Decentralised system for demand-oriented collection of food waste - Assessment of biomethane potential, pathogen development and microbial community structure.

Enormous amounts of food waste (FW) are produced worldwide, requiring efficient disposal strategies, both economically and ecologically. Anaerobic digestion to produce biomethane is among the most promising strategies, but requires proper solutions for storage and delivery of the waste material. Here, a decentralized system for demand-oriented FW storage and its practical usability was assessed. FW was stored under batch and fed-batch strategies at 5 °C, 20 °C and 30 °C for 28 days. The results showed that FW can be stored without cooling since bacterially produced lactic acid rapidly stabilized the material and inactivated pathogens. While FW storage worked well under all storage conditions and strategies, 16S analysis revealed a distinct microbiota, which was highly characteristic for each storage temperature. Moreover, FW storage had no negative impact on methane yield and stored FW contained readily degradable substances for demand-oriented biogas production.

Biomethane potential of industrial paper wastes and investigation of the methanogenic communities involved.

BACKGROUND: Cellulose-containing waste products from the agricultural or industrial sector are potentially one of the largest sources of renewable energy on earth. In this study, the biomethane potential (BMP) of two types of industrial paper wastes, wood and pulp residues (WR and PR, respectively), were evaluated under both mesophilic and thermophilic conditions, and various pretreatment methods were applied in the attempt to increase the methane potential during anaerobic digestion. The methanogenic community composition was investigated with denaturing gradient gel electrophoresis (DGGE) and the ANAEROCHIP microarray, and dominant methanogens were quantitated using quantitative PCR. RESULTS: All pretreatments investigated in this study with the exception of the alkaline pretreatment of PR were found to increase the BMP of two paper industry wastes. However, the low recalcitrance level of the PR resulted in the pretreatments being less effective in increasing BMP when compared with those for WR. These results were supported by the physico-chemical data. A combined application of ultrasound and enzymatic pretreatment was found to be the best strategy for increasing methane yields. The retention time of substrates in the reactors strongly influenced the BMP of wastes subjected to the different pretreatments. In sludges from both paper wastes subjected to the various pretreatments, mixotrophic Methanosarcina species were found to dominate the community, accompanied by a consortium of hydrogenotrophic genera. CONCLUSIONS: Pretreating industrial paper wastes could be a potentially viable option for increasing the overall degradation efficiency and decreasing reactor retention time for the digestion of complex organic matter such as lignocellulose or hemicellulose. This would help reduce the environmental burden generated from paper production. Although there were minor differences in the methanogenic communities depending on the temperature of anaerobic digestion, there was little effect of substrate and pretreatment type on the community composition. Thus, methanogen community dynamics would not seem to be an appropriate indicator regarding BMP in the AD processes investigated.

Co-composting of biowaste and wood ash, influence on a microbially driven-process.

A trial at semi-industrial scale was conducted to evaluate the effect of wood ash amendment on communal biowaste in a composting process and on the final composts produced. For this purpose, three treatments including an unamended control (C0) and composts with additions of 6% (C6), and 12% (C12) of wood ash (w/w) were studied, and physico-chemical parameters as well as microbial activity and community composition were investigated. At the end of the process, composts were tested for toxicity and quality, and microbial physiological activity. The influence of ash addition on compost temperature, pH, microbial activity and composition was stronger during the early composting stages and diminished with time, whereby composts became more similar. Using the COMPOCHIP microarray, a reduction in the pathogenic genera Listeria and Clostridium was observed, which together with the temperature increases of the composting process helped in the hygienisation of composts. Lactobacillus species were also affected, such that reduced hybridisation signals were observed with increased ash addition, due to the increased pH values in amended composts. Organic matter mineralisation was also increased through ash addition, and no negative effects on the composting process were observed. The nutrient content of the final products was increased through the addition of ash, and no toxic effects were observed. Nonetheless, greater concentrations of heavy metals were found in composts amended with more ash, which resulted in a downgrading of the compost quality according to the Austrian Compost Ordinance. Thus, regulation of both input materials and end-product quality is essential in optimising composting processes.

Methane yields and methanogenic community changes during co-fermentation of cattle slurry with empty fruit bunches of oil palm.

The biomethane potential and structural changes of the methanogenic community in a solid-state anaerobic digestion process co-digesting cattle slurry and empty fruit bunches were investigated under mesophilic (37°C) and thermophilic (55°C) conditions. Phylogenetic microarrays revealed the presence of two hydrogenotrophic genera (Methanoculleus and Methanobrevibacter) and one acetoclastic genus (Methanosarcina). Methanosarcina numbers were found to increase in both mesophilic and thermophilic treatments of empty fruit bunches. Methanobrevibacter, which dominated in the cattle slurry, remained constant during anaerobic digestion (AD) at 37°C and decreased in numbers during digestion at 55°C. Numbers of Methanoculleus remained constant at 37°C and increased during the thermophilic digestion. Physicochemical data revealed non-critical concentrations for important monitoring parameters such as total ammonia nitrogen, free ammonia nitrogen and volatile fatty acids in all treatments after AD. The biomethane potential of empty fruit bunches was higher under thermophilic conditions than under mesophilic conditions.

Investigation into the effect of high concentrations of volatile fatty acids in anaerobic digestion on methanogenic communities.

A study was conducted to determine whether differences in the levels of volatile fatty acids (VFAs) in anaerobic digester plants could result in variations in the indigenous methanogenic communities. Two digesters (one operated under mesophilic conditions, the other under thermophilic conditions) were monitored, and sampled at points where VFA levels were high, as well as when VFA levels were low. Physical and chemical parameters were measured, and the methanogenic diversity was screened using the phylogenetic microarray ANAEROCHIP. In addition, real-time PCR was used to quantify the presence of the different methanogenic genera in the sludge samples. Array results indicated that the archaeal communities in the different reactors were stable, and that changes in the VFA levels of the anaerobic digesters did not greatly alter the dominating methanogenic organisms. In contrast, the two digesters were found to harbour different dominating methanogenic communities, which appeared to remain stable over time. Real-time PCR results were inline with those of microarray analysis indicating only minimal changes in methanogen numbers during periods of high VFAs, however, revealed a greater diversity in methanogens than found with the array.

Changes in the microbial communities during co-composting of digestates.

Anaerobic digestion is a waste treatment method which is of increasing interest worldwide. At the end of the process, a digestate remains, which can gain added value by being composted. A study was conducted in order to investigate microbial community dynamics during the composting process of a mixture of anaerobic digestate (derived from the anaerobic digestion of municipal food waste), green wastes and a screened compost (green waste/kitchen waste compost), using the COMPOCHIP microarray. The composting process showed a typical temperature development, and the highest degradation rates occurred during the first 14 days of composting, as seen from the elevated CO2 content in the exhaust air. With an exception of elevated nitrite and nitrate levels in the day 34 samples, physical-chemical parameters for all compost samples collected during the 63 day process indicated typical composting conditions. The microbial communities changed over the 63 days of composting. According to principal component analysis of the COMPOCHIP microarray results, compost samples from the start of the experiment were found to cluster most closely with the digestate and screened compost samples. The green waste samples were found to group separately. All starting materials investigated were found to yield fewer and lower signals when compared to the samples collected during the composting experiment.

Coffee husk composting: an investigation of the process using molecular and non-molecular tools.

Various parameters were measured during a 90-day composting process of coffee husk with cow dung (Pile 1), with fruit/vegetable wastes (Pile 2) and coffee husk alone (Pile 3). Samples were collected on days 0, 32 and 90 for chemical and microbiological analyses. C/N ratios of Piles 1 and 2 decreased significantly over the 90 days. The highest bacterial counts at the start of the process and highest actinobacterial counts at the end of the process (Piles 1 and 2) indicated microbial succession with concomitant production of compost relevant enzymes. Denaturing gradient gel electrophoresis of rDNA and COMPOCHIP microarray analysis indicated distinctive community shifts during the composting process, with day 0 samples clustering separately from the 32 and 90-day samples. This study, using a multi-parameter approach, has revealed differences in quality and species diversity of the three composts.

Biotic and abiotic processes contribute to successful anaerobic degradation of cyanide by UASB reactor biomass treating brewery waste water.

In contrast to the general aerobic detoxification of industrial effluents containing cyanide, anaerobic cyanide degradation is not well understood, including the microbial communities involved. To address this knowledge gap, this study measured anaerobic cyanide degradation and the rearrangements in bacterial and archaeal microbial communities in an upflow anaerobic sludge blanket (UASB) reactor biomass treating brewery waste water using bio-methane potential assays, molecular profiling, sequencing and microarray approaches. Successful biogas formation and cyanide removal without inhibition were observed at cyanide concentrations up to 5 mg l(-1). At 8.5 mg l(-1) cyanide, there was a 22 day lag phase in microbial activity, but subsequent methane production rates were equivalent to when 5 mg l(-1) was used. The higher cumulative methane production in cyanide-amended samples indicated that part of the biogas was derived from cyanide degradation. Anaerobic degradation of cyanide using autoclaved UASB biomass proceeded at a rate more than two times lower than when UASB biomass was not autoclaved, indicating that anaerobic cyanide degradation was in fact a combination of simultaneous abiotic and biotic processes. Phylogenetic analyses of bacterial and archaeal 16S rRNA genes for the first time identified and linked the bacterial phylum Firmicutes and the archaeal genus Methanosarcina sp. as important microbial groups involved in cyanide degradation. Methanogenic activity of unadapted granulated biomass was detected at higher cyanide concentrations than reported previously for the unadapted suspended biomass, making the aggregated structure and predominantly hydrogenotrophic nature of methanogenic community important features in cyanide degradation. The combination of brewery waste water and cyanide substrate was thus shown to be of high interest for industrial level anaerobic cyanide degradation.

Treatment alternatives of slaughterhouse wastes, and their effect on the inactivation of different pathogens: a review.

Slaughterhouse wastes are a potential reservoir of bacterial, viral, prion and parasitic pathogens, capable of infecting both animals and humans. A quick, cost effective and safe disposal method is thus essential in order to reduce the risk of disease following animal slaughter. Different methods for the disposal of such wastes exist, including composting, anaerobic digestion (AD), alkaline hydrolysis (AH), rendering, incineration and burning. Composting is a disposal method that allows a recycling of the slaughterhouse waste nutrients back into the earth. The high fat and protein content of slaughterhouse wastes mean however, that such wastes are an excellent substrate for AD processes, resulting in both the disposal of wastes, a recycling of nutrients (soil amendment with sludge), and in methane production. Concerns exist as to whether AD and composting processes can inactivate pathogens. In contrast, AH is capable of the inactivation of almost all known microorganisms. This review was conducted in order to compare three different methods of slaughterhouse waste disposal, as regards to their ability to inactivate various microbial pathogens. The intention was to investigate whether AD could be used for waste disposal (either alone, or in combination with another process) such that both energy can be obtained and potentially hazardous materials be disposed of.

Effects of pH and microbial composition on odour in food waste composting.

A major problem for composting plants is odour emission. Slow decomposition during prolonged low-pH conditions is a frequent process problem in food waste composting. The aim was to investigate correlations between low pH, odour and microbial composition during food waste composting. Samples from laboratory composting experiments and two large scale composting plants were analysed for odour by olfactometry, as well as physico-chemical and microbial composition. There was large variation in odour, and samples clustered in two groups, one with low odour and high pH (above 6.5), the other with high odour and low pH (below 6.0). The low-odour samples were significantly drier, had lower nitrate and TVOC concentrations and no detectable organic acids. Samples of both groups were dominated by Bacillales or Actinobacteria, organisms which are often indicative of well-functioning composting processes, but the high-odour group DNA sequences were similar to those of anaerobic or facultatively anaerobic species, not to typical thermophilic composting species. High-odour samples also contained Lactobacteria and Clostridia, known to produce odorous substances. A proposed odour reduction strategy is to rapidly overcome the low pH phase, through high initial aeration rates and the use of additives such as recycled compost.

Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants.

To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full-scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real-time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year.

Characterisation of source-separated household waste intended for composting.

Large-scale composting of source-separated household waste has expanded in recent years in the Nordic countries. One problem can be low pH at the start of the process. Incoming biowaste at four composting plants was characterised chemically, physically and microbiologically. The pH of food waste ranged from 4.7 to 6.1 and organic acid concentration from 24 to 81 mmol kg(-1). The bacterial diversity in the waste samples was high, with all samples dominated by Gammaproteobacteria, particularly Pseudomonas and Enterobacteria (Escherichia coli, Klebsiella, Enterobacter). Lactic acid bacteria were also numerically important and are known to negatively affect the composting process because the lactic acid they produce lowers the pH, inhibiting other bacteria. The bacterial groups needed for efficient composting, i.e. Bacillales and Actinobacteria, were present in appreciable amounts. The results indicated that start-up problems in the composting process can be prevented by recycling bulk material and compost.

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.

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.

Design and testing of real-time PCR primers for the quantification of Methanoculleus, Methanosarcina, Methanothermobacter, and a group of uncultured methanogens.

In this study, 16S rRNA gene primers were designed to complement the suite of already available PCR primers for the detection of different methanogens involved in biogas production through anaerobic digestion by SYBR Green real-time PCR. Primers designed for use in TaqMan real-time PCR for the organisms Methanosaeta, Methanosarcina, and Methanoculleus have been described previously; however, we found that (i) the Methanoculleus primers were not specific to members of the genus and that (ii) the Methanosarcina primers did not work specifically with SYBR Green real-time PCR. Thus, we designed new primers for these and other methanogens, and we optimized SYBR Green real-time PCR assays. Primers were tested by end-point and real-time PCR, and they were found to work specifically and sensitively. Application of these primers will allow the detection and quantification of Methanoculleus, Methanosarcina, Methanothermobacter, and a group of yet uncultured archaea from anaerobic habitats.

Plant and animal wastes composting: effects of the N source on process performance.

The aim of this work was to evaluate the impact of different N-rich animal wastes on the composting of ligno-cellulosic wastes by a range of classical and novel methods, with particular emphasis on microbial community composition. Two composting mixtures were prepared by adding to a mixture of cotton carding wastes and wheat straw: (i) meat and bone meal and (ii) blood meal and horn and hoof meal. Composts were analyzed using physico-chemical and biochemical properties, as well as nucleic acid microarrays. Results showed that physico-chemical and biochemical parameters differentiated composts depending on their degree of stability, while microarray hybridization discriminated compost samples according to the starting materials used in the compost production. Microarray analysis indicated not only the presence in the composts of bacteria involved in N(2) fixation and plant disease suppression, but also the presence of Acinetobacter calcoaceticus that is suspected to trigger an autoimmune response related to bovine spongiform encephalopathy. The present work highlights the importance of using parameters addressing different properties of the composting matrix for a proper evaluation of the process performance.

Application of COMPOCHIP microarray to investigate the bacterial communities of different composts.

A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts.

Molecular analysis of bacterial community succession during prolonged compost curing.

The compost environment consists of complex organic materials that form a habitat for a rich and diverse microbial community. The aim of this research was to study the dynamics of microbial communities during the compost-curing phase. Three different methods based on 16S rRNA gene sequence were applied to monitor changes in the microbial communities: (1) denaturing gradient gel electrophoresis of PCR-generated rRNA gene fragments; (2) partial rRNA gene clone libraries; and (3) a microarray of oligonucleotide probes targeting rRNA gene sequences. All three methods indicated distinctive community shifts during curing and the dominant species prevailing during the different curing stages were identified. We found a successional transition of different bacterial phylogenetic groups during compost curing. The Proteobacteria were the most abundant phylum in all cases. The Bacteroidetes and the Gammaproteobacteria were ubiquitous. During the midcuring stage, Actinobacteria were dominant. Different members of nitrifying bacteria and cellulose and macromolecule-degrading bacteria were found throughout the curing process. In contrast, pathogens were not detected. In the cured compost, bacterial population shifts were still observed after the compost organic matter and other biochemical properties had seemingly stabilized.

Identification of anammox bacteria in a full-scale deammonification plant making use of anaerobic ammonia oxidation.

The existence of anaerobic ammonia-oxidizing (anammox) bacteria was postulated in the late 1970s. Approximately 20 years later, these lithotrophic members of the nitrogen cycle were identified as deep-branching members of the planctomycetes. Recently, full-scale implementation of biological deammonification was successfully achieved in the DEMON reactor at the wastewater treatment plant in Strass, Austria. The sludge of this reactor contains red granules and brownish flocs that can be physically separated. The two fractions yielded different banding patterns in denaturing gradient gel electrophoresis of PCR products obtained with primer sets targeting the 16S rRNA genes of planctomycetes. Comparative analysis of partial sequences of almost full-length 16S rRNA gene clones obtained from the granules and flocs confirms the differences in the community composition of the two fractions. The sequences retrieved from the red granules were 93% similar to those of Candidatus Brocadia anammoxidans, a bacterium known to catalyze the anaerobic ammonia oxidation.

Microbial community related to volatile organic compound (VOC) emission in household biowaste.

Malodorous emissions and potentially pathogenic microorganisms which develop during domestic organic waste collection are not only a nuisance but may also pose health risks. The aim of the present study was to determine whether the presence of specific microorganisms in biowastes is directly related to the composition of the emitted volatile organic compounds (VOCs). The succession of microbial communities during 16 days of storage in organic waste collection bins was studied by denaturing gradient gel electrophoresis (DGGE) of amplified 16S ribosomal DNA in parallel with a classical cultivation and isolation approach. Approximately 60 different bacterial species and 20 different fungal species were isolated. Additionally, some bacterial species were identified through sequencing of excised DGGE bands. Proton transfer reaction mass spectrometry (PTR-MS) was used to detect VOCs over the sampling periods, and co-inertia analyses of VOC concentrations with DGGE band intensities were conducted. Positive correlations, indicating production of the respective VOC or enhancement of microbial growth, and negative correlations, indicating the use of, or microbial inhibition by the respective compound, were found for the different VOCs. Measurement of the VOC emission pattern from a pure culture of Lactococcus lactis confirmed the positive correlations for the protonated masses 89 (tentatively identified as butyric acid), 63 (tentatively identified as dimethylsulfide), 69 (likely isoprene) and 73 (likely butanone).