Michael J. Klug

Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure

Analysis of fatty acid methyl ester (FAME) profiles extracted from soils is a rapid and inexpensive procedure that holds great promise in describing soil microbial community structure without traditional reliance on selective culturing, which seems to severely underestimate community diversity. Interpretation of FAME profiles from environmental samples can be difficult because many fatty acids are common to different microorganisms and many fatty acids are extracted from each soil sample. We used principal components (PCA) and cluster analyses to identify similarities and differences among soil microbial communities described using FAME profiles. We also used PCA to identify particular FAMEs that characterized soil sample clusters. Fatty acids that are found only or primarily in particular microbial taxa-marker fatty acids-were used in conjunction with these analyses. We found that the majority of 162 soil samples taken from a conventionally-tilled corn field had similar FAME profiles but that about 20% of samples seemed to have relatively low, and that about 10% had relatively high, bacterial:fungal ratios. Using semivariance analysis we identified 21:0 iso as a new marker fatty acid. Concurrent use of geostatistical and FAME analyses may be a powerful means of revealing other potential marker FAMEs. When microbial communities from the same samples were cultured on R2A agar and their FAME profiles analyzed, there were many differences between FAME profiles of soil and plated communities, indicating that profiles of FAMEs extracted from soil reveal portions of the microbial community not culturable on R2A. When subjected to PCA, however, a small number of plated communities were found to be distinct due to some of the same profile characteristics (high in 12:0 iso, 15:0 and 17:1 ante A) that identified soil community FAME profiles as distinct. Semivariance analysis indicated that spatial distributions of soil microbial populations are maintained in a portion of the microbial community that is selected on laboratory media. These similarities between whole soil and plated community FAME profiles suggest that plated communities are not solely the result of selection by the growth medium, but reflect the distribution, in situ, of the dominant, culturable soil microbial populations.

SOIL RESOURCES, MICROBIAL ACTIVITY, AND PRIMARY PRODUCTION ACROSS AN AGRICULTURAL ECOSYSTEM

The degree to which soil resource availability is linked to patterns of microbial activity and plant productivity within ecosystems has important consequences for our understanding of how ecosystems are structured and for the management of systems for agricultural production. We studied this linkage in a 48-ha site in southwest Michigan, USA, that had been cultivated and planted to row crops for decades. Prior to seeding the site to genetically identical soybean plants (Glycine max) in early spring, we removed soil samples from ≈600 locations; plant biomass was harvested from these same locations later in the season. Soil samples were analyzed for physical properties (texture, bulk density), chemical properties (moisture, pH, total C, total N, inorganic N), and biological attributes (microbial biomass, microbial population size, respiration potential, and nitrification and N-mineralization potentials). Plant analyses included biomass and C and N contents. Soil resource variability across this long-cultiva...

Model for the distribution of sulfate reduction and methanogenesis in freshwater sediments

Abstract A model, based on the in situ physiological characteristics of methanogens and sulfate reducers, was developed to describe the distribution of methanogenesis and sulfate reduction in freshwater sediments. The model predicted the relative importance of methane production and sulfate reduction in lakes of various trophic status and generated profiles of sulfate, acetate, methanogenesis, and sulfate reduction comparable to the profiles that are expected based on field studies. The model indicated that at sulfate concentrations greater than 30μM a sulfate-reducing zone develops because sulfate reducers maintain acetate concentrations too low for methanogens to grow. At lower sulfate concentrations a methanogenic zone develops because the dual limitations of low sulfate concentrations and acetate consumption by methanogens prevents sulfate reducers from growing. The model and a compilation of previously published field data indicate that, within the reported range of sulfate concentrations, the relative importance of methanogenesis and sulfate reduction in freshwater sediments is primarily dependent upon the rates of organic matter decomposition.

Nutrient dynamics, bacterial populations, and mosquito productivity in tree hole ecosystems and microcosms

Water-filled treeholes provide an experimental setting for examining pro- cesses within an ecosystem, and influences of external factors on those processes. Using a limnological, experimental approach involving both natural tree holes and laboratory mi- crocosms of the tree hole ecosystem, we identified and studied interacting, biotic processes, including dynamics of bacterial populations and variation in concentration of inorganic nutrients in tree hole water, and density-dependent competition for food among larvae of the mosquito Aedes triseriatus. We characterized the influence of external factors (inputs of leaf detritus and stemflow) on those processes. Analyses of water samples over time showed that tree hole water was rich and dynamic in nutrients (nitrite, nitrate, ammonium, phosphate, and sulfate); ammonium was the dominant form ofinorganic nitrogen. Variation in nutrient concentrations in microcosms depended upon exogenous inputs (leaf detritus and stemflow water), dilution of nutrients by stemflow, nutrient cycling processes (nitri- fication, denitrification, and sulfate reduction), and ammonium excretion by mosquito larvae. The densities of bacteria in tree hole water, obtained using direct counts of DAPI- fluorochrome stained samples and epifluorescence microscopy, ranged from 2.0 x 106 to 6.0 x 107 cells/mL, and in microcosms from 4.6 x 105 to 2.6 x 108 cells/mL. Experi- mentation involving microcosms revealed that bacterial abundance was reduced by mos- quito feeding and stemflow flushing. Further experiments showed that stemflow flushing increased mosquito productivity from microcosms several-fold and released mosquitoes from density-dependent competition. This effect was likely related to nutrient input and the simultaneous removal of toxic metabolites owing to inputs of stemflow water. We conclude that disturbance by a physical factor, stemflow, has a major influence on the interactions of nutrient dynamics, bacterial populations, and mosquito productivity in temperate tree-hole ecosystems.

Influence of diet on the structure and function of the bacterial hindgut community of crickets

The effect of diets varying in carbohydrate and protein content on the structure and function of the hindgut microbiota of crickets was evaluated by determining bacterial densities, fermentation activity, and guanine plus cytosine (G + C) profiles of the DNA extracted from the microbial hindgut community. DNA isolated from the gut community was fractionated and quantified according to G + C content as a comprehensive, coarse‐level measure of the composition and structure of the community. The bacterial densities measured by direct counts were not significantly different among the four diets. The crickets were initially reared in the laboratory on cricket chow, which resulted in a hindgut community dominated by bacteria with a G + C content between 32% and 57%. Crickets shifted to an alfalfa diet showed a similar hindgut community G + C profile, although microbial populations with DNA between 35% and 45% G + C were more abundant in alfalfa‐ than chow‐fed crickets. The apparent complexity of the gut community was reduced in crickets fed beet‐pulp and protein‐based diets compared to those fed chow and alfalfa, and was dominated by populations with a low percentage G + C content. Hindgut communities in crickets fed pulp and protein diets also showed a decrease in hydrogen and carbon dioxide production, suggesting that these diets affected the biochemical activity of the hindgut community. The protein‐based diet resulted in a decrease in the rate of evolution of volatile fatty acids, while the ratio of butyrate production to acetate and propionate production was significantly higher in these crickets. Our results show the emergence of a new microbial community structure concomitant with changes in microbial biochemical activity due to shifts in the cricket’s dietary regime.

The contribution of hindgut bacteria to dietary carbohydrate utilization by crickets (Orthoptera: Gryllidae)

Abstract 1. 1. Growth rates of germfree house crickets fluctuated during development more than their conventional counterparts in response to an alternating diet regime. 2. 2. Gut bacteria increased the digestive efficiency of soluble plant polysaccharides for crickets on each diet treatment and further, dampened the effect of diet change on the digestion of that pool of carbohydrates. 3. 3. Comparisons of carbohydrase activity in different gut segments and in germfree and conventional crickets, showed that bacteria in the anterior hindgut supply most of the conventional insects degradative capabilities against a heterogenous class of soluble plant storage/structural polysaccharides. 4. 4. Gut bacteria in crickets aid the insects ability to utilize a wider range of dietary carbohydrates by responding to changes in dietary sources with induced production of enzymes and/or changes in population levels.

Temporal variations in parameters reflecting terminal-electron-accepting processes in an aquifer contaminated with waste fuel and chlorinated solvents

Abstract A fundamental issue in aquifer biogeochemistry is the means by which solute transport, geochemical processes, and microbiological activity combine to produce spatial and temporal variations in redox zonation. In this paper, we describe the temporal variability of TEAP conditions in shallow groundwater contaminated with both waste fuel and chlorinated solvents. TEAP parameters (including methane, dissolved iron, and dissolved hydrogen) were measured to characterize the contaminant plume over a 3-year period. We observed that concentrations of TEAP parameters changed on different time scales and appear to be related, in part, to recharge events. Changes in all TEAP parameters were observed on short time scales (months), and over a longer 3-year period. The results indicate that (1) interpretations of TEAP conditions in aquifers contaminated with a variety of organic chemicals, such as those with petroleum hydrocarbons and chlorinated solvents, must consider additional hydrogen-consuming reactions (e.g., dehalogenation); (2) interpretations must consider the roles of both in situ (at the sampling point) biogeochemical and solute transport processes; and (3) determinations of microbial communities are often necessary to confirm the interpretations made from geochemical and hydrogeological measurements on these processes.

Relationships between bacterial productivity and organic carbon at a soil—stream interface

Microbial communities at soil-stream interfaces may be particularly important in regulating amounts and forms of nutrients that leave upland soils and enter stream ecosystems. While microbial communities are thought to be responsible for key nutrient transformations within near-stream sediments, there is relatively little mechanistic information on factors that control microbial activities in these areas. In this study, we examine the roles of dissolved organic carbon (DOC) vs. particulate organic carbon (POC) as potential controls on rates of bacterial productivity (measured as incorporation of [3H]thymidine into bacterial DNA) and amounts of bacterial biomass (measured as fatty acid yield) in sediments along a transect perpendicular to a soil–stream interface. We hypothesized that spatial patterns in bacterial productivity would vary in response to strong and persistent patterns in pore-water concentrations of DOC that were observed along a soil-stream transect throughout a 2-year period. Our results did not support the existence of such a link between pore-water DOC and bacterial productivity. In contrast, we found bacterial productivity and biomass were related to small-scale spatial variations in sediment POC on 3 of 4 sample dates. While our results indicate that total bacterial productivity in near-stream sediments is not consistently linked to spatial variations in pore-water DOC, it is likely that DOC and POC are not mutually exclusive and the relative contribution of DOC and POC to sedimentary microbes varies temporally and spatially in different riparian habitats.

Bacterial and Fungal Biomass Responses to Feeding by Larval Aedes triseriatus (Diptera: Culicidae)

Abstract We investigated the effect of different densities (0, 20, or 40) of developing larval Aedes triseriatus (Say) on bacterial abundance, bacterial productivity, and leaf fungal biomass in a microcosm experiment. Larvae in the low-density treatment developed normally, but larvae at the high density were significantly slower to develop. Both bacterial abundance (direct microscopic counts) and bacterial productivity (3H-leucine incorporation rates) on leaf material were significantly lower in the presence of larvae. Bacterial abundance in the water column did not change significantly with treatment, but bacterial productivity varied with time and declined significantly at both larval densities. Bacteria on the walls and bottom of the containers also were less abundant and significantly less productive in the presence of larvae. Aside from presence/absence effects, there was no clear evidence that larval impacts were density-dependent. Leaf-associated fungal biomass, as measured by ergosterol levels, varied with time but was not significantly affected by any treatment, suggesting most fungal tissue was incorporated in the leaf matrix and unavailable to larvae. Based upon estimated biomass accrual and respiration of larvae, it appears that bacterial biomass and production were insufficient to account for carbon demands of growing larvae. Because fungal biomass and leaf mass likely contributed little to gross larval demands, other carbon sources (e.g., protozoa and extracellular microbial components) were probably used by larvae. Although apparently insufficient for all larval carbon demands, bacterial and leaf fungal biomass may be adequate for other larval nutritional needs (i.e., nitrogen and essential lipids).

Relative Values of Oxygen, Nitrate, and Sulfate to Terminal Microbial Processes in the Sediments of Lake Superior

The vertical distributions of the terminal electron acceptors oxygen, nitrate, and sulfate, and the rates of associated microbial processes (aerobic respiration, denitrification, and sulfate reduction) were determined in sediment core samples retrieved from Lake Superior with the Johnson Sea Link II. Direct measurements with microelectrodes showed the penetration depth of oxygen into the sediment was 8 to 24 mm, while the depth distribution of nitrate varied greatly among the sampled sites. Based on our process rate measurements, oxygen supported respiration accounted for at least 94% of the carbon metabolism in these sediments, with the remainder being processed largely through denitrification. Sulfate reduction was found to be an insignificant process.