Biodegradation of different PET variants from food containers by Ideonella sakaiensis.

The accumulation of macro-, micro- and nano-plastic wastes in the environment is a major global concern, as these materials are resilient to degradation processes. However, microorganisms have evolved their own biological means to metabolize these petroleum-derived polymers, e.g., Ideonella sakaiensis has recently been found to be capable of utilizing polyethylene terephthalate (PET) as its sole carbon source. This study aims to prove its potential capacity to biodegrade two commercial PET materials, obtained from food packaging containers. Plastic pieces of different crystallinity were simultaneously introduced to Ideonella sakaiensis during a seven-week lasting investigation. Loss in weight, appearance of plastics, as well as growth of Ideonella sakaiensis-through quantitative real-time PCR-were determined. Both plastics were found enzymatically attacked in a two-stage degradation process, reaching biodegradation capacities of up to 96%. Interestingly, the transparent, high crystallinity PET was almost fully degraded first, followed by the colored low-crystallinity PET. Results of quantitative real-time PCR-based gene copy numbers were found in line with experimental results, thus underlining its potential of this method to be applied in future studies with Ideonella sakaiensis.

Extraction of Cofactor F420 for Analysis of Polyglutamate Tail Length from Methanogenic Pure Cultures and Environmental Samples.

The cofactor F420 plays a central role as a hydride carrier in the primary and secondary metabolism of many bacterial and archaeal taxa. The cofactor is best known for its role in methanogenesis, where it facilitates thermodynamically difficult reactions. As the polyglutamate tail varies in length between different organisms, length profile analyses might be a powerful tool for distinguishing and characterizing different groups and pathways in various habitats. Here, the protocol describes the extraction and optimization of cofactor F420 detection by applying solid-phase extraction combined with high-performance liquid chromatography analysis independent of cultural or molecular biological approaches. The method was applied to gain additional information on the expression of cofactor F420 from microbial communities in soils, anaerobic sludge, and pure cultures and was evaluated by spiking experiments. Thereby, the study succeeded in generating different F420 tail-length profiles for hydrogenotrophic and acetoclastic methanogens in controlled methanogenic pure cultures as well as from environmental samples such as anaerobic digester sludge and soils.

Lignin intermediates lead to phenyl acid formation and microbial community shifts in meso- and thermophilic batch reactors.

BACKGROUND: Lignin intermediates resulting from lignocellulose degradation have been suspected to hinder anaerobic mineralisation of organic materials to biogas. Phenyl acids like phenylacetate (PAA) are early detectable intermediates during anaerobic digestion (AD) of aromatic compounds. Studying the phenyl acid formation dynamics and concomitant microbial community shifts can help to understand the microbial interdependencies during AD of aromatic compounds and may be beneficial to counteract disturbances. RESULTS: The length of the aliphatic side chain and chemical structure of the benzene side group(s) had an influence on the methanogenic system. PAA, phenylpropionate (PPA), and phenylbutyrate (PBA) accumulations showed that the respective lignin intermediate was degraded but that there were metabolic restrictions as the phenyl acids were not effectively processed. Metagenomic analyses confirmed that mesophilic genera like Fastidiosipila or Syntrophomonas and thermophilic genera like Lactobacillus, Bacillus, Geobacillus, and Tissierella are associated with phenyl acid formation. Acetoclastic methanogenesis was prevalent in mesophilic samples at low and medium overload conditions, whereas Methanoculleus spp. dominated at high overload conditions when methane production was restricted. In medium carbon load reactors under thermophilic conditions, syntrophic acetate oxidation (SAO)-induced hydrogenotrophic methanogenesis was the most important process despite the fact that acetoclastic methanogenesis would thermodynamically be more favourable. As acetoclastic methanogens were restricted at medium and high overload conditions, syntrophic acetate oxidising bacteria and their hydrogenotrophic partners could step in for acetate consumption. CONCLUSIONS: PAA, PPA, and PBA were early indicators for upcoming process failures. Acetoclastic methanogens were one of the first microorganisms to be impaired by aromatic compounds, and shifts to syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in thermophilic reactors. Previously assumed associations of specific meso- and thermophilic genera with anaerobic phenyl acid formation could be confirmed.

Proposal of Thermoactinomyces mirandus sp. nov., a filamentous, anaerobic bacterium isolated from a biogas plant.

We isolated a filamentous, thermophilic, and first anaerobic representative of the genus Thermoactinomyces, designated strain AMNI-1T, from a biogas plant in Tyrol, Austria and report the results of a phenotypic, genetic, and phylogenetic investigation. Strain AMNI-1T was observed to form a white branching mycelium that aggregates into pellets when grown in liquid medium. Cells could primarily utilize lactose, glucose, and mannose as carbon and energy sources, with acetate accelerating and yeast extract being mandatory for growth. The optimum growth temperature and pH turned out to be 55 °C and pH 7.0, respectively, with an optimum NaCl concentration of 0-2% (w/v). 16S rRNA gene sequence comparison indicated that the genetic relatedness between strain AMNI-1T and Thermoactinomyces intermedius, Thermoactinomyces khenchelensis, and Thermoactinomyces vulgaris was less than 97%. The G + C content of the genomic DNA was 44.7 mol%. The data obtained suggest that the isolate represents a novel and first anaerobic species of the genus Thermoactinomyces, for which the name Thermoactinomyces mirandus is proposed. The type strain is AMNI-1T (= DSM 110094T = LMG 31503T). The description of the genus Thermoactinomyces is emended accordingly.

Soil-Derived Inocula Enhance Methane Production and Counteract Common Process Failures During Anaerobic Digestion.

Although soil-borne methanogens are known to be highly diverse and adapted to extreme environments, their application as potential (anaerobic) inocula to improve anaerobic digestion has not been investigated until now. The present study aimed at evaluating if soil-derived communities can be beneficial for biogas (methane, CH4) production and endure unfavorable conditions commonly associated with digestion failure. Nine study sites were chosen and tested for suitability as inoculation sources to improve biogas production via in situ measurements (CH4 fluxes, physical and chemical soil properties, and abundance of methanogens) and during a series of anaerobic digestions with (a) combinations of both sterile or unsterile soil and diluted fermenter sludge, and (b) pH-, acetate-, propionate-, and ammonium-induced disturbance. Amplicon sequencing was performed to assess key microbial communities pivotal for successful biogas production. Four out of nine tested soil inocula exerted sufficient methanogenic activity and repeatedly allowed satisfactory CH4/biogas production even under deteriorated conditions. Remarkably, the significantly highest CH4 production was observed using unsterile soil combined with sterile sludge, which coincided with both a higher relative abundance of methanogens and predicted genes involved in CH4 metabolism in these variants. Different bacterial and archaeal community patterns depending on the soil/sludge combinations and disturbance variations were established and these patterns significantly impacted CH4 production. Methanosarcina spp. seemed to play a key role in CH4 formation and prevailed even under stressed conditions. Overall, the results provided evidence that soil-borne methanogens can be effective in enhancing digestion performance and stability and, thus, harbor vast potential for further exploitation.

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

The influence of cattle grazing on methane fluxes and engaged microbial communities in alpine forest soils.

Recent dynamics and uncertainties in global methane budgets necessitate a dissemination of current knowledge on the controls of sources and sinks of atmospheric methane. Forest soils are considered to be efficient methane sinks; however, as they are microbially mediated they are sensitive to anthropogenic influences and tend to switch from being sinks to being methane sources. With regard to global changes in land use, the present study aimed at (i) investigating the influence of grazing on flux rates of methane in forest soils, (ii) deducing possible (a)biotic factors regulating these fluxes, and (iii) gaining an insight into the complex interactions between methane-cycling microorganisms and ecosystem functioning. Here we show that extensive grazing significantly mitigated the soil's sink strength for atmospheric methane through alterations of both microbial activity and community composition. In situ flux measurements revealed that all native, non-grazed areas were net methane consumers, while the adjacent, grazed areas were net methane producers. Whereas neither parent material nor soil properties including moisture and organic matter showed any correlation to the ascertained fluxes, significantly higher archaeal abundances at the grazed study sites indicated that small inputs of methanogens associated with cattle grazing may be sufficient to sustainably increase methane emissions.

Biological Pretreatment Strategies for Second-Generation Lignocellulosic Resources to Enhance Biogas Production.

With regard to social and environmental sustainability, second-generation biofuel and biogas production from lignocellulosic material provides considerable potential, since lignocellulose represents an inexhaustible, ubiquitous natural resource, and is therefore one important step towards independence from fossil fuel combustion. However, the highly heterogeneous structure and recalcitrant nature of lignocellulose restricts its commercial utilization in biogas plants. Improvements therefore rely on effective pretreatment methods to overcome structural impediments, thus facilitating the accessibility and digestibility of (ligno)cellulosic substrates during anaerobic digestion. While chemical and physical pretreatment strategies exhibit inherent drawbacks including the formation of inhibitory products, biological pretreatment is increasingly being advocated as an environmentally friendly process with low energy input, low disposal costs, and milder operating conditions. Nevertheless, the promising potential of biological pretreatment techniques is not yet fully exploited. Hence, we intended to provide a detailed insight into currently applied pretreatment techniques, with a special focus on biological ones for downstream processing of lignocellulosic biomass in anaerobic digestion.

Abundance and potential metabolic activity of methanogens in well-aerated forest and grassland soils of an alpine region.

Although methanogens were recently discovered to occur in aerated soils, alpine regions have not been extensively studied for their presence so far. Here, the abundance of archaea and the methanogenic guilds Methanosarcinales, Methanococcales, Methanobacteriales, Methanomicrobiales and Methanocella spp. was studied at 16 coniferous forest and 14 grassland sites located at the montane and subalpine belts of the Northern Limestone Alps (calcareous) and the Austrian Central Alps (siliceous) using quantitative real-time PCR. Abundance of archaea, methanogens and the methanogenic potentials were significantly higher in grasslands than in forests. Furthermore, methanogenic potentials of calcareous soils were higher due to pH. Methanococcales, Methanomicrobiales and Methanocella spp. were detected in all collected samples, which indicates that they are autochthonous, while Methanobacteriales were absent from 4 out of 16 forest soils. Methanosarcinales were absent from 10 out of 16 forest soils and 2 out of 14 grassland soils. Nevertheless, together with Methanococcales they represented the majority of the 16S rRNA gene copies quantified from the grassland soils. Contrarily, forest soils were clearly dominated by Methanococcales. Our results indicate a higher diversity of methanogens in well-aerated soils than previously believed and that pH mainly influences their abundances and activities.

Biological pre-treatment: Enhancing biogas production using the highly cellulolytic fungus Trichoderma viride.

With regard to renewable sources of energy, bioconversion of lignocellulosic biomass has long been recognized as a desirable endeavor. However, the highly heterogeneous structure of lignocellulose restricts the exploitation of its promising potential in biogas plants. Hence, effective pre-treatment methods are decisive prerequisites to overcome these challenges in order to improve the utilization ratio of (ligno) cellulosic substrates during fermentation. In the present study, the application of Trichoderma viride in an aerobic upstream process prior to anaerobic digestion led up to a threefold increase in the yield of methane and total gas in a lab-scale investigation. Due to its highly efficient cellulolytic activities, T. viride seemed to be responsible for an improved nutrient availability that positively influenced the anaerobic microbiocenosis. Aerobic pre-treatment of organic matter with T. viride is therefore a promising solution to achieve higher methane yields and degradation performances without any additional energy demand, nor undesired by-product inhibition.