Marta Goberna

16S rDNA analysis reveals low microbial diversity in community level physiological profile assays

The metabolic diversity of microbial communities is fundamental for the multiple soil functions mediated by microorganisms. Community level physiological profiles (CLPPs) based on sole C source oxidation have been used as a fast and reproducible tool to study soil microbial functional diversity because the utilisation of available carbon is the key factor governing microbial growth in soil. Our aim was to assess the phylogenetic affiliation of the microorganisms responsible for C consumption after inoculating Biolog plates. For this purpose, two semi-arid Mediterranean forest soils with significantly different patterns of C consumption and microbial community structure were used. Following the inoculation of the Biolog plates, suspensions from seven wells were sampled after 1, 2 and 7 d of incubation. DNA was extracted and the microbial communities analysed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing of excised bands. Despite major differences in the microbial communities of the soils studied, their DGGE banding patterns after incubation were similar for all the analysed C source suspensions. Microorganisms belonging to beta-Proteobacteria (Ralstonia sp. and Burkholderia sp.) and alpha-Proteobacteria (Rhizobium sp.) were dominant. These opportunists had a competitive advantage under the conditions at which the CLPPs were analysed. This study reveals that significantly different CLPP patterns can be generated on the basis of only 3-4 genera, as reflected by PCR-DGGE analysis. Also for this reason, CLPPs based on incubations of soil suspensions should just be used as a screening method and always be accompanied by other techniques for community analysis.

Effect of Biowaste Sludge Maturation on the Diversity of Thermophilic Bacteria and Archaea in an Anaerobic Reactor

ABSTRACT Prokaryotic diversity was investigated near the inlet and outlet of a plug-flow reactor. After analyzing 800 clones, 50 bacterial and 3 archaeal phylogenetic groups were defined. Clostridia (>92%) dominated among bacteria and Methanoculleus (>90%) among archaea. Significant changes in pH and volatile fatty acids did not invoke a major shift in the phylogenetic groups. We suggest that the environmental filter imposed by the saline conditions (20 g liter−1) selected a stable community of halotolerant and halophilic prokaryotes.

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

ABSTRACT The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter−1) and ammonia N concentrations (1.3 g liter−1). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH4 production (rPearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H2 partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH4 production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H2 scavengers while yielding the highest CH4 production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.

Population dynamics at digester overload conditions

Two different case studies concerning potential overload situations of anaerobic digesters were investigated and mathematically modelled by means of the Anaerobic Digestion Model No. 1 (ADM1). The first scenario included a digester failure at a municipal WWTP which occurred during revision works of the upstream digester within a two-step digestion system when the sludge was directly by-passed to the 2nd-step reactor. Secondly, the non-occurrence of a highly expected upset situation in a lab-scale digester fed with cattle manure was investigated. ADM1 was utilized to derive indicators which were used to investigate the relationship between digester stability and biomass population dynamics. Conventional design parameters such as the organic loading rate appeared unsuitable for process description under dynamic conditions. Indicators reflecting the biokinetic state (e.g. F(net)/M(net) or the VFA/alkalinity ratio) are more adequate for the assessment of the stability of reactors in transient situations.

Microbes in Aerobic and Anaerobic Waste Treatment

This chapter gives an overview of the materials and chemical compounds that are the subject of microbial degradation under both aerobic and anaerobic conditions. Bacteria, fungi, and archaea that are responsible for degradation or for specific phases of a degradation process are indicated. Special attention is given to two major processes of organic waste recycling involving microorganisms – composting and anaerobic digestion for biogas production. The use of classical and novel tools for investigating the involved microbiota is discussed. Also, aspects of nutrient and greenhouse gas balances are addressed. The chapter concludes by emphasizing that with microbial action, an environmentally sound recycling of organic residues is possible, and that this should be encouraged by waste management policies.

Continuous-feeding vermicomposting as a recycling management method to revalue tomato-fruit wastes from greenhouse crops

Huge quantities of discarded fruits generated from greenhouse crops represent a worldwide environmental problem. The aim of this work was to assess the efficiency of vermicomposting as a recycling management option for biotransforming tomato-fruit wastes from greenhouses into an organic nutrient-rich product available for agricultural purposes. A pilot vermireactor was constructed. It was provided with a manure layer, where an initial population of Eisenia fetida was introduced and fed continuously at a high organic loading rate (13.6 kg TOC m(-3)wk(-1)) for 150 days. Vermicompost chemical and enzymatic parameters as well as the bacterial and fungal community structure were determined for 210 days (vermicomposting plus a maturation period). Earthworm biomass increased after 90 days, and then declined due to increasing pH, electrical conductivity and ammonium concentration. The temporal patterns of dehydrogenase, β-glucosidase, protease and urease were related to earthworm growth and the stabilization of organic matter. Bacterial DGGE profiles differed between the period of degradation of labile substrates and the maturation step. Fungal communities at the stage of maximum earthworm biomass differed most, suggesting a gut passage effect. The end product was chemically stable and enriched in nutrients, demonstrating that tomato-fruit wastes can be successfully vermicomposted into a valuable soil amendment. We suggest continuous-feeding vermicomposting as an environmentally sound management option for greenhouse wastes.

Burning Fire-Prone Mediterranean Shrublands: Immediate Changes in Soil Microbial Community Structure and Ecosystem Functions

Wildfires subject soil microbes to extreme temperatures and modify their physical and chemical habitat. This might immediately alter their community structure and ecosystem functions. We burned a fire-prone shrubland under controlled conditions to investigate (1) the fire-induced changes in the community structure of soil archaea, bacteria and fungi by analysing 16S or 18S rRNA gene amplicons separated through denaturing gradient gel electrophoresis; (2) the physical and chemical variables determining the immediate shifts in the microbial community structure; and (3) the microbial drivers of the change in ecosystem functions related to biogeochemical cycling. Prokaryotes and eukaryotes were structured by the local environment in pre-fire soils. Fire caused a significant shift in the microbial community structure, biomass C, respiration and soil hydrolases. One-day changes in bacterial and fungal community structure correlated to the rise in total organic C and NO3−–N caused by the combustion of plant residues. In the following week, bacterial communities shifted further forced by desiccation and increasing concentrations of macronutrients. Shifts in archaeal community structure were unrelated to any of the 18 environmental variables measured. Fire-induced changes in the community structure of bacteria, rather than archaea or fungi, were correlated to the enhanced microbial biomass, CO2 production and hydrolysis of C and P organics. This is the first report on the combined effects of fire on the three biological domains in soils. We concluded that immediately after fire the biogeochemical cycling in Mediterranean shrublands becomes less conservative through the increased microbial biomass, activity and changes in the bacterial community structure.

Microbes at Work

Vermicomposting, a very efficient method of converting solid organic waste into an environmentally-friendly, useful and valuable resource, is an accelerated process that involves bio-oxidation and stabilization of the waste as a result of the interactions between some species of earthworms and microorganisms. Although microorganisms are the main agents for biochemical decomposition of organic matter, earthworms are critical in the process of vermicomposting. Complex interactions among the organic matter, microorganisms, earthworms and other soil invertebrates result in the fragmentation, bio-oxidation and stabilization of the organic matter.

Abiotic stress tolerance and competition-related traits underlie phylogenetic clustering in soil bacterial communities

Soil bacteria typically coexist with close relatives generating widespread phylogenetic clustering. This has been ascribed to the abiotic filtering of organisms with shared ecological tolerances. Recent theoretical developments suggest that competition can also explain the phylogenetic similarity of coexisting organisms by excluding large low-competitive clades. We propose that combining the environmental patterns of traits associated with abiotic stress tolerances or competitive abilities with phylogeny and abundance data, can help discern between abiotic and biotic mechanisms underlying the coexistence of phylogenetically related bacteria. We applied this framework in a model system composed of interspersed habitats of highly contrasted productivity and comparatively dominated by biotic and abiotic processes, i.e. the plant patch-gap mosaic typical of drylands. We examined the distribution of 15 traits and 3290 bacterial taxa in 28 plots. Communities showed a marked functional response to the environment. Conserved traits related to environmental stress tolerance (e.g. desiccation, formation of resistant structures) were differentially selected in either habitat, while competition related traits (e.g. organic C consumption, formation of nutrient-scavenging structures) prevailed under high resource availability. Phylogenetic clustering was stronger in habitats dominated by biotic filtering, suggesting that competitive exclusion of large clades might underlie the ecological similarity of co-occurring soil bacteria.

Design and development of the ANAEROCHIP microarray for investigation of methanogenic communities

The aim of this study was to design a microarray targeting methanogens found in anaerobic digesters, and to apply this chip together with a cloning approach to investigate the methanogenic community present in an anaerobic digester. Oligonucleotide probes were designed based on sequence differences in the 16S rRNA genes in order to target microorganisms in situ. For microarray hybridisations, DNA was subjected to PCR amplification of the 16S rRNA gene and Cy5-labeled. The microarray was tested with pure cultures, and of the 1854 individual probe-target hybridisation reactions performed, there were only 28 false positive (1.5%) and 16 false negative signals (0.86%). The sensitivity of the array was also tested, and it was found that when 0.4pg of DNA from a pure culture was subjected to PCR amplification, signals above the detection limit were obtained. Also, the application of 25ng of PCR product from a pure culture to an array resulted in detectable signals. The ANAEROCHIP was hybridised with DNA from an anaerobic sludge. Strong hybridisation signals were obtained for Methanoculleus, and weaker signals, in decreasing order, were obtained for Methanosarcina, Methanobacterium, Methanobrevibacter, and Methanosphaera. In order to check the results obtained with the microarray, the archaeal community structure of the same digester was analysed by 16S rRNA gene cloning and sequencing. Community structure was determined by restriction digestion of almost 200 clones and by sequencing of the 15 different resulting patterns. Methanoculleus was the dominant (84.1%) microorganism in the anaerobic sludge, and Methanobrevibacter (5.8%), Methanobacterium (3.7%), Methanosarcina (2.1%), Methanosphaera (1.6%), an uncultured archaeon (1.6%) and Methanothermobacter (1%) were also detected. These results showed the microarray to be a suitable tool for studying methanogenic communities in sludge.