarkable, because the amplitude of diversity erosion generated by the dilution approach was huge (level of bacterial species loss was estimated to be around 99.99% for the highest dilution). Our results demonstrate that the vast diversity of the soil microbiota makes soil ecosystem functioning largely insensitive to biodiversity erosion even for functions performed by specialized groups.

Bacteria↗

Propionate oxidation by and methanol inhibition of anaerobic ammonium-oxidizing bacteria.

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min(-1) mg of protein(-1)) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO(2), with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.

Bacteria, Anaerobic↗

Current and future applications of flow cytometry in aquatic microbiology.

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

Animals↗

Microbial colonization and decomposition of carex litter in an arctic lake.

The decomposition and microbial colonization of Carex leaf litter were examined in an arctic lake in Alaska during the summer of 1978. Dried leaf segments in screen bags were placed at various locations and depths for 13 and 26 days. Weight loss varied from 24.15 to 33.56% and from 27.69 to 65.01% after 13 and 26 days, respectively. Abiotic controls lost approximately 19.5% with no subsequent change. Weight loss significantly correlated with microbial colonization as measured by alkaline phosphatase activity (r = 0.780), cellulase activity (r = 0.613), heterotrophic CO(2) fixation (r = 0.835), and acetate incorporation into microbial lipids (r = 0.618). Alkaline phosphatase activity correlated with cellulase activity (r = 0.889), and heterotrophic CO(2) fixation correlated with acetate incorporation into lipids (r = 0.712). Weight loss after 26 days inversely correlated with the logarithm of the depth of incubation regardless of whether incubation occurred on the sediment surface or in the water column. These findings suggest that a rapid initial period of microbial colonization is driven by nutrients derived from the litter and that the rate of these processes is controlled by a factor(s) inversely related to the logarithm of depth, such as light intensity, primary production, or turbulence.

Journal Article↗

Determination of the effect of aluminium sulphate on natural microbial coenoses in experiment.

The methods of determining the effect of chemical compounds on the processes of microbial self-cleaning of waters are mainly based on the calculation of the number of indicator microorganisms grown after being incubated on complex nutrient media following exposure of the waters with the substances under study. These methods examine the reaction of specific microorganisms under conditions rather different from those really existing in the water reservoir. The author proposes to use the method of heterotrophic assimilation of CO2 for the determination of the activity of natural bacterial associations in the study of the effect on them of both multicomponent sewage waters of industrial plants and specific chemical compounds. The determination of the values of bacterial productivity according to heterotrophic assimilation of CO2 by natural microbial coenosis was used as the criterion of the effect of the toxicant. Toxicity of the substance under study is evaluated according to its inhibitory effect on the production of natural bacteriocoenosis (test object). Field of application: water toxicology, supervision of toxicity of aqueous media, evaluation of the quality of water. Standard experiment examining the effect of aluminium sulphate on natural bacterial coenosis is considered an example. It has been demonstrated that the effect of aluminium sulphate begins to be felt in concentrations starting from 1 mg X l-1. Statistical analysis of results was carried out by calculating the mean value and confidence intervals. The investigations have shown that application of the method of heterotrophic assimilation of CO2 for these purposes is feasible and of considerable interest with a view to its high sensitivity and precision.

Alum Compounds↗

Physico-chemical factors affecting the E. coli removal in a rotating biological contactor (RBC) treating UASB effluent.

The removal mechanism of E. coli from UASB effluent using a Rotating Biological Contractor (RBC) has been investigated. Preliminary batch experiments in a RBC indicate a first-order removal kinetics. Variation in the dissolved oxygen concentration and E. coli counts over the depth of the RBC has been recorded and indicates that the RBC is not a completely mixed reactor. Therefore batch experiments were carried out in a beaker where the different operating conditions can be controlled. Factors affecting the removal of E. coli via a biofilm system as stirring, dissolved oxygen concentration, pH, and addition of cationic polymer were investigated. The results obtained indicated that the most important removal mechanism of E. coli in the biofilm is the adsorption process, followed by sedimentation. Die-off is a relatively minor removal mechanism in an RBC system. Higher removal rate of E. coli was observed in an aerobic compared to an anaerobic biofilm system. Variation of dissolved oxygen concentration (3.3-8.7 mgl(-1)) and pH-values between 6.5 and 9.3 did not exert any significant effect on the removal rate of the E. coli by the heterotrophic biofilm. A rapid adsorption of E. coli to the biofilm occurred during the first days after adding the cationic polymer, after which the adsorption slowed down.

Bioreactors↗

Use of artificial neural networks to evaluate the effectiveness of riverbank filtration.

Riverbank filtration (RBF) is a low-cost water treatment technology in which surface water contaminants are removed or degraded as the infiltrating water moves from the river/lake to the pumping wells. The removal or degradation of contaminants is a combination of physicochemical and biological processes. This paper illustrates the development and application of three types of artificial neural networks (ANNs) to estimate the effectiveness of two RBF facilities in the US. The feed-forward back-propagation network (BPN) and radial basis function network (RBFN) model prediction results produced excellent agreement with measured data at a correlation coefficient above 0.99 for filtrate water quality parameters, including temperature as well as turbidity, heterotrophic bacteria, and coliform removal. In comparison, the fuzzy inference system network (FISN) predicted only temperature and bacteria removal with reasonable accuracy. It is shown that the predictive performances of the ANNs depend on the model structure and model inputs.

Bacteria↗

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea.

The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions. The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the world's largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors. Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific 'ladderane' membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.

Anaerobiosis↗

Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport.

The respiratory pathways of glycolysis, the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain are ubiquitous throughout nature. They are essential for both energy provision in heterotrophic cells and a wide range of other physiological functions. Although the series of enzymes and proteins that participate in these pathways have long been known, their regulation and control are much less well understood. Further complexity arises due to the extensive interaction among these pathways in particular, and also between cytosolic and mitochondrial metabolism in general. These interactions include those between mitochondrial function in the photosynthetic and photorespiratory processes, amino-acid biosynthesis and the regulation of cellular redox. Recently, a wide range of molecular and biochemical strategies have been adopted to elucidate the functional significance of these interactions.

Biological Transport↗

Reduction of nitric oxide by denitrifying bacteria.

Two heterotrophic denitrifying bacteria, Paracoccus denitrificans and Pseudomonas denitrificans, have been shown to utilize nitric oxide (NO) as a terminal electron acceptor and succinate, yeast extract, and heat/alkali pretreated municipal sewage sludge as carbon and energy sources. Complete removal of NO (0.50%) from a feed gas sparged into the cultures was observed. It is suggested that reduction of NO may be a common feature of denitrifying bacteria and that a microbial process to dispose of NO(x) may be economically viable.

Nitric Oxide↗

Liberation of amino acids by heterotrophic nitrogen fixing bacteria.

Large amounts of amino acids are produced by nitrogen-fixing bacteria such as Azotobacter, Azospirillum, Rhizobium, Mesorhizobium and Sinorhizobium when growing in culture media amended with different carbon and nitrogen sources. This kind of bacteria live in close association with plant roots enhanced plant growth mainly as a result of their ability to fix nitrogen, improving shoot and root development suppression of pathogenic bacteria and fungi, and increase of available P concentration. Also, it has been strongly evidenced that production of biologically substances such as amino acids by these rhizobacteria are involved in many of the processes that explain plant-grown promotion. This paper reviews literature concerning amino acids production by nitrogen-fixing bacteria. The role of amino acids in microbial interactions in the rhizosphere and establishment of plant bacterial association is also discussed.

Amino Acids↗

Inactivation of a predicted leader peptidase prevents photoautotrophic growth of Synechocystis sp. strain PCC 6803.

To establish the role of the two putative type I leader peptidases (LepB1 and LepB2) encoded in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we generated independent knockout mutants for both genes by introducing kanamycin resistance cassettes into the two open reading frames (sll0716 [lepB1] and slr1377 [lepB2], respectively). Although the insertion was successful in both instances, it was not possible to select homozygous mutant cells for lepB2, suggesting that the function of this gene is essential for cell viability. In contrast, LepB1 is apparently essential only for photoautotrophic growth, because homozygous lepB1::Km(r) cells could be propagated under heterotrophic conditions. They were even capable to some extent of photosynthetic oxygen evolution. However, the photosynthetic activity decreased gradually with extended incubation in the light and was particularly affected by high light intensities. Both features were indicative of photooxidative damage, which was probably caused by inefficient replacement of damaged components of the photosynthetic machinery due to the lack of a leader peptidase removing the signal peptides from photosynthetic precursor proteins. Indeed, processing of the PsbO precursor polypeptide to the corresponding mature protein was significantly affected in the mutant, and reduced amounts of other proteins that are synthesized as precursors with signal peptides accumulated in the cells. These results strongly suggest that LepB1 is important for removal of the signal peptides after membrane transport of the components of the photosynthetic machinery, which in turn is a prerequisite for the biogenesis of a functional photosynthetic electron transport chain.

Bacterial Proteins↗

Microbial reduction of sulfur dioxide with anaerobically digested municipal sewage biosolids as electron donors.

A concentrated stream of sulfur dioxide (SO2) is produced by regeneration of the sorbent in certain new regenerable processes for the desulfurization of flue gas. We have previously proposed that this SO2 can be converted to elemental sulfur for disposal or byproduct recovery using a microbial/Claus process. In this process, two-thirds of the SO2-reducing gas stream would be contacted with a mixed culture containing sulfate-reducing bacteria (SRB), where SO2 would act as an electron acceptor with reduction to hydrogen sulfide (H2S). This H2S could then be recombined with the remaining SO2 and sent to a Claus unit to produce elemental sulfur. The sulfate-reducing bacterium, Desulfovibrio desulfuricans, has been immobilized by coculture with flocforming heterotrophs from an anaerobic digester, resulting in a SO2-reducing floc that may be collected from the effluent of a continuous reactor for recycle by gravity sedimentation. The carbon and energy source for these cultures was anaerobically digested municipal sewage solids. The maximum specific activity for SO2 reduction in these cultures, in terms of dry weight of D. desulfuricans biomass, was 9.1 mmol of SO2/h.g. The stoichiometry with respect to the electron donor was 15.5 mg of soluble COD/mmol of SO2 reduced.

Anaerobiosis↗

Enumeration of bacteriophages and host bacteria in sewage and the activated-sludge treatment process.

Bacteriophage populations in an activated-sludge sewage treatment plant were enumerated. A newly developed assay for quantitation of total phages, employing direct electron microscopic counts, was used in conjunction with the plaque assay. The total concentration of phages was significantly higher in reactor mixed liquor and effluent than in influent sewage, indicating a net production of phages within the reactor. Maximum total phage concentrations in the fluid phase of sewage, activated-sludge mixed liquor, and reactor effluent were 2.2 x 10(7), 9.5 x 10(7), and 8.4 x 10(7)/ml, respectively. Conditions were optimized for isolation of predominant heterotrophic aerobic bacteria from sewage and mixed liquor. Blending at ice water temperatures was superior to ultrasound or enzyme treatments for maximum release of viable bacteria from microbial floc. A solidified extract of mixed liquor was superior to standard media for cultivating maximum numbers of heterotrophic bacteria. The highest culture counts for sewage and mixed liquor were 1.4 x 10(7) and 1.3 x 10(9)/ml, respectively, which represented only 3 and 6.8% of the total microscopic cell counts. Only 3 out of 48 dominant bacterial isolates from either mixed liquor or sewage were hosts for phages present in the system. The sum of phage populations infecting these three hosts accounted for, at best, 3.8% (sewage) and 0.2% (mixed liquor) of the total number of phages present. Generally, specific phage titers were lower in mixed liquor than in sewage, indicating that these hosts were not responsible for the net production of phages in the reactor. This study emphasizes the limitations of the plaque assay for ecological studies of phages, and it suggests that bacteria responsible for phage production in activated-sludge mixed liquor are either minor components of the heterotrophic population, floc-producing strains, or members of other physiological groups.

Bacteria↗

Regulation of autotrophic and heterotrophic carbon dioxide fixation in Hydrogenomonas facilis.

After growth on various carbon sources, sonic extracts of Hydrogenomonas facilis contained ribulosediphosphate (RuDP) carboxylase and phosphoribulokinase (Ru5-P kinase). After very short sonic treatment, a reductive adenosine triphosphate (ATP)-dependent incorporation of (14)CO(2) was also detectable. Reduced nicotinamide adenine dinucleotide (NADH(2)) served as reductant 30-fold more effectively than reduced nicotinamide adenine dinucleotide phosphate (NADPH(2)). Adenosine 5'-phosphate (AMP) and adenosine 5'-pyrophosphate (ADP) inhibited Ru5-P kinase and NADH(2)-, ATP-dependent CO(2) fixation. The levels and duration of CO(2) fixation suggested that it is a cyclic process. The requirement of reduced pyridine nucleotide and ATP and the sensitivity of fixation to AMP and ADP support the conjecture that it occurs via the Calvin cycle. After thorough study of variables affecting catalysis, specific activities (millimicromoles of substrate disappearing per milligram of protein) at 30 C were determined for RuDP carboxylase (C), Ru5-P kinase (K) and ATP-, NADH(2)- dependent CO(2) fixation (CO(2) F) after growth autotrophically on fructose, glucose, ribose, glutamate, lactate, succinate, and acetate. Values for these growth modes were, respectively-for C: 67.3, 51.1, 51.4, 24.6, 2.05, 10.2, 2.25, 1.4; for K: 24.7, 24.0, 23.2, 14.2, 12.8, 12.9, 13.4, 2.8; and for CO(2) F: 4.54, 4.83, 3.10, 2.87, 0.85, 1.51, 0.24, 0.41. The qualitative parallel between values for RuDP carboxylase and CO(2) fixation suggests that one major control point in fixation is the step catalyzed by RuDP carboxylase.

Adenine Nucleotides↗

[Microbiological processes in the Lost City vent field, mid-Atlantic ridge].

Microbiological and biogeochemical measurements showed that the intensities of CO2 assimilation, methane oxidation, and sulfate reduction in the Lost City vent field (30 degrees N) reach 3.8 microg C/(1 day), 0.06 microg C/(1 day), and 117 microg S/(1 day), respectively. On the surface of the carbonate structures occurring in this field, two varieties of bacterial mats were found. The first variety, which is specific to the Lost City alkaline vent field, represents jelly bacterial mats dominated by slime-producing bacteria of several morphotypes. This mat variety also contains chemolithotrophic and heterotrophic microorganisms, either microaerobic or anaerobic. The intensities of CO2 assimilation, methane oxidation, and sulfate reduction in this variety reach 747 microg C/(dm3 day), 0.02 microg C/(dm3 day), and 28,000 microg S/(dm3 day), respectively. Bacterial mats of the second variety are formed by nonpigmented filamentous sulfur bacteria, which are close morphologically to Thiothrix. The intensities of CO2 assimilation, methane oxidation, and sulfate reduction in the second mat variety reach 8.2 microg C/(dm3 day), 5.8 microg C/(dm3 day), and 17,000 microg S/(dm3 day), respectively. These data suggest the existence of subsurface microflora in the Lost City vent field.

Atlantic Ocean↗

Processing dewatered sewage sludge using electrokinetic technology.

High content of heavy metals and presence of pathogens in the dewatered sewage sludge have been the main obstacles for land application of sewage sludge-made fertilizer. The aim of this study was to examine the effects of the innovative electrokinetic (EK) technology on removal of heavy metals from sewage sludge, on the reduction of pathogens, and on sludge chemical characteristics. The results showed that the removal efficiencies for Zn, Cu, Ni, Cr, As and Pb were 94.9%, 95.4%, 89.7%, 67.8%, 31.2% and 18.7%, respectively. Acidification pretreatment of the dewatered sludge for 29 h decreased the content of heterotrophic bacteria from 1.5 x 10(8) c.f.u./g of wet sludge to 1.1 x 10(4) c.f.u./g of wet sludge. Although the initial content of total coliforms and fecal coliforms in sewage sludge were 5.8 x 10(5) c.f.u./g of wet sludge and 4.0 x 10(5) c.f.u./g of wet sludge, respectively, no viable cells were detected. Minor losses of K and N were detected, but the loss of P was found to be significant in EK treated sewage sludge. The treated sludge was technically considered as very stable based on the carbon dioxide evolution rate.

Bioreactors↗

An examination of the carbon isotope effects associated with amino acid biosynthesis.

Stable carbon isotope ratios (delta(13)C) were determined for alanine, proline, phenylalanine, valine, leucine, isoleucine, aspartate (aspartic acid and asparagine), glutamate (glutamic acid and glutamine), lysine, serine, glycine, and threonine from metabolically diverse microorganisms. The microorganisms examined included fermenting bacteria, organotrophic, chemolithotrophic, phototrophic, methylotrophic, methanogenic, acetogenic, acetotrophic, and naturally occurring cryptoendolithic communities from the Dry Valleys of Antarctica. Here we demonstrated that reactions involved in amino acid biosynthesis can be used to distinguish amino acids formed by life from those formed by nonbiological processes. The unique patterns of delta(13)C imprinted by life on amino acids produced a biological bias. We also showed that, by applying discriminant function analysis to the delta(13)C value of a pool of amino acids formed by biological activity, it was possible to identify key aspects of intermediary carbon metabolism in the microbial world. In fact, microorganisms examined in this study could be placed within one of three metabolic groups: (1) heterotrophs that grow by oxidizing compounds containing three or more carbon-to-carbon bonds (fermenters and organotrophs), (2) autotrophs that grow by taking up carbon dioxide (chemolitotrophs and phototrophs), and (3) acetoclastic microbes that grow by assimilation of formaldehyde or acetate (methylotrophs, methanogens, acetogens, and acetotrophs). Furthermore, we demonstrated that cryptoendolithic communities from Antarctica grouped most closely with the autotrophs, which indicates that the dominant metabolic pathways in these communities are likely those utilized for CO(2 )fixation. We propose that this technique can be used to determine the dominant metabolic types in a community and reveal the overall flow of carbon in a complex ecosystem.

Amino Acids↗