Added Carbon Source Research Articles

Sulfate-reducing bacterial community response to carbon source amendments in contaminated aquifer microcosms

Microbial sulfate reduction is an important metabolic activity in many reduced habitats. However, little is known about the sulfate-reducing communities inhabiting petroleum hydrocarbon (PHC)-contaminated freshwater aquifer sediments. The purpose of this study was to identify the groups of sulfate-reducing bacteria (SRB) selectively stimulated when sediment from a PHC-contaminated freshwater aquifer was incubated in sulfate-reducing aquifer microcosms that were amended with specific carbon sources (acetate, butyrate, propionate, lactate, and citrate). After 2 months of incubation, the SRB community was characterized using phospholipid fatty acid (PLFA) analysis combined with multivariate statistics as well as fluorescence in situ hybridization (FISH). Molybdate was used to specifically inhibit SRB in separate microcosms to investigate the contribution of non-SRB to carbon source degradation. Results indicated that sulfate reduction in the original sediment was an important process but was limited by the availability of sulfate. Substantially lower amounts of acetate and butyrate were degraded in molybdate treatments as compared to treatments without molybdate, suggesting that SRB were the major bacterial group responsible for carbon source turnover in microcosms. All of the added carbon sources induced changes in the SRB community structure. Members of the genus Desulfobulbus were present but not active in the original sediment but an increase of the fatty acids 15:1ω6c and 17:1ω6c and FISH results showed an enrichment of these bacteria in microcosms amended with propionate or lactate. The appearance of cy17:0 revealed that bacteria affiliated with the Desulfobacteriaceae were responsible for acetate degradation. Desulfovibrio and Desulfotomaculum spp. were not important populations within the SRB community in microcosms because they did not proliferate on carbon sources usually favored by these organisms. Metabolic, PLFA, and FISH results provided information on the SRB community in a PHC-contaminated freshwater environment, which exhibited stimulation patterns similar to other (e.g. marine) environments.

Anaerobic degradation of oleic acid by suspended and granular sludge: identification of palmitic acid as a key intermediate

The aim of the present work was to study the maximum potential methane production in batch assays of sludge samples taken along the operation of two EGSB reactors (RI inoculated with granular sludge and RII inoculated with suspended sludge) fed with increasing oleic acid concentrations between 2 and 8 gCOD/l (HRT = 1 day). After removing the residual substrate, the sludge was incubated in batch vials without any added carbon source. A maximum methane production rate of 152+/-21 mlCH4(STP)/gVS.day was obtained for the suspended sludge taken on day 70, when oleate at a concentration of 2 g COD/l was fed with a co-substrate (50% COD). The maximum plateau achieved in the methane production curve was 1145+/-307 mlCH4(STP)/gVS, obtained for the suspended sludge taken on day 162, when oleate was fed as the sole carbon source at 6 g COD/I. The methanization rate of the adsorbed substrate was enhanced under stirring conditions and was inhibited by adding oleic acid. Extraction and GC analysis confirmed that the main adsorbed substrate was palmitate, and not oleate. Accumulated palmitate adsorbed onto the sludge and further beta-oxidation was inhibited when in the presence of oleic acid. If oleic acid was removed from the medium beta-oxidation proceeded with methane production. Suspended sludge was more efficient than granular sludge.

13C-discrimination during microbial respiration of added C3-, C4- and 13C-labelled sugars to a C3-forest soil.

We tested whether 13C-discrimination during microbial respiration, or during CO2 sampling in the field, can explain changes observed in the δ13C of emitted CO2 that follow the addition of C4-sucrose, as a microbial substrate, to the soil of a C3-ecosystem. We approached this problem by adding C3-glucose (δ13C=-23.4‰), C4-sucrose (-10.8‰) or 13C-labelled glucose (103.7‰) to the intact mor layer, the upper organic soil (-26.5‰, bulk soil organic matter), of a boreal Pinus sylvestris L. forest. If 13C-discrimination is significant, it should generate illusory differences in the calculated contributions from the added C and endogenous C3-C to total soil respiration, when C4-sucrose or 13C-labelled glucose is added. Further, if discrimination occurs, we should also be able to detect a shift in the δ13C of respired CO2 after the addition of C3-glucose. The addition of the three sugar solutions gave similar increases in soil respiration (up to a doubling 1h after the additions), while the addition of water gave no increase in respiration. There was no change in δ13C of the emitted CO2 after additions of H2O or C3-glucose. In contrast, the addition of C4-sucrose and 13C-labelled glucose gave δ13C values of evolved CO2 that were 4.5‰ and 30.3‰ higher than the pre-sugar values, respectively. The calculated respiration rates of the added carbon sources, C4-C or 13C-labelled C, were very similar. Also, we found very similar sugar-induced increases in respiration of endogenous C3-C in the plots supplied with C4-sucrose and 13C-labelled glucose, accounting for about 50% of the total increase in respiration 1h after addition. Our results confirm that any microbial 13C-discrimination during respiration is minor.

Activity and diversity of sulfate-reducing bacteria in a petroleum hydrocarbon-contaminated aquifer.

Microbial sulfate reduction is an important metabolic activity in petroleum hydrocarbon (PHC)-contaminated aquifers. We quantified carbon source-enhanced microbial SO(4)(2-) reduction in a PHC-contaminated aquifer by using single-well push-pull tests and related the consumption of sulfate and added carbon sources to the presence of certain genera of sulfate-reducing bacteria (SRB). We also used molecular methods to assess suspended SRB diversity. In four consecutive tests, we injected anoxic test solutions (1,000 liters) containing bromide as a conservative tracer, sulfate, and either propionate, butyrate, lactate, or acetate as reactants into an existing monitoring well. After an initial incubation period, 1,000 liters of test solution-groundwater mixture was extracted from the same well. Average total test duration was 71 h. We measured concentrations of bromide, sulfate, and carbon sources in native groundwater as well as in injection and extraction phase samples and characterized the SRB population by using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE). Enhanced sulfate reduction concomitant with carbon source degradation was observed in all tests. Computed first-order rate coefficients ranged from 0.19 to 0.32 day(-1) for sulfate reduction and from 0.13 to 0.60 day(-1) for carbon source degradation. Sulfur isotope fractionation in unconsumed sulfate indicated that sulfate reduction was microbially mediated. Enhancement of sulfate reduction due to carbon source additions in all tests and variability of rate coefficients suggested the presence of specific SRB genera and a high diversity of SRB. We confirmed this by using FISH and DGGE. A large fraction of suspended bacteria hybridized with SRB-targeting probes SRB385 plus SRB385-Db (11 to 24% of total cells). FISH results showed that the activity of these bacteria was enhanced by addition of sulfate and carbon sources during push-pull tests. However, DGGE profiles indicated that the bacterial community structure of the dominant species did not change during the tests. Thus, the combination of push-pull tests with molecular methods provided valuable insights into microbial processes, activities, and diversity in the sulfate-reducing zone of a PHC-contaminated aquifer.

Ethanol‐Stimulated Bioremediation of Nitrate‐Contaminated Ground Water

AbstractExtensive characterization of a nitrate‐contaminated site at a Canadian airport revealed that natural attenuation of the plume was limited due to aerobic conditions. However, nitrate removal rates as high as 1.4 mg‐N/(L/day) were observed in batch tests carried out under denitrifying conditions with an added carbon source. On the basis of these results, a field denitrification pilot test using a setup of vertical withdrawal and injection wells was carried out. Ethanol was used as a carbon source to stimulate the growth and activity of indigenous denitrifying bacterial populations. Denitrification rates of up to 1.2 mg‐N/(L/day) were achieved during the test. The test confirmed the feasibility of the technology, which uses the indigenous bacterial populations for denitrification and transforms the aquifer into a subsurface bioreactor.

Effect of food-grade surfactant on bioremediation of explosives-contaminated soil

The use of native soil bacteria to biodegrade explosives-contaminated soil under co-metabolic conditions has been demonstrated. The addition of food-grade surfactants could improve the process by enhancing the rates of explosives desorption from soil, thereby increasing the bioavailability of explosives for microbial degradation. The objective of this study was to decrease residence time in the slurry reactor, thereby increasing output and reducing clean-up costs. In this study, Tween 80 (monooleate), served not only as a surfactant but also as the carbon substrate for the soil microorganisms. Four 2 l soil slurry reactors were operated in batch mode with soil containing 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). The results indicated that TNT and RDX were removed in all reactors except the control (no added carbon source). The reactor enriched with surfactant and molasses performed better than reactors with either molasses or surfactant alone. The TNT and its metabolite, 4-amino-2,6-dinitrotoluene were removed faster in the reactor with surfactant plus molasses (35 days) than in the reactor with molasses alone as carbon source (45 days). A radiolabeling study of the mass balance of TNT in the slurry reactors showed substantial mineralization of TNT to CO 2.

Root mucilage from pea and its utilization by rhizosphere bacteria as a sole carbon source.

Plant roots secrete a complex polysaccharide mucilage that may provide a significant source of carbon for microbes that colonize the rhizosphere. High molecular weight mucilage was separated by high-pressure liquid chromatography gel filtration from low molecular weight components of pea root exudate. Purified pea root mucilage generally was similar in sugar and glycosidic linkage composition to mucilage from cowpea, wheat, rice, and maize, but appeared to contain an unusually high amount of material that was similar to arabinogalactan protein. Purified pea mucilage was used as the sole carbon source for growth of several pea rhizosphere bacteria, including Rhizobium leguminosarum 8401 and 4292, Burkholderia cepacia AMMD, and Pseudomonas fluorescens PRA25. These species grew on mucilage to cell densities of three- to 25-fold higher than controls with no added carbon source, with cell densities of 1 to 15% of those obtained on an equal weight of glucose. Micromolar concentrations of nod gene-inducing flavonoids specifically stimulated mucilage-dependent growth of R. leguminosarum 8401 to levels almost equaling the glucose controls. R. leguminosarum 8401 was able to hydrolyze p-nitrophenyl glycosides of various sugars and partially utilize a number of purified plant polysaccharides as sole carbon sources, indicating that R. leguminosarum 8401 can make an unexpected variety of carbohydrases, in accordance with its ability to extensively utilize pea root mucilage.

Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta.

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of beta-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.

Redox modulation of chloroplast DNA replication in Chlamydomonas reinhardtii.

We constructed a plasmid probe containing DNA sequences unique for chloroplast (Cp) genome and nuclear genome of Chlamydomonas reinhardtii. Using this probe and quantitative Southern blot analyses, we determined the content ratio of Cp DNA/nuclear DNA in total DNA isolated from cells grown in different conditions. Algal cells grown photoheterotrophically with acetate as an added carbon source contain the highest amount of Cp DNA compared with cells grown in other conditions tested. We investigated the effect of nitrogen limitation, 5-fluorodeoxyuridine treatment, cadmium exposure, photoautotrophic growth, and heterotrophic growth in darkness. The change in the Cp/nuclear DNA ratio in cells shifted from one growth condition to another depended on cell division; Cp DNA content in undivided cells remained constant. Therefore, the reduction of Cp DNA content was attributed by under replication rather than selective degradation of Cp DNA. Cells with low Cp DNA content often contained less reduced glutathione, suggesting the possible effect of redox status. Low Cp DNA content was detected in cells treated with inhibitors that block electron flow of photosystems and in mutants with PS I defective phenotype. On the basis of these data, we propose that in C. reinhardtii, Cp DNA replication be modulated by redox status.

Removal of Perchlorate from Ground Water by Hydrogen‐Utilizing Bacteria

AbstractAn autotrophic consortium of bacteria, utilizing hydrogen and bicarbonate as electron and carbon sources, respectively, for the reduction of perchlorate has been enriched. The efficiency of this consortium for perchlorate reduction in a packed‐bed bioreactor is described. The use of hydrogen as an electron source for microbial biodegradation of pollutants in a packed‐bed bioreactor is advantageous because hydrogen does not promote vigorous biomass growth, resulting in less clogging of the system, and hydrogen gas reduces the need for post‐reactor water cleanup. The consortium, comprised of four bacteria, was capable of removing nitrate and perchlorate simultaneously from mineral salts medium in a batch culture. A 120‐mL bioreactor was designed to test the capacity of the consortium to remove perchlorate in a flow‐through system. At a flow rate of 1 mL min−1, perchlorate levels naturally occurring in San Gabriel Valley ground water (0.740 mg L−1) were completely removed to less than 0.004 mg L−1, which is the detection limit. However, as the flow rate was increased, the removal efficiency decreased. Factors affecting the rate of perchlorate removal included nonuniform distribution of biomass in the column, unstable pH of the ground water, and limited delivery of hydrogen to the bacteria. This bioreactor also completely removes perchlorate from ground water at a flow rate of 1 mL min−1 without an added carbon source, presumably using the bicarbonate naturally present in the ground water.

Surfactant‐Enhanced Bioremediation of Soil Contaminated with 2,4,6‐Trinitrotoluene in Soil Slurry Reactors

The addition of food‐grade surfactants could improve the use of native soil bacteria to biodegrade explosives‐contaminated soil under co‐metabolic conditions by enhancing the rates of explosives' desorption from soil, thus increasing the bioavailability of explosives for microbial degradation. The objective of this study is to decrease residence time in the reactor, thereby increasing output and reducing cleanup costs. In this study, Tween 80 (monooleate) served not only as a surfactant but also as the carbon substrate for soil microorganisms. Four 2‐L soil slurry reactors were operated in batch mode with soil containing 2,4,6‐trinitrotoluene (TNT). Results indicated that TNT was removed in all reactors except the control (no added carbon source). The reactor enriched with surfactant and molasses performed better than reactors with either molasses or surfactant alone. The TNT and its metabolite, 4‐amino‐2,6‐dinitrotoluene, were removed faster in the reactor with surfactant plus molasses (35 days) than in the reactor with molasses alone as carbon source (45 days). A radiolabeling study of the mass balance of TNT in the slurry reactors showed substantial mineralization of TNT to carbon dioxide.

Survival of, and plasmid transfer between modified Enterobacter cloacae strains under long-term starvation conditions in a continuous-flow system

Continuous-flow, packed-bed column reactors, which provide an experimental model of a soil profile, were used to investigate survival of, and plasmid transfer between, strains of Enterobacter cloacae. When columns, inoculated with nutrient-sufficient donor and recipient strains, were provided with a minimal salts medium with no added carbon source, transconjugant cells appeared in their effluents. During the first few days of such experiments, the concentration of cells in the effluent declined but then the donor population stabilized, while the recipient and transconjugant populations continued to decrease. The results indicate that the amount of nutrient required to maintain and transfer plasmids is very low. No transconjugants were observed in the effluent from columns inoculated with pre-starved donor and recipient strains.

In-House Composting to Reduce Larval House Fly, Musca domestica L., Populations

Hen manure from high-rise houses was treated biweekly with mechanical agitation alone or in combination with added cardboard, hay, or sawdust. Compared to non-agitated, non-treated control manure, house fly numbers were reduced in agitated treatments and in those with added carbon sources in concert with lower manure moisture, higher manure temperature, and greater ammonia levels. Chemical composition of the manure was altered by the agitation and addition of composting materials, increasing dry matter, and decreasing the nitrogen concentration. Adding hay or sawdust increased both the carbon and carbon-to-nitrogen ratio of the final products. All the composted and agitated manure treatments appeared to reduce the bulk density and improve the consistency of raw manure; however, ammonia respiratory protection for the skid loader operator is necessary during in-house agitation, and may be a health concern for the hens as well.

Utilisation of an electro-optical method to investigate the amidase activity of microbial cells

The electro-optical (EO) properties of a microbial cell suspension of Brevibacterium sp. strain 13 PA were examined during acrylamide and acrylic acid metabolism and during culture of cells in a mineral medium without added carbon sources. The dependence of the suspension turbidity changes due to cellular orientation on the frequency of an orienting electric field (orientational spectra, OSs) over the range of 10–10000 kHz were used. Alterations in the OSs, depending on cellular amidase activity, were recorded during growth with acrylamide as the role source of carbon. However, no amidase activity was observed in cells growing with acrylic acid as the sole carbon source. Growth on the carbon-free mineral medium resulted in a reduction in amidase activity. It is apparent that the OS changes reflect the corresponding changes in cell polarisability, which may be associated with acrylamide hydrolysis. The data generated from these studies can be taken as a basis for the EO determination of the enzyme activity of microbial biocatalysts.

Utilisation of an electro-optical method to investigate the amidase activity of microbial cells

The electro-optical (EO) properties of a microbial cell suspension of Brevibacterium sp. strain 13 PA were examined during acrylamide and acrylic acid metabolism and during culture of cells in a mineral medium without added carbon sources. The dependence of the suspension turbidity changes due to cellular orientation on the frequency of an orienting electric field (orientational spectra, OSs) over the range of 10–10000 kHz were used. Alterations in the OSs, depending on cellular amidase activity, were recorded during growth with acrylamide as the role source of carbon. However, no amidase activity was observed in cells growing with acrylic acid as the sole carbon source. Growth on the carbon-free mineral medium resulted in a reduction in amidase activity. It is apparent that the OS changes reflect the corresponding changes in cell polarisability, which may be associated with acrylamide hydrolysis. The data generated from these studies can be taken as a basis for the EO determination of the enzyme activity of microbial biocatalysts.

Survival of Inoculants in Polluted Sediments: Effect of Strain Origin and Carbon Source Competition

Inoculation of polluted environments with specialized strains is an important strategy in bioremediation. Especially the efficacy and safety of genetically engineered biodegradative microorganisms could be improved further, if host organisms with predictable survival in specific target ecosystems were identified and could be engineered to carry catabolic gene cassettes. Here we empirically tested whether strains originally isolated from the target ecosystem (indigenous inoculants) had superior survival rates compared to strains derived from geographically and ecologically different locations (nonindigenous inoculants). We experimentally tested this approach by isolating various strains of Pseudomonas and related genera from a highly polluted river sediment (Spittelwasser River) and following their survival in sediment slurries from the same river in comparison with that of nonindigenous strains. Both indigenous and nonindigenous inoculants grew and maintained cell densities of between 107 and 109 cfu ml−1 in autoclaved sediment slurries for up to 70 days, indicating that they were not negatively affected by abiotic factors, especially potentially toxic pollutants. In live sediment slurries, however, all inoculants died out at varying rates. If live sediment slurries were amended with a carbon source that the inoculant could degrade, most strains were able to grow, regardless of their original source. In some cases, high cell densities of the inoculant were maintained for up to 70 days, even after exhaustion of the added carbon source. Reisolating these strains and monitoring their survival in sediment slurries from a different sampling date did not reveal increased survival rates compared to the original strains. It is concluded that the use of indigenous isolates for bioremediation and as hosts for constructing genetically engineered organisms does not provide any advantage in dynamic, highly competitive environments. For safety reasons, therefore, a limited number of well-characterized strains whose pathogenic potential is known and for which ecological information can be obtained, should be used. To ensure survival of the inoculant, either the site must be engineered to provide a temporary advantage for the introduced strain, or the inoculant must be able to exploit a specific niche better than the indigenous microbial community.

Effect of application solvents on heterotrophic and nitrifying populations in soil microcosms

AbstractAgricultural practices may cause contamination of soil and ground water with a combination of organic compounds (pesticides and fuel) and nitrogen fertilizers. In coupled microcosm studies that monitored the mineralization of naphthalene and the nitrification of ammonia, it was observed that the solvent (dichloromethane) used to apply naphthalene to the soil inhibited nitrification, although there was no effect on naphthalene mineralization. Further studies were performed with a series of application solvents: methanol, acetonitrile, trichloromethane, and dichloromethane. Soil and solvent were allowed to equilibrate with ambient air for various times before capping and incubation of microcosms. Results indicated that dichloromethane equilibrated for 5 mins inhibited nitrification for at least 3 weeks relative to the control (water). Acetonitrile and trichloromethane similarly inhibited nitrification. Methanol and dichloromethane equilibrated for 60 mins also significantly delayed nitrification, although to a lesser extent. Inhibition of nitrification was not permanent, and nitrification activity was eventually restored in all systems tested. None of the solvents inhibited mineralization of the added carbon source. These results indicate that special care must be taken to ensure that applications solvents do not affect the activity of sensitive microbial populations, such as the nitrifiers, that may be part of a study.

Effect of plant photosynthesis, carbon sources and ammonium availability on nitrogen fixation rates in the rhizosphere of Zostera noltii

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 12:285-290 (1997) - doi:10.3354/ame012285 Effect of plant photosynthesis, carbon sources and ammonium availability on nitrogen fixation rates in the rhizosphere of Zostera noltii Welsh DT, Bourguès S, de Wit R, Auby I Rates of nitrogen fixation (measured as acetylene reduction) in the rhizosphere of the seagrass Zostera noltii were highly dependent upon plant photosynthetic activity being significantly stimulated at elevated CO2 concentrations and by light, both in the short-term and over diurnal cycles. Stimulation by light became insignificant when 5 mM sucrose was added to the sediment porewater, indicating that in the absence of added carbon sources, light stimulation was due to direct inputs of plant photosynthate to the rhizosphere. Addition of a range of carbon sources to the rhizosphere sediment stimulated rates of acetylene reduction, with this stimulation being significant for sucrose and lactate. Surprisingly, whilst low additions of ammonium to the sediment porewater (10 to 50 μM) inhibited 50% of acetylene reduction activity, approximately 30% of this activity persisted in the presence of 1 mM ammonium chloride; this indicating that in at least a proportion of the N-fixing community, nitrogenase activity was not regulated in the short term by the availability of alternative nitrogen sources. Acetylene reduction · Nitrogen fixation · Rhizosphere · Zostera noltii · Plant photosynthesis · Root exudates · Carbon sources · Ammonium Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 12, No. 3. Publication date: May 29, 1997 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 1997 Inter-Research.

Factors influencing the induction of endo-xylanase by Thermoascus aurantiacus

Fourteen carbohydrates were screened for their ability to induce xylanase in the newly isolated thermophilic fungus, Thermoascus aurantiacus. Among the carbohydrates screened, beech wood xylan, birch wood glucuronoxylan and methyl β- d-xylopyranoside (MXP) appeared to induce xylanase. Low levels of constitutive enzymes were produced on all the compounds even in the absence of an added carbon source to the basal medium. A long lag period for enzyme induction was observed with all the inducers. This lag period was markedly reduced when the mycelia were starved prior to the addition of the inducer. The most powerful non-metabolizable inducer, MXP, induced the highest xylanase activity in a concentration up to 2.5 mg ml −1. The optimum initial pH and culture temperature for induced synthesis of xylanase were around pH 4.0 and 50°C, respectively. For pronounced synthesis of the enzyme organic nitrogen, in addition to an appropriate inducer, was necessary. The surfactant, Tween-80, had enhanced the enzyme secretion into the culture fluid. AgNO 3, CoNO 3, HgCl 2, KMnO 4, MnCl 2 and SDS showed moderate to strong inhibitory effects on both the synthesis and activity of xylanase, while CuSO 4, EDTA, SnCl and ZnCl 2 showed a strong inhibitory effect on xylanase activity only. The enzyme induction was repressed by high concentration of glucose and this could not be reversed by exogenous cyclic-adenosine 3′,5′-monophosphate (cAMP). Mixture of inducer (MXP) and repressor (glucose) produced a delayed induction compared with solutions of inducers only. The repression, however, was completely restored after the depletion of glucose level from the medium to the non-repressive level (approx. 0.2 mg ml −1).

Effect of water and nitrogen additions on free-living nitrogen fixer populations in desert grass root zones.

In this study we measured changes in population levels of free-living N2-fixing bacteria in the root zones of potted Bouteloua eriopoda and Sporobolus flexuosus plants as well as the photosynthetic indices of the plants in response to added nitrogen, added water, and added water plus nitrogen treatments. In addition, N2 fixer population changes in response to added carbon source and nitrogen were measured in plant-free soil columns. There were significant increases in the numbers of N2 fixers associated with both plant species in the water and the water plus nitrogen treatments. Both treatments increased the photosynthetic index, suggesting that plant exudates were driving N2 fixer population changes. Population increases were greatest in the water plus nitrogen treatments, indicating that added nitrogen was synergistic with added water and suggesting that nitrogen addition spared bacteria the metabolic cost of N2 fixation, allowing greater reproduction. Plant-free column studies demonstrated a synergistic carbon-nitrogen effect when carbon levels were limiting (low malate addition) but not when carbon was abundant (high malate), further supporting this hypothesis. The results of this study indicate the presence of N2 fixer populations which interact with plants and which may play a role in the nitrogen balance of desert grasslands.