Jay L. Garland

Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization

Profiles of potential utilization of 95 separate C sources by microbial communities can be readily generated from direct incubation of environmental samples in BIOLOG microplates. Color formation from a redox indicator dye is used to quantify the degree of C source utilization. I aimed to examine different analytical approaches for classifying microbial communities based on these profiles. Specifically, the relative effects of average rate of color development versus the pattern of relative C source utilization on the classification of rhizosphere samples from different crop types were evaluated. The average rate of color development was correlated to the density of total (acridine-orange) bacterial cells (R2 = 0.52) and active (5-cyano-2,3-ditolyl tetrazolium chloride) bacterial cells (R2 = 0.70) inoculated into the plate. Analysis of plates with different rates of color development after a specific incubation period resulted in samples with variation in the overall extent of color development (expressed as average well color development, or AWCD). Classification of these samples using principal component analysis was significantly influenced by the variation in AWCD, resulting in the classification of samples based on the density of inoculum rather than the pattern of C source utilization. The effect of variation in AWCD was eliminated by normalizing data prior to ordination, or by using an alternative ordination technique, detrended correspondence analysis. Variation in AWCD can be limited through multiple-plate readings and subsequent selection of plates with a common reference point in AWCD. The specific AWCD used for analysis does not appear important for classification purposes; consistent discrimination of rhizosphere samples from different crop types was apparent for analysis across a wide range of AWCD (0.25–1.00 abs. units). The specific differences in C source utilization between rhizosphere sample types did depend on the set point used for analysis due to the differences in the rate of color formation among wells. Results suggest that single-plate readings can be used to classify samples, but only if potential differences in AWCD are accounted for in the data analysis. Repeated plate readings will provide a more complete understanding of differences in C source utilization among samples.

Patterns of potential C source utilization by rhizosphere communities

Abstract Patterns of potential C source utilization by microbial communities were tested as a means for differentiating among and within rhizosphere samples of different crops. Utilization of 95 separate sole C sources was determined by inoculating microbial suspensions into BIOLOG plates and quantifying color production from a redox-sensitive dye. Suspensions were produced by shaking excised root samples from wheat, white potato, soybean and sweetpotato grown in hydroponic culture within controlled environmental growth chambers. Distinctive patterns of C source utilization were apparent for each crop type, and the differences among crops were consistent for experiments repeated over 2 years. A temporal shift in C source utilization related to plant development state was observed in soybean. Results indicate that this rapid method is effective for detecting plant dependent differences in rhizosphere communities, and changes in response to plant developmental state.

Relative effectiveness of kinetic analysis vs single point readings for classifying environmental samples based on community-level physiological profiles (CLPP)

The relative effectiveness of average-well-color-development-normalized single-point absorbance readings (AWCD) vs the kinetic parameters mu(m), lambda, A, and integral (AREA) of the modified Gompertz equation fit to the color development curve resulting from reduction of a redox sensitive dye from microbial respiration of 95 separate sole carbon sources in microplate wells was compared for a dilution series of rhizosphere samples from hydroponically grown wheat and potato ranging in inoculum densities of 1 x 10(4)-4 x 10(6) cells ml-1. Patterns generated with each parameter were analyzed using principal component analysis (PCA) and discriminant function analysis (DFA) to test relative resolving power. Samples of equivalent cell density (undiluted samples) were correctly classified by rhizosphere type for all parameters based on DFA analysis of the first five PC scores. Analysis of undiluted and 1:4 diluted samples resulted in misclassification of at least two of the wheat samples for all parameters except the AWCD normalized (0.50 abs. units) data, and analysis of undiluted, 1:4, and 1:16 diluted samples resulted in misclassification for all parameter types. Ordination of samples along the first principal component (PC) was correlated to inoculum density in analyses performed on all of the kinetic parameters, but no such influence was seen for AWCD-derived results. The carbon sources responsible for classification differed among the variable types with the exception of AREA and A, which were strongly correlated. These results indicate that the use of kinetic parameters for pattern analysis in CLPP may provide some additional information, but only if the influence of inoculum density is carefully considered.

Impact of dilution on microbial community structure and functional potential: comparison of numerical simulations and batch culture experiments

ABSTRACT A series of microcosm experiments was performed using serial dilutions of a sewage microbial community to inoculate a set of batch cultures in sterile sewage. After inoculation, the dilution-defined communities were allowed to regrow for several days and a number of community attributes were measured in the regrown assemblages. Based upon a set of numerical simulations, community structure was expected to differ along the dilution gradient; the greatest differences in structure were anticipated between the undiluted–low-dilution communities and the communities regrown from the very dilute (more than 10−4) inocula. Furthermore, some differences were expected among the lower-dilution treatments (e.g., between undiluted and 10−1) depending upon the evenness of the original community. In general, each of the procedures used to examine the experimental community structures separated the communities into at least two, often three, distinct groups. The groupings were consistent with the simulated dilution of a mixture of organisms with a very uneven distribution. Significant differences in community structure were detected with genetic (amplified fragment length polymorphism and terminal restriction fragment length polymorphism), physiological (community level physiological profiling), and culture-based (colony morphology on R2A agar) measurements. Along with differences in community structure, differences in community size (acridine orange direct counting), composition (ratio of sewage medium counts to R2A counts, monitoring of each colony morphology across the treatments), and metabolic redundancy (i.e., generalist versus specialist) were also observed, suggesting that the differences in structure and diversity of communities maintained in the same environment can be manifested as differences in community organization and function.

Community-Level Physiological Profiling Performed with an Oxygen-Sensitive Fluorophore in a Microtiter Plate

ABSTRACT Community-level physiological profiling based upon fluorometric detection of oxygen consumption was performed on hydroponic rhizosphere and salt marsh litter samples by using substrate levels as low as 50 ppm with incubation times between 5 and 24 h. The rate and extent of response were increased in samples acclimated to specific substrates and were reduced by limiting nitrogen availability in the wells.

Comparison of aerobically-treated and untreated crop residue as a source of recycled nutrients in a recirculating hydroponic system

This study compared the growth of potato plants on nutrients recycled from inedible potato biomass. Plants were grown for 105 days in recirculating, thin-film hydroponic systems containing four separate nutrient solution treatments: (1) modified half-strength Hoaglands (control), 2) liquid effluent from a bioreactor containing inedible potato biomass, 3) filtered (0.2 micrometer) effluent, and 4) the water soluble fraction of inedible potato biomass (leachate). Approximately 50% of the total nutrient requirement in treatments 2-4 were provided (recycled) from the potato biomass. Leachate had an inhibitory effect on leaf conductance, photosynthetic rate, and growth (50% reduction in plant height and 60% reduction in tuber yield). Plants grown on bioreactor effluent (filtered or unfiltered) were similar to the control plants. These results indicated that rapidly degraded, water soluble organic material contained in the inedible biomass, i.e., material in leachate, brought about phytotoxicity in the hydroponic culture of potato. Recalcitrant, water soluble organic material accumulated in all nutrient recycling treatments (650% increase after 105 days), but no increase in rhizosphere microbial numbers was observed.

Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter monitoring for direct detection of bacterial agents

Aim:  To incorporate into the lambda phage genome, a luxI‐based acyl‐homoserine lactone (AHL) synthase genetic construct and exploit the autoamplified power of quorum sensing to translate a phage infection event into a chemical signature detectable by a lux‐based bioluminescent bioreporter, with focus towards facile detection of microbial pathogens.

Culturability as an indicator of succession in microbial communities

Successional theory predicts that opportunistic species with high investment of energy in reproduction and wide niche width will be replaced by equilibrium species with relatively higher investment of energy in maintenance and narrower niche width as communities develop. Since the ability to rapidly grow into a detectable colony on nonselective agar medium could be considered as characteristic of opportunistic types of bacteria, the percentage of culturable cells may be an indicator of successional state in microbial communities. The ratios of culturable cells (colony forming units on R2A agar) to total cells (acridine orange direct microscopic counts) and culturable cells to active cells (reduction of 5-cyano-2,3-ditolyl tetrazolium chloride) were measured over time in two types of laboratory microcosms (the rhizosphere of hydroponically grown wheat and aerobic, continuously stirred tank reactors containing plant biomass) to determine the effectiveness of culturabilty as an index of successional state. The culturable cell:total cell ratio in the rhizosphere decreased from approximately 0.25 to less than 0.05 during the first 30-50 days of plant growth, and from 0.65 to 0.14 during the first 7 days of operation of the bioreactor. The culturable cell:active cell ratio followed similar trends, but the values were consistently greater than the culturable cell:total cell ratio, and even exceeded I in early samples. Follow-up studies used a cultivation-independent method, terminal restriction fragment length polymorphisms (TRFLP) from whole community DNA, to assess community structure. The number of TRFLP peaks increased with time, while the number of culturable types did not, indicating that the general decrease in culturability is associated with a shift in community structure. The ratio of respired to assimilated C-14-labeled amino acids increased with the age of rhizosphere communities, supporting the hypothesis that a shift in resource allocation from growth to maintenance occurs with time. Results from this work indicate that the percentage of culturable cells may be a useful method for assessing the successional state of microbial communities.

Distribution of Microbial Communities Associated with the Dominant High Marsh Plants and Sediments of the United States East Coast

Microbial communities in the sediment and associated with the dominant type of standing dead plant were collected from the high marsh zones of 10 sites along the eastern coast of the United States from Maine to Florida. Microbial community composition was examined using T-RFLP, and bacterial and fungal abundance was determined microscopically. Within the sediment, community composition was strongly correlated with latitude, indicating that biogeographical factors are important determinants of sediment community composition, whereas abundance was positively and strongly correlated with sediment organic matter content. A strong biogeographical effect was observed for both bacterial and fungal abundance on standing dead plants, but there was no clear relationship between community composition and latitude. Microbial community composition was more similar among plants of the same type (i.e., related plant species) suggesting that plant type (i.e., substrate quality) is primarily responsible for the determining community composition on standing dead plants.

Effect of Microbial Species Richness on Community Stability and Community Function in a Model Plant-Based Wastewater Processing System

Microorganisms will be an integral part of biologically based waste processing systems used for water purification or nutrient recycling on long-term space missions planned by the National Aeronautics and Space Administration. In this study, the function and stability of microbial inocula of different diversities were evaluated after inoculation into plant-based waste processing systems. The microbial inocula were from a constructed community of plant rhizosphere-associated bacteria and a complexity gradient of communities derived from industrial wastewater treatment plant-activated sludge. Community stability and community function were defined as the ability of the community to resist invasion by a competitor (Pseudomonas fluorescens 5RL) and the ability to degrade surfactant, respectively. Carbon source utilization was evaluated by measuring surfactant degradation and through Biolog and BD oxygen biosensor community level physiological profiling. Community profiles were obtained from a 16S–23S rDNA intergenic spacer region array. A wastewater treatment plant-derived community with the greatest species richness was the least susceptible to invasion and was able to degrade surfactant to a greater extent than the other complexity gradient communities. All communities resisted invasion by a competitor to a greater extent than the plant rhizosphere isolate constructed community. However, the constructed community degraded surfactant to a greater extent than any of the other communities and utilized the same number of carbon sources as many of the other communities. These results demonstrate that community function (carbon source utilization) and community stability (resistance to invasion) are a function of the structural composition of the community irrespective of species richness or functional richness.