Sessions

Sessions - Microbial Ecology I and II

Microbial community profiling of fungi and bacteria in an in-vessel composter

Roberts, Michael S. (1), Klamer, Morten (2), Frazier, C. (1) and GARLAND, JAY L. (1)

(1) Dynamac Corporation, Kennedy Space Center, FL, (2)Department of Biology, University of Central Florida, USA

Microbial community dynamics within complex systems such as compost are poorly understood due to an abundance of diverse microbial types and an inability to define the conditions necessary for their cultivation. We have applied traditional plating techniques in combination with Terminal Restriction Fragment Length Polymorphism analysis (T-RFLP) to monitor changes in fungal and bacterial community composition during composting in a 28-L rotating drum composter equipped with thermal control and on-line monitoring. The effect of starting C:N ratio and temperature upon diversity was investigated by composting alfalfa (Medicago sativa) and aspen shavings mixed to yield initial C:N ratios of 15 to 30. Compost temperature was regulated to evaluate pathogen survival in response to three thermal profiles: short-term thermophilic (55 C;3 days), constant-term thermophilic (55 C;9 days), and no thermal control (self-heating). Changes in fungal biomass and total microbial biomass were estimated by the ergosterol technique and by phospholipid fatty acid analysis (PLFA), respectively. Molecular estimates of bacterial and fungal diversity were estimated from primers targeting small subunit rDNA and the Intergenic Transcribed Spacer (ITS) region, respectively. Plating, ergosterol analysis, and T-RFLP profiling each indicated a shift in fungal community composition from day 0 to days 30/90. Approximately 50% of fungal community T-RFLP peaks could be assigned to species isolated from compost but 5% of isolate ITS signatures were not detected by T-RFLP community profiling. Both techniques revealed the same pattern of species richness. No changes in microbial community composition resulting from initial C:N or thermal treatments were detected at days 30 and 90. Principal component analyses of T-RFLP profiles revealed that fungal diversity declined more rapidly and recovered more slowly in response to thermophilic treatments than bacterial diversity.

Microbiological and Chemical Characterization During Composting of Cattle Manure and Forestry Wastes - A Case Study in Madeira Island

ROCHAL, M. (1), Cordeiro, N. (1), Cunha Queda, A.C.F. (2), Capela, R. (1)

(1) Universidade da Madeira, Departamento de Biologia, ICCA- Centro de Investigacao em Ciencias Agrarias, Campus Universitario de Penteada (2)Instituto Superior de Agronomia, Departmento de Quimica Agricola e Ambiental, Tapada da Ajuda, Portugal

Large quantities of forestry wastes are produced every year in Madeira Island, as well as appreciable quantities of cattle manure. The composting of these two otherwise landfilled wastes, is therefore an important recycling option that will reduce their environmental impact. The main objective of this work was to study the dynamic of several physical-chemical parameters during the composting of cattle manure and forest residues, as well as the quantitative and qualitative characterization of the microbial populations, which are responsible for the biodegradation of the materials used. The composting trials were performed in piles using several turning frequencies. The results indicate that the turning frequencies had a significant effect on the evolution of the operational parameters, especially on temperature, O₂ and CO₂ contents, moisture, electrical conductivity and, nutrient availability. Losses of nitrogen by volatilisation of ammonia were not detected, and therefore, we could obtain composts with high nitrogen levels. The microorganisms identified are in agreement with the evolution of the operational parameters followed, especially the nitrogen dynamics. The identification of free-living nitrogen fixing bacteria, as well as, autotrophic and heterotrophic nitrifying bacteria, allowed us to justify the high nitrogen concentrations found in the composts. The biodiversity indexes showed that the composts reached biological stabilization.

Microbial Community Structure During Composting of Waste Feathers Using Terminal Restriction Fragment Length Polymorphisms of 16S rDNA

(1) S.M. TIQUIA, (1) H.M. Keener, (1) D. Elwell, (2) J.M. Ichida, (3) E.H. Burt Jr., (1) F.C. Michel Jr

(1) Dept. of Food, Ag, and Bio Engineering, The Ohio State University, (2) Dept. of Botany/Microbiology, Ohio Wesleyan University, (3) Dept. of Zoology, Ohio Wesleyan University, USA

The disposal of dead poultry and waste feathers is a major concern for processing plants and poultry farms in the United States. Waste feathers generated from the poultry industry account for 5 to 7% of the total weight of a mature chicken. Composting is one of the more economical and environmentally safe methods to recycle feather waste generated from poultry industry. Feathers by weight consist of 90% crude keratin protein and 15% N. However, the keratin in waste feathers is very resistant to degradation and may require the addition of bacterial inocula to enhance the degradation process during composting. Bacillus licheneformis and Streptomyces spp. are known keratin degraders found on feathers. However, the effects of the addition of these bacterial inocula during feather composting has not previously been studied. In this study, terminal restriction fragment length polymorphisms (T-RFLP) of 16S rDNA genes were analyzed to follow microbial community changes during feather composting with bacterial inocula. Waste feathers were co-composted with poultry litter and straw in 200-L compost vessels. One set of compost vessels contained was inoculated with Bacillus licheneformis and Streptomyces sp. previously shown to degrade feathers in pure culture while a control set was not inoculated. The results revealed that addition of bacterial inocula did not enhance the rate of CO2 evolution during waste feather composting. Furthermore, the microbial community structure was very similar (Sab= 70-85%) in the inoculated and uninoculated waste feathers. Bacillus licheneformis and Streptomyces sp. were also detected in waste feathers without bacterial inocula, by T-RFLP method using HhaI, MspI, and RsaI digestions. Thus these organisms already apparently exist in the co-composting material (poultry litter and feathers). The indigenous microorganisms in the uninoculated compost vessels may have developed rapidly, to the extent that composting in these vessels proceeded at equal rates (based on CO2 evolution and temperature patterns) as in the inoculated compost vessels. Such a finding is of remarkable significance in the waste feather composting since this indicates that the feather composting does not require inoculation.

The Influence of Process Management and Microbial Community Structure On the Cultivation of A Biological Control Agent in Compost

JEAN S. VANDERGHEYNST (1), Todd M. Dooley (2), Megan N. Marshall (1)

(1) Department of Biological and Agricultural Engineering, UC Davis, (2) Altran Corporation,San Francisco, CA, USA

Utilization of compost as a cultivation system and carrier for biological control agents offers opportunities for both the recycling of organic wastes and reduction in pesticide application. Understanding the growth of biological control agents in compost is critical to the success of this application. Compost inoculation studies were carried out in a model system using the biological control agent Trichoderma harzianum to determine the influence of degree of decomposition, microbial competition and compost management upon inoculation on T. harzianum colonization. Compost was produced from grape pomace and rice straw in aerated, one-liter reactors and inoculations were done after 3, 7 and 13 days of composting. Temperature was managed at 26C, 30C and 35C after inoculation. Compost samples were analyzed for microbial community structure and biomass by phospholipid fatty acid analysis and by denaturing gradient gel electrophoresis profiling of small subunit ribosomal DNA genes amplified from DNA extracted directly from compost samples. T. harzianum colonization was measured by plating serial dilutions of compost samples on selective media. T. harzianum colonization of non-sterile compost samples was poor when both available substrate and microbial biomass levels were high. Poor colonization of non-sterile compost samples was also observed when pH was high. In summary the potential for microbial competition, temperature and pH played a significant role in the colonization of compost samples by the biological control agent T. harzianum

Specific Microbial Communities Inhabiting Maturing Compost

Susanne KLAMMER (1), & Heribert Insam (1)

(1) Institute of Microbiology, University of Innsbruck, Technikerstr. 25, A-6020 Innsbruck, Austria

The succession of microbial populations during the turnover of organic matter in a composting process follows typical patterns, which have traditionally been revealed by isolation techniques. Molecular analyses have improved the understanding of community succession, but so far no direct link between compost maturity and the occurrence of specific organisms could be made. The aim of this investigation was to show if the microbial community composition can serve as an indicator of compost maturity, or reveal information on the quality of the product. Further analysis of genetic fingerprints compared with general quality parameters used so far included the determination of the taxonomic affiliation of putative dominating bacteria by cloning and sequencing of different bands extracted from denaturing gradient gels (DGGEs). The detailed analysis and comparison of different mature composts originating from diverse starting materials and processed under different running conditions showed to what extent substrates determine the emerging microbial community.

Impact of Compost and Manure Amendments On Bacterial Populations in the Rhizospheres of Tomato and Cabbage

B. MCSPADDEN-GARDENER (1), S.A. Miller(1), M. Kleinhenz (2), D. Doohan (2), and P. Grewal (3)

(1) Dept. of Plant Pathology, (2) Dept. of Horticulture and Crop Science, (3) Dept. of Entomology, The Ohio State University-OARDC, Wooster, OH 44691, USA

Addition of organic matter to soil generally increases the total abundance and activities of soil microbial populations. However, the relative abundance of different beneficial and pathogenic populations may also change. The impacts of adding raw and composted dairy manure on soil and rhizosphere microbial communities, plant disease, nematode diversity, crop quality, and weed presure are being examined in a vegetable rotation that is transitioning to organic production. The population size and colonization frequency of culturable Pseudomonas spp., including those with biocontrol capacity, were determined. Differences were observed in the relative abundance of indigenous biocontrol populations containing the genes for 2,4-diacetylphloroglucinol and pyoluteorin biosynthesis. Changes in uncultured bacterial populations also were monitored, using FT-ARDRA. The potential impact of crop-specific and treatment-specific differences in the observed microbial populations will be discussed.

Effect of Compost Amendment On Microbial Community in Soil, Rhizosphere and Roots

DROR MINZ(1); Stefan J. Green(1,2); Larissa Kautsky(1); Ehud Inbar(1,2) and Yitzhak Hadar(2)

(1) Soil, Water and Environmental Sciences, Volcani Research Center, Bet Dagan; (2) Hebrew University of Jerusalem, Rehovot, Israel

A combination of biochemical, chemical, microbiological and molecular tools were employed to investigate the effect of application of compost on the microbiology of soil, rhizosphere and root systems. Short and long term shifts in microbial community composition and functions were noted in all systems. Changes in biomass, respiration and enzymatic activity were correlated with microbial community composition profiles as determined by Denaturing Gradient Gel Electrophoresis (DGGE) of PCR-amplified DNA fragments and the sequencing of relevant bands. Addition of compost caused a substantial shift in community composition in the soil, mainly with regard to members of the Cytophaga-Flavobacterium-Bacteroides group, suggesting their involvement in degradation of compost compounds in the soil. Compost microorganisms dominated the roots and rhizosphere. An increase of diversity of microorganisms following compost amendment and reduced diversity following chemical fertilization were observed.

Nucleic Acid Techniques to Characterize Microbial Communities in Composting Material

Institute for Agroecology, Federal Research Center for Agriculture (FAL), Braunschweig, Germany

Composting processes are characterized by successions of microbial communities selected by energy yielding substrates and by physico-chemical conditions, such as pH, temperature or water potential. For optimizing process conditions it would be desirable to allow the identification of dominant microorganisms in the communities and identify their key metabolic activities. Recent methodological developments in PCR-based detection techniques have allowed to analyze the diversity of microbial communities in composting material, independent of cultivation. Genes, encoding for the small subunit rRNA (SSU rRNA) of ribosomes have been found extremely useful in this context since they can be PCR-amplified from DNA directly extracted from compost material. The diversity of PCR products can then be characterized by cloning in sequencing or, prior to these steps, by generating genetic profiles, e.g., by single strand conformation polymorphism (SSCP). We have developed and used this technique (PCR-SSCP) to characterize microbial community structures during a hot composting of agricultural waste. The technique allowed to study the succession of different phylogenetic groups of the microbial community in parallel, i.e., Eubacteria, Actinomycetes and Fungi. Dominant members of each group could be identified after cloning and sequencing. Our approach is also useful to study the diversity of smaller phylogenetic groups which are known to contribute with specific activities to the composting process, e.g., the diversity of ammonium-oxidizing bacteria. The PCR-SSCP methodology can potentially be extended to study the diversity of functional genes, such those encoding for cellulases or enzymes involved in the nitrogen cycle. Another possibility to detect specific activities is based on RT-PCR of mRNA, directly extracted from the compost material. A protocol which allows the detect specific gene expression of fungal cells in environmental substrates was developed.

Systematic and functional characterization of fungal biodiversity in compost and vermicompost

ANTONELLA ANASTASI (1), Giovanna C. Varese (1), Samuele Voyron (1) and Silvano Scannerini (1)

Composting is an increasingly applied technology in waste management; the high organic matter content and the biological activity of compost makes it effective for use in a variety of fields including agriculture, revegetation, bioremediation. Fungi play an important role in composting and in compost applications especially for their ability to use complex carbon sources. The mycoflora of a quality compost (made exclusively from plant debris by a thermophilic process) and of a vermicompost (made by the action of earthworms on plant and animal wastes - mesophilic process) was investigated from a taxonomic and functional point of view. The soil dilution plate technique was applied on 3 media at 3 temperatures for isolation and further identification of fungal entities and enzymatic activities (amylase, cellulase, chitinase, esterase, ligninase, pectinase, phosphatase, plastic degradation and xylanase) of most of the species from both composts were evaluated using a semiquantitative method on 15 substrates. Important differences in quali-quantitative species composition occurred between the two composts. On the whole 208 total fungal entities were isolated: 53 exclusively from green compost, 79 exclusively from vermicompost, 76 in common to both. This taxonomic diversity is reflected in the metabolic potential. Cellulase, protease and lipase activities were significantly higher in green compost than in vermicompost. The opposite was true for lignin and plastic polymer degradation that prevailed in vermicompost. These structural and functional characterizations are important to define the best fields of application of these products and their quality from an hygienic point of view.