Experimental testing of hypotheses for temperature- and pH-based niche specialization of ammonia oxidizing archaea and bacteria.

Investigation of niche specialization in microbial communities is important in assessing consequences of environmental change for ecosystem processes. Ammonia oxidizing bacteria (AOB) and archaea (AOA) present a convenient model for studying niche specialization. They coexist in most soils and effects of soil characteristics on their relative abundances have been studied extensively. This study integrated published information on the influence of temperature and pH on AOB and AOA into several hypotheses, generating predictions that were tested in soil microcosms. The influence of perturbations in temperature was determined in pH 4.5, 6 and 7.5 soils and perturbations in pH were investigated at 15°C, 25°C and 35°C. AO activities were determined by analysing changes in amoA gene and transcript abundances, stable isotope probing and nitrate production. Experimental data supported major predictions of the effects of temperature and pH, but with several significant discrepancies, some of which may have resulted from experimental limitations. The study also provided evidence for unpredicted activity of AOB in pH 4.5 soil. Other discrepancies highlighted important deficiencies in current knowledge, particularly lack of consideration of niche overlap and the need to consider combinations of factors when assessing the influence of environmental change on microbial communities and their activities.

Nitrous oxide from soil denitrification: factors controlling its biological production.

Increasing concentrations of nitrate, nitrite, and molecular oxygen enhanced production of nitrous oxide relative to molecular nitrogen during denitrification in soils. Soil acidity interacted with nitrate to increase the ratio of nitrous oxide to molecular nitrogen. In response to anoxic conditions, nitrous oxide production initially increased but nitrous oxide was then consumed, a pattern which resulted from the sequential synthesis of nitrogenous oxide reductases.

Coupling transport and biodegradation of VOCs in surface and subsurface soils.

Volatile organic chemicals present at Superfund sites preferentially partition into the soil gas and may be available for microbial degradation. A simple mass transfer model for biodegradation for volatile substrates has been developed for the aerobic decomposition of aromatic and aliphatic hydrocarbons. The mass transfer analysis calculates diffusive fluxes from soil gas through water and membrane films and into the cell. This model predicts an extreme sensitivity of potential biodegradation rates to the air-water partition coefficients of the compounds. Aromatic hydrocarbons are removed rapidly while the aliphatic hydrocarbons are much slower by orders of magnitude. Furthermore, oxygen transfer is likely to limit aromatic hydrocarbon degradation rates. The model presents results that cast doubt on the practicality of using methane or propane for the co-metabolic destruction of trichloroethylene in a gas phase bioreactor. Toluene as a primary substrate has better mass transfer characteristics to achieve more efficient trichloroethylene degradation. Hence, in sites where these contaminants coexist, bioremediation could be improved.

Water Content Mediated Microaerophilic Toluene Biodegradation in Arid Vadose Zone Materials.

We investigated the conditions promoting toluene biodegradation for gasoline-contaminated near-surface (0.6 m depth) and subsurface (4.7 to 5.0 m depth) vadose zone soils sampled from an arid environment. At both depths, water addition was required for toluene biodegradation to occur. In near-surface samples, no inorganic nutrient addition was necessary and (i) biodegradation was fastest at 0.0 MPa, (ii) biodegradation rates decreased with decreasing water potential down to ?1.0 MPa, and (iii) biodegradation was undetectable at ?1.5 MPa. For subsurface material, toluene depletion was stimulated either by slurrying with a nutrient solution or by adjusting the moisture content to 20% (0.0 MPa) with nutrient solution and lowering the oxygen concentration (to effectively 1 mg L-1 in the aqueous phase). Thus, in the subsurface material, toluene depletion was microaerobic and nutrient-limited, occurring only under low oxygen and with inorganic nutrient addition. Our studies implicate microaerophily as an important characteristic of the toluene-degrading communities in these dry soils, with soil water as a primary controller of oxygen availability.

Enhanced phenanthrene biodegradation in soil by slender oat root exudates and root debris.

To investigate the mechanisms by which slender oat (Avena barbata Pott ex Link) enhances phenanthrene biodegradation, we analyzed the impacts of root exudates and root debris on phenanthrene biodegradation and degrader community dynamics. Accelerated phenanthrene biodegradation rates occurred in soils amended with slender oat root exudates as well as combined root debris + root exudate as compared with unamended controls. Root exudates significantly enhanced phenanthrene biodegradation in rhizosphere soils, either by increasing contaminant bioavailability and/or increasing microbial population size and activity. A modified most probable number (MPN) method was used to determine quantitative shifts in heterotrophic and phenanthrene degrader communities. During the first 4 to 6 d of treatment, heterotrophic populations increased in all amended soils. Both root debris-amended and exudate-amended soil then maintained larger phenanthrene degrader populations than in control soils later in the experiment after much of the phenanthrene had been utilized. Thus, root amendments had a greater impact over time on phenanthrene degraders than heterotrophs resulting in selective maintenance of degrader populations in amended soils compared with controls.

Seasonal dynamics of microbial community composition and function in oak canopy and open grassland soils.

Soil microbial communities are closely associated with aboveground plant communities, with multiple potential drivers of this relationship. Plants can affect available soil carbon, temperature, and water content, which each have the potential to affect microbial community composition and function. These same variables change seasonally, and thus plant control on microbial community composition may be modulated or overshadowed by annual climatic patterns. We examined microbial community composition, C cycling processes, and environmental data in California annual grassland soils from beneath oak canopies and in open grassland areas to distinguish factors controlling microbial community composition and function seasonally and in association with the two plant overstory communities. Every 3 months for up to 2 years, we monitored microbial community composition using phospholipid fatty acid (PLFA) analysis, microbial biomass, respiration rates, microbial enzyme activities, and the activity of microbial groups using isotope labeling of PLFA biomarkers (13C-PLFA). Distinct microbial communities were associated with oak canopy soils and open grassland soils and microbial communities displayed seasonal patterns from year to year. The effects of plant species and seasonal climate on microbial community composition were similar in magnitude. In this Mediterranean ecosystem, plant control of microbial community composition was primarily due to effects on soil water content, whereas the changes in microbial community composition seasonally appeared to be due, in large part, to soil temperature. Available soil carbon was not a significant control on microbial community composition. Microbial community composition (PLFA) and 13C-PLFA ordination values were strongly related to intra-annual variability in soil enzyme activities and soil respiration, but microbial biomass was not. In this Mediterranean climate, soil microclimate appeared to be the master variable controlling microbial community composition and function.

Response of microbial community composition and function to soil climate change.

Soil microbial communities mediate critical ecosystem carbon and nutrient cycles. How microbial communities will respond to changes in vegetation and climate, however, are not well understood. We reciprocally transplanted soil cores from under oak canopies and adjacent open grasslands in a California oak-grassland ecosystem to determine how microbial communities respond to changes in the soil environment and the potential consequences for the cycling of carbon. Every 3 months for up to 2 years, we monitored microbial community composition using phospholipid fatty acid analysis (PLFA), microbial biomass, respiration rates, microbial enzyme activities, and the activity of microbial groups by quantifying (13)C uptake from a universal substrate (pyruvate) into PLFA biomarkers. Soil in the open grassland experienced higher maximum temperatures and lower soil water content than soil under the oak canopies. Soil microbial communities in soil under oak canopies were more sensitive to environmental change than those in adjacent soil from the open grassland. Oak canopy soil communities changed rapidly when cores were transplanted into the open grassland soil environment, but grassland soil communities did not change when transplanted into the oak canopy environment. Similarly, microbial biomass, enzyme activities, and microbial respiration decreased when microbial communities were transplanted from the oak canopy soils to the grassland environment, but not when the grassland communities were transplanted to the oak canopy environment. These data support the hypothesis that microbial community composition and function is altered when microbes are exposed to new extremes in environmental conditions; that is, environmental conditions outside of their "life history" envelopes.

Redox fluctuation structures microbial communities in a wet tropical soil.

Frequent high-amplitude redox fluctuation may be a strong selective force on the phylogenetic and physiological composition of soil bacterial communities and may promote metabolic plasticity or redox tolerance mechanisms. To determine effects of fluctuating oxygen regimens, we incubated tropical soils under four treatments: aerobic, anaerobic, 12-h oxic/anoxic fluctuation, and 4-day oxic/anoxic fluctuation. Changes in soil bacterial community structure and diversity were monitored with terminal restriction fragment length polymorphism (T-RFLP) fingerprints. These profiles were correlated with gross N cycling rates, and a Web-based phylogenetic assignment tool was used to infer putative community composition from multiple fragment patterns. T-RFLP ordinations indicated that bacterial communities from 4-day oxic/anoxic incubations were most similar to field communities, whereas those incubated under consistently aerobic or anaerobic regimens developed distinctly different molecular profiles. Terminal fragments found in field soils persisted either in 4-day fluctuation/aerobic conditions or in anaerobic/12-h treatments but rarely in both. Only 3 of 179 total fragments were ubiquitous in all soils. Soil bacterial communities inferred from in silico phylogenetic assignment appeared to be dominated by Actinobacteria (especially Micrococcus and Streptomycetes), "Bacilli," "Clostridia," and Burkholderia and lost significant diversity under consistently or frequently anoxic incubations. Community patterns correlated well with redox-sensitive processes such as nitrification, dissimilatory nitrate reduction to ammonium (DNRA), and denitrification but did not predict patterns of more general functions such as N mineralization and consumption. The results suggest that this soil's indigenous bacteria are highly adapted to fluctuating redox regimens and generally possess physiological tolerance mechanisms which allow them to withstand unfavorable redox periods.

Mechanisms for soil moisture effects on activity of nitrifying bacteria.

Moisture may limit microbial activity in a wide range of environments including salt water, food, wood, biofilms, and soils. Low water availability can inhibit microbial activity by lowering intracellular water potential and thus reducing hydration and activity of enzymes. In solid matrices, low water content may also reduce microbial activity by restricting substrate supply. As pores within solid matrices drain and water films coating surfaces become thinner, diffusion path lengths become more tortuous, and the rate of substrate diffusion to microbial cells declines. We used two independent techniques to evaluate the relative importance of cytoplasmic dehydration versus diffusional limitations in controlling rates of nitrification in soil. Nitrification rates in shaken soil slurries, in which NH(inf4)(sup+) was maintained at high concentrations and osmotic potential was controlled by the addition of K(inf2)SO(inf4), were compared with rates in moist soil incubations, in which substrate supply was controlled by the addition of NH(inf3) gas. Comparison of results from these techniques demonstrated that diffusional limitation of substrate supply and adverse physiologic effects associated with cell dehydration can explain all of the decline in activity of nitrifying bacteria at low soil water content. However, the relative importance of substrate limitation and dehydration changes at different water potentials. For the soil-microbial system we worked with, substrate limitation was the major inhibiting factor when soil water potentials were greater than -0.6 MPa, whereas adverse physiological effects associated with cell dehydration were more inhibiting at water potentials of less than -0.6 MPa.

Metabolic status of bacteria and fungi in the rhizosphere of ponderosa pine seedlings.

We determined the quantity and metabolic status of bacteria and fungi in rhizosphere and nonrhizosphere soil from microcosms containing ponderosa pine seedlings. Rhizosphere soil was sampled adjacent to coarse, fine, or young roots. The biovolume and metabolic status of bacterial and fungal cells was determined microscopically and converted to total and active biomass values. Cells were considered active if they possessed the ability to reduce the artificial electron acceptor 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (INT) to visible intracellular deposits of INT formazan. A colorimetric assay of INT formazan production was also used to assess dehydrogenase activity. INT-active microorganisms made up 44 to 55% of the microbial biomass in the soils studied. The proportion of fungal biomass that exhibited INT-reducing activity (40 to 50%) was higher than previous estimates of the active proportion of soil fungi determined by using fluorescein diacetate. Comparison between soils from different root zones revealed that the highest total and INT-active fungal biomass was adjacent to fine mycorrhizal roots, whereas the highest total and active bacterial biomass was adjacent to the young growing root tips. These observations suggest that fungi are enhanced adjacent to the fine roots compared with the nonrhizosphere soil, whereas bacteria are more responsive than fungi to labile carbon inputs in the young root zone. Colorimetric dehydrogenase assays detected gross differences between bulk and rhizosphere soil activity but were unable to detect more subtle differences due to root types. Determination of total and INT-active biomass has increased our understanding of the role of spatial compartmentalization of bacteria and fungi in rhizosphere carbon flow.

Relationship between Desiccation and Exopolysaccharide Production in a Soil Pseudomonas sp.

The relationship between desiccation and the production of extracellular polysaccharides (EPS) by soil bacteria was investigated by using a Pseudomonas species isolated from soil. Cultures subjected to desiccation while growing in a sand matrix contained more EPS and less protein than those growing at high water potential, suggesting that resources were allocated to EPS production in response to desiccation. Desiccation did not have a significant effect on activity as measured by reduction of iodonitrotetrazolium. Purified EPS produced by the Pseudomonas culture contained several times its weight in water at low water potential. Sand amended with EPS held significantly more water and dried significantly more slowly than unamended sand, implying that an EPS matrix may buffer bacterial colonies from some effects of desiccation. We conclude that bacteria may use EPS production to alter their microenvironment to enhance survival of desiccation.

Differential effects of permeating and nonpermeating solutes on the fatty acid composition of Pseudomonas putida.

We examined the effect of reduced water availability on the fatty acid composition of Pseudomonas putida strain mt-2 grown in a defined medium in which the water potential was lowered with the permeating solutes NaCl or polyethylene glycol (PEG) with a molecular weight of 200 (PEG 200) or the nonpermeating solute PEG 8000. Transmission electron microscopy showed that -1.0-MPa PEG 8000-treated cells had convoluted outer membranes, whereas -1.0-MPa NaCl-treated or control cells did not. At the range of water potential (-0.25 to -1.5 MPa) that we examined, reduced water availability imposed by PEG 8000, but not by NaCl or PEG 200, significantly altered the amounts of trans and cis isomers of monounsaturated fatty acids that were present in whole-cell fatty acid extracts. Cells grown in basal medium or under the -0.25-MPa water potential imposed by NaCl or PEG 200 had a higher trans:cis ratio than -0.25-MPa PEG 8000-treated cells. As the water potential was lowered further with PEG 8000 amendments, there was an increase in the amount of trans isomers, resulting in a higher trans:cis ratio. Similar results were observed in cells grown physically separated from PEG 8000, indicating that these changes were not due to PEG toxicity. When cells grown in -1.5-MPa PEG 8000 amendments were exposed to a rapid water potential increase of 1.5 MPa or to a thermodynamically equivalent concentration of the permeating solute, NaCl, there was a decrease in the amount of trans fatty acids with a corresponding increase in the cis isomer. The decrease in the trans/cis ratio following hypoosomotic shock did not occur in the presence of the lipid synthesis inhibitor cerulenin or the growth inhibitors chloramphenicol and rifampicin, which indicates a constitutively operating enzyme system. These results indicate that thermodynamically equivalent concentrations of permeating and nonpermeating solutes have unique effects on membrane fatty acid composition.

Temporal change in nitrous oxide and dinitrogen from denitrification following onset of anaerobiosis.

Similar temporal patterns were found in three mineral soils for the composition of the gaseous products of denitrification following the onset of anaerobic conditions. During the early period of anaerobiosis (0 up to 1 to 3 h), N(2) was the dominant product of denitrification. The NO(3) --> N(2)O activity then increased, but was not accompanied by a corresponding increase in N(2)O-reducing activity. This resulted in a relatively extended period of time (1 to 3 up to 16 to 33 h) during which N(2)O was a major product. Eventually (after 16 to 33 h), an increase in N(2)O-reducing activity occurred without a comparable increase in the N(2)O-producing activity. The increase in the rate of N(2)O reduction did not occur in the presence of chloramphenicol and required the presence of N(2)O or NO(3) during the preceding anaerobic incubation. During the final period (16 to 33, up to 48 h), N(2) was generally the sole product of denitrification, since the rate of N(2)O reduction exceeded the rate of N(2)O production. A similar sequential pattern was also found for a culture of a denitrifying Flavobacterium sp. shifted to anaerobic growth. A staggered synthesis of the enzymes in the denitrification sequence apparently occurred in response to anoxia, which caused first a net production of N(2)O followed by consumption of N(2)O.

Biodegradation of metal-nitrilotriacetate complexes by a Pseudomonas species: mechanism of reaction.

A nitrilotriacetate (NTA)-degrading Pseudomonas species was shown to degrade Ca, Mn, Mg, Cu, Zn, Cd, Fe, and Na chelates of NTA at nearly equal rates when the appropriate metal concentrations are low enough to avoid toxicity from the freed metal. Ni-NTA, however, was not degraded. When higher concentrations of metal-NTA substrates were used, soil stimulated degradation of Cu, Zn, and Cd complexes, probably as a result of binding toxic freed metals. The metal associated with the NTA substrate does not appear to be transported into the cell, since metals do not accumulate in the cells and the presence of NTA reduces metal toxicity. The data are consistent with the hypothesis that an envelope-associated component, probably a transport protein involved in binding, is responsible for the disassociation of the metal from the NTA. Both soil and this NTA-degrading organism destabilize the metal-NTA complex, which suggests that in the natural environment both would act to limit mobilization of metals as soluble NTA chelates. Crude soluble enzyme preparations degrade Fe-, Mn-, and Na-NTA complexes but not Cu-NTA.

Salt stress control of intracellular solutes in streptomycetes indigenous to saline soils.

Actinomycetes were isolated from a number of saline and saline-sodic California soils. From these isolates, two species of Streptomyces (S. griseus and S. californicus) were selected to assess their physiological response to salinity. NaCl was more inhibitory to growth rates and specific growth yields than were equivalent concentrations of KCl. Intracellular concentrations of the free amino acid pool increased in response to salt stress. Whereas the neutral free amino acids proline, glutamine, and alanine accumulated as salinity increased, concentrations of the acidic free amino acids glutamate and aspartate were reduced. Accumulation of free amino acids by streptomycetes under salt stress suggests a response typical of procaryotes, although the specific amino acids involved differ from those associated with other gram-positive bacteria. Above a salinity threshold of about 0.75 M (-3.8 MPa), there was little further intracellular accumulation of free amino acids, whereas accumulation of K salts sharply increased.

Pathway of degradation of nitrilotriacetate by a Pseudomonas species.

The pathway of degradation of nitrilotriacetate (NTA) was determined by using cell-free extracts and a 35-fold purification of NTA monooxygenase. The first step in the breakdown was an oxidative cleavage of the tertiary amine by the monooxygenase to form the aldo acid, glyoxylate, and the secondary amine, iminodiacetate (IDA). NTA N-oxide acted as a substrate analog for induction of the monooxygenase and was slowly metabolized by the enzyme, but was not an intermediate in the pathway. No intermediate before IDA was found, but an unstable alpha-hydroxy-NTA intermediate was postulated. IDA did undergo cleavage in the presence of the purified monooxygenase to give glyoxylate and glycine, but was not metabolized in cell-free extracts. Glyoxylate was further metabolized by cell-free extracts to yield CO2 and glycerate or glycine, products also found from NTA metabolism. Of the three bacterial isolates in which the NTA pathway has been studied, two strains, one isolated from a British soil and ours from a Michigan soil, appear to be almost identical.

Proline transport increases growth efficiency in salt-stressed Streptomyces griseus.

Streptomyces griseus synthesizes proline for osmoregulation under salt stress. Uptake of exogenous [14C]proline and internal synthesis of proline were quantified in cells growing at salt concentrations from 0 to 1 M NaCl. Externally supplied proline accounted for an increased proportion of the intracellular pool of free proline as salt concentration was increased, but neither the concentration nor the composition of the internal amino acid pool was substantially altered by supply of exogenous proline. Uptake of exogenous proline significantly increased the specific growth yield of S. griseus growing under salt stress; the increased yield was proportional to reductions in proline synthesis.

Fungal toxicity of mobilized soil aluminum and manganese.

Inhibition of Aspergillus flavus growth from spores was used as a simple bioassay for toxicity of Al and Mn mobilized by simulated acid precipitation. Al was identified as being toxic in soil leachates resulting from acid inputs of pH less than 2.7. Inhibition by Mn was not detectable. The addition of fluoride significantly reduced Al toxicity, suggesting that biotoxicity of Al is partially dependent on the anionic composition of the soil solution.

Water stress effects on toluene biodegradation by Pseudomonas putida.

We quantified the effects of matric and solute water potential on toluene biodegradation by Pseudomonas putida mt-2, a bacterial strain originally isolated from soil. Across the matric potential range of 0 to -1.5 MPa, growth rates were maximal for P. putida at -0.25 MPa and further reductions in the matric potential resulted in concomitant reductions in growth rates. Growth rates were constant over the solute potential range 0 to -1.0 MPa and lower at -1.5 MPa. First order toluene depletion rate coefficients were highest at 0.0 MPa as compared to other matric water potentials down to -1.5 MPa. Solute potentials down to -1.5 MPa did not affect first order toluene depletion rate coefficients. Total yield (protein) and carbon utilization efficiency were not affected by water potential, indicating that water potentials common to temperate soils were not sufficiently stressful to change cellular energy requirements. We conclude that for P. putida: (1) slightly negative matric potentials facilitate faster growth rates on toluene but more negative water potentials result in slower growth, (2) toluene utilization rate per cell mass is highest without matric water stress and is unaffected by solute potential, (3) growth efficiency did not differ across the range of matric water potentials 0.0 to -1.5 MPa.

Identification of heterotrophic nitrification in a sierran forest soil.

A potential for heterotrophic nitrification was identified in soil from a mature conifer forest and from a clear-cut site. Potential rates of NO(2) production were determined separately from those of NO(3) by using acetylene to block autotrophic NH(4) oxidation and chlorate to block NO(2) oxidation to NO(3) in soil slurries. Rates of NO(2) production were similar in soil from the forest and the clear-cut site and were strongly inhibited by acetylene. The rate of NO(3) production was much greater than that of NO(2) production, and NO(3) production was not significantly affected by acetylene or chlorate. Nitrate production was partially inhibited by cycloheximide, but was not significantly reduced by streptomycin. Neither the addition of ammonium nor the addition of peptone stimulated NO(3) production. N labeling of the NH(4) pool demonstrated that NO(3) was not coming from NH(4). The potential for heterotrophic nitrification in these forest soils was greater than that for autotrophic nitrification.