Effect of temperature and temperature fluctuation on thermophilic anaerobic digestion of cattle manure

The influence of temperature, 50 and 60 degrees C, at hydraulic retention times (HRTs) of 20 and 10 days, on the performance of anaerobic digestion of cow manure has been investigated in completely stirred tank reactors (CSTRs). Furthermore, the effect of both daily downward and daily upward temperature fluctuations has been studied. In the daily downward temperature fluctuation regime the temperatures of each reactor was reduced by 10 degrees C for 10 h while in the daily upward fluctuation regime the temperature of each reactor was increased 10 degrees C for 5 h. The results show that the methane production rate at 60 degrees C is lower than that at 50 degrees C at all experimental conditions of imposed HRT except when downward temperature fluctuations were applied at an HRT of 10 days. It also was found that the free ammonia concentration not only affects the acetate-utilising bacteria but also the hydrolysis and acidification process. The upward temperature fluctuation affects the maximum specific methanogenesis activity more severely as compared to imposed downward temperature fluctuations. The results clearly reveal the possibility of using available solar energy at daytime to heat up the reactor(s) without the need of heat storage during nights, especially at an operational temperature of 50 degrees C and at a 20 days HRT, and without the jeopardising of the overheating.     

Mesophilic versus thermophilic anaerobic digestion of cattle manure: methane productivity and microbial ecology

In this study, productivity and physicochemical and microbiological (454 sequencing) parameters, as well as environmental criteria, were investigated in anaerobic reactors to contribute to the ongoing debate about the optimal temperature range for treating animal manure, and expand the general knowledge on the relation between microbiological and physicochemical process indicators. For this purpose, two reactor sizes were used (10 m³ and 16 l), in which two temperature conditions (35°C and 50°C) were tested. In addition, the effect of the hydraulic retention time was evaluated (16 versus 20 days).Thermophilic anaerobic digestion showed higher organic matter degradation (especially fiber), higher pH and higher methane (CH₄) yield, as well as better percentage of ultimate CH₄ yield retrieved and lower residual CH₄ emission, when compared with mesophilic conditions. In addition, lower microbial diversity was found in the thermophilic reactors, especially for Bacteria, where a clear intensification towards Clostridia class members was evident.Independent of temperature, some similarities were found in digestates when comparing with animal manure, including low volatile fatty acids concentrations and a high fraction of Euryarchaeota in the total microbial community, in which members of Methanosarcinales dominated for both temperature conditions; these indicators could be considered a sign of process stability.     

Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystem

The dynamics of bacterial and archaeal populations of a laboratory-scale anaerobic digestor were investigated during a crisis period of the process reflected by an accumulation of acetate. A culture-independent approach based on single strand conformation polymorphism (SSCP) analysis of total 16S rDNA and 16S rRNA amplification products was used. A spirochete and a Synergistes sp. showed high and changing activity levels during the study. A Clostridium sp. showed a transient increase in presence and activity concomitant with the highest acetate concentrations. A major shift in the most active archaeal populations from hydrogenotrophic to acetoclastic methanogens preceded the recovery of the reactor.     

Single strand conformation polymorphism monitoring of 16S rDNA Archaea during start-up of an anaerobic digester

A laboratory-scale continuously mixed anaerobic digester was inoculated with a mix of anaerobic sludge and fed with glucose. The start-up strategy was progressive and chemical analyses were done to evaluate digester performance from day 1 to day 107. In parallel, Archaeal community dynamics were monitored by SSCP analysis of the V3 region of 16S rDNA genes and further characterized by partial sequencing of 16S rDNA genes. At day 1 the inoculum contained at least five distinct Archaeal peaks close to known methanogenic species. The dominant peak was very close to Methanosaeta concilli, the remaining species being members of the Methanobacteriales and Methanomicrobiales. A rapid shift of the Archaeal population was observed during the experiment. At day 21 Methanobacterium formicicum, which was not detected at day 1, became the dominant methanogenic species in the bioreactor and remained so until the end of the experiment.     

Dynamic simulation of cyclic batch anaerobic digestion of cattle manure

Cyclic batch reactors with periodical feeds and extractions, are often used in cattle manure anaerobic digestion. The dynamic behavior of this type of reactor was simulated in this study. The kinetic model developed by I. Angelidaki et al. [Biotechnol. Bioeng. 42 (1993) 159], together with microbial growth kinetics, conventional material balances for an ideally cyclic batch reactor, liquid-gas interactions, and liquid phase equilibrium chemistry were used in this study. The model showed good agreement with the experimental data of R.I. Mackie and M.P. Bryant [Appl. Microbiol. Biotechnol. 43 (1995) 346], and R. Borja et al. [Chem. Eng. J. 54 (1994) B9]. The effects of hydraulic retention time (HRT), organic loading rate, reactant concentrations, feeding interval, and initial conditions such as pH and ammonia concentration on process performance can be evaluated by the dynamic model. Also simulation results show that the equilibrium conditions can be considered for CO2 distribution between liquid and gas phases, especially for processes with long retention times.     

Comparison of bacterial community structure and dynamics during the thermophilic composting of different types of solid wastes: anaerobic digestion residue,pig manure and chicken manure

This study investigated the impact of composting substrate types on the bacterial community structure and dynamics during composting processes. To this end, pig manure (PM), chicken manure (CM), a mixture of PM and CM (PM + CM), and a mixture of PM, CM and anaerobic digestion residue (ADR) (PM + CM + ADR) were selected for thermophilic composting. The bacterial community structure and dynamics during the composting process were detected and analysed by polymerase chain reaction–denaturing gradient gel electrophoresis (DGGE) coupled with a statistic analysis. The physical-chemical analyses indicated that compared to single-material composting (PM, CM), co-composting (PM + CM, PM + CM + ADR) could promote the degradation of organic matter and strengthen the ability of conserving nitrogen. A DGGE profile and statistical analysis demonstrated that co-composting, especially PM + CM + ADR, could improve the bacterial community structure and functional diversity, even in the thermophilic stage. Therefore, co-composting could weaken the screening effect of high temperature on bacterial communities. Dominant sequencing analyses indicated a dramatic shift in the dominant bacterial communities from single-material composting to co-composting. Notably, compared with PM, PM + CM increased the quantity of xylan-degrading bacteria and reduced the quantity of human pathogens.     

Comparison of two-stage thermophilic (68 degrees C/55 degrees C) anaerobic digestion with one-stage thermophilic (55 degrees C) digestion of cattle manure

A two-stage 68 degrees C/55 degrees C anaerobic degradation process for treatment of cattle manure was studied. In batch experiments, an increase of the specific methane yield, ranging from 24% to 56%, was obtained when cattle manure and its fractions (fibers and liquid) were pretreated at 68 degrees C for periods of 36, 108, and 168 h, and subsequently digested at 55 degrees C. In a lab-scale experiment, the performance of a two-stage reactor system, consisting of a digester operating at 68 degrees C with a hydraulic retention time (HRT) of 3 days, connected to a 55 degrees C reactor with 12-day HRT, was compared with a conventional single-stage reactor running at 55 degrees C with 15-days HRT. When an organic loading of 3 g volatile solids (VS) per liter per day was applied, the two-stage setup had a 6% to 8% higher specific methane yield and a 9% more effective VS-removal than the conventional single-stage reactor. The 68 degrees C reactor generated 7% to 9% of the total amount of methane of the two-stage system and maintained a volatile fatty acids (VFA) concentration of 4.0 to 4.4 g acetate per liter. Population size and activity of aceticlastic methanogens, syntrophic bacteria, and hydrolytic/fermentative bacteria were significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. The density levels of methanogens utilizing H2/CO2 or formate were, however, in the same range for all reactors, although the degradation of these substrates was significantly lower in the 68 degrees C reactor than in the 55 degrees C reactors. Temporal temperature gradient electrophoresis profiles (TTGE) of the 68 degrees C reactor demonstrated a stable bacterial community along with a less divergent community of archaeal species.     

Effects of mixing on methane production during thermophilic anaerobic digestion of manure: lab-scale and pilot-scale studies

The effect of mixing on anaerobic digestion of manure was evaluated in lab-scale and pilot-scale experiments at 55 degrees C. The effect of continuous (control), minimal (mixing for 10 min prior to extraction/feeding) and intermittent mixing (withholding mixing for 2h prior to extraction/feeding) on methane production was investigated in three lab-scale continuously stirred tank reactors. On comparison to continuous mixing, intermittent and minimal mixing strategies improved methane productions by 1.3% and 12.5%, respectively. Pilot-scale studies also supported the lab-scale results with an average 7% increase in biogas yields during intermittent mixing compared to continuous mixing. The effect of mixing intensities (minimal, gentle or vigorous) in batch assays at 55 degrees C showed that when the process was overloaded by high substrate to inoculum ratio (40/60), gentle (35 times per minute) or minimal mixing (10 min mixing before feeding) was advantageous compared to vigorous mixing (110 times per minute). On the other hand, under low substrate to inoculum ratio (10/90), gentle mixing was the best. The study thus indicated that mixing schemes and intensities have some effect on anaerobic digestion of manures.     

Rapid start-up of thermophilic anaerobic digestion with the turf fraction of MSW as inoculum

This study aims to determine suitable start-up conditions and inoculum sources for thermophilic anaerobic digestion. Within days of incubation MSW at 55 °C, methane was produced at a high rate. In an attempt to narrow down which components of typical MSW contained the thermophilic methanogens, vacuum cleaner dust, banana peel, kitchen waste, and garden waste were tested as inoculum for thermophilic methanogenesis with acetate as the substrate. Results singled out grass turf as the key source of thermophilic acetate degrading methanogenic consortia. Within 4 days of anaerobic incubation (55 °C), anaerobically incubated grass turf samples produced methane accompanied by acetate degradation enabling successful start-up of thermophilic anaerobic digestion. Other essential start-up conditions are specified. Stirring of the culture was not conducive for successful start-up as it resulted specifically in propionate accumulation.     

Illumina sequencing of bacterial 16S rDNA and 16S rRNA reveals seasonal and species-specific variation in bacterial communities in four moss species

In order to better understand the factors that influence bacterial diversity and community composition in moss-associated bacteria, a study of bacterial communities in four moss species collected in three seasons was carried out via high-throughput sequencing of 16S rDNA and 16S rRNA. Moss species included Cratoneuron filicinum, Pylaisiella polyantha, Campyliadelphus polygamum, and Grimmia pilifera, with samples collected in May, July, and October 2015 from rocks at Beijing Songshan National Nature Reserve. In total, the bacterial richness and diversity were high regardless of moss species, sampling season, or data source (DNA vs. RNA). Bacterial sequences were assigned to a total of 558 OTUs and 279 genera in 16 phyla. Proteobacteria and Actinobacteria were the two most abundant phyla, and Cellvibrio, Lapillicoccus, Jatrophihabitans, Friedmanniella, Oligoflexus, and Bosea the most common genera in the samples. A clustering algorithm and principal coordinate analysis revealed that C. filicinum and C. polygamum had similar bacterial communities, as did P. polyantha and G. pilifera. Metabolically active bacteria showed the same pattern in addition to seasonal variation: bacterial communities were most similar in summer and autumn, looking at each moss species separately. In contrast, DNA profiles lacked obvious seasonal dynamics. A partial least squares discriminant analysis identified three groups of samples that correlated with differences in moss species resources. Although bacterial community composition did vary with the sampling season and data source, these were not the most important factors influencing bacterial communities. Previous reports exhibited that mosses have been widely used in biomonitoring of air pollution by enriching some substances or elements in the moss-tag technique and the abundant moss associated bacteria might also be important components involved in the related biological processes. Thus, this survey not only enhanced our understanding of the factors which influence microbial communities in mosses but also would be helpful for better use and development of the moss-tag technique in the environmental biomonitoring.     

Study of bacterial communities in Antarctic coastal waters by a combination of 16S rRNA and 16S rDNA sequencing

An ecological study on distribution of Antarctic bacterial communities was determined by 16S-based phylogenetic analyses of clone libraries derived from RNA and DNA extracted from two different marine areas and compared between each other. Superficial seawater samples were collected from four stations in Ross Sea, three of them located in Rod Bay and one in Evans Cove; for each station two clone libraries (16S rDNA and 16S rRNA) were prepared and evident divergences between DNA and RNA libraries of each site were obtained. Of all phylotypes 93.6% were found in RNA libraries; in contrast, only 31 phylotypes (70.5%) were retrieved from total microbial community (DNA libraries). DNA and RNA sequences related to gamma-Proteobacteria and Bacteroidetes groups, typical for Antarctic sea-ice bacterial communities, were detected in analysed sites. 16S rDNA and rRNA libraries derived from the two different areas were enriched by picophytoplanktonic 16S sequences of plastid and mitochondrion origins, reflecting that the algal blooms occurred during sampling (Antarctic summer 2003). The finding in Rod Bay libraries of high percentage of DNA clones apparently affiliated with beta-Proteobacteria typical for activated sludges and well water could be explained by the presence of a sewage depuration system at this site. Obtained results clearly demonstrate that combination of 16S rDNA and 16S rRNA gene sequencing is preferred approach to have a more reliable vision on the composition of microbial communities.     

16S rDNA sequence diversity of a culture-accessible part of an anaerobic digestor bacterial community

A bacterial culture-based inventory with 16S rDNA identification of the isolates was carried out on an anaerobic digestor microbial ecosystem to compare to the 16S rDNA sequences directly retrieved from the ecosystem by a molecular inventory previously made in our laboratory. Twenty OTUs (Operational Taxonomic Units) belonging to five of the major bacterial groups were identified from 338 isolated colonies. The sequences of 13 of the 20 OTUs were not closely related to any hitherto published sequences (less than 96% sequence identity). Six OTUs out of 20 were found to have sequences similar to sequences of the molecular inventory. Despite the biases expected to be associated with the molecular and culture-based methods, the distribution of the isolated OTUs into the different bacterial phyla was similar to that of the molecular OTUs.     

Evaluation of Anammox and denitrification during anaerobic digestion of poultry manure

Two approaches based on ne w process development and biological nitrogen transformation were investigated in a bench study for removing nitrogen as N2 gas from poultry waste while stabilizing the wastes. The process, known as "Anammox", was explored in batch anaerobic culture using serum bottles. The Anammox process involves the use of nitrite as an electron acceptor in the bacterially mediated oxidation of ammonia to yield N2. Studies are described wherein nitrite was added to poultry waste and the effects on ammonium levels were monitored. About 13-22% ammonium removal was observed with the inoculation of returned activated sludge, and the total ammonium reduction was not proportional to the reduction of nitrite, thereby suggesting that Anammox was less competitive under the conditions in our studies. The addition of nitrite and nitrate was not inhibitory to the process based on gas generation and COD reduction. The classical nitrogen removal process of nitrification followed with denitrification offers a more reliable basis for nitrogen removal from poultry wastes.     

Effects of ammonia nitrogen of H2 and CH4 production during anaerobic digestion of dairy cattle manure

A number of researchers have verified the inhibitory effects of elevated H2 concentrations on various anaerobic fermentation processes. The objective of this work was to investigate the potential for using hydrogen gas production to predict upsets in anaerobic digesters operating on dairy cattle manure. In an ammonia nitrogen overload experiment, urea was added to the experimental digesters to obtain increased ammonia concentrations (600, 1,500, or 3,000 mg N/l). An increase in urea concentration resulted in an initial cessation of H2 production followed by an increase in H2 formation. Additions of 600, 1,500, or 3,000 mg N/l initially resulted in the reduction of biogas H2 concentrations. After 24 h, the H2 concentration increased in the 600 and 1,500 mg N/l digesters, but production remained inhibited in the 3,000 mg N/l digesters. Both methane and total biogas production decreased following urea addition. Volatile solids reduction also decreased during these periods. The digester effluent pH and alkalinity increased due to the increased NH4 formed with added urea. Based on these results, changes in H2 concentration could be a useful parameter for monitoring changes due to increased NH3 in dairy cattle manure anaerobic digesters.     

Characterization of start-up performance and archaeal community shifts during anaerobic self-degradation of waste-activated sludge

Successful start-up strategy for anaerobic digestion of waste-activated sludge using internal inoculum and relationship between the shift of methanogenic community and the digester performance during start-up was investigated. Combination of TS control of inoculum and batch operation during early days enabled the successful start-up operation without serious volatile fatty acid accumulation, followed by the stable continuous operation. However, the propionate degradation was rate-limiting step during the batch operation. The results of real-time quantitative polymerase chain reaction analysis suggested that there was a correlation between the population of the genus Methanosarcina and the methane production rate coupled with acetate consumption during batch operation, and the results of terminal-restriction fragment length polymorphism (T-RFLP) revealed that the increasing intensity of T-RF peaks of hydrogenotrophic methanogens was associated with a decrease in the level of C3-acids.     

16S rDNA fingerprinting of rhizosphere bacterial communities associated with healthy and Phytophthora infected avocado roots

Molecular techniques employing 16S rDNA profiles generated by PCR-DGGE were used to detect changes in bacterial community structures of the rhizosphere of avocado trees during infection by Phytophthora cinnamomi and during repeated bioaugmentation with a disease suppressive fluorescent pseudomonad. When the 16S rDNA profiles were analyzed by multivariate analysis procedures, distinct microbial communities were shown to occur on healthy and infected roots. Bacterial communities from healthy roots were represented by simple DNA banding profiles, suggestive of colonization by a few predominant species, and were approximately 80% similar in structure. In contrast, roots that were infected with Phytophthora, but which did not yet show visible symptoms of disease, were colonized by much more variable bacterial communities that had significantly different community structures from those of healthy roots. Root samples from trees receiving repeated applications of the disease suppressive bacterium Pseudomonas fluorescens st. 513 were free of Phytophthora infection, and had bacterial community structures that were similar to those of nontreated healthy roots. Sequence analysis of clones generated from four predominant bands cut from the DGGE gels revealed the presence of pseudomonads, as well as several previously unidentified bacteria. Differentiation of 16S rDNA profiles for healthy and infected roots suggests that rhizosphere bacterial community structure may serve as an integrative indicator of changes in chemical and biological conditions in the plant rhizosphere during the infection process.     

Effect of pre-aeration and inoculum on the start-up of batch thermophilic anaerobic digestion of municipal solid waste

In this study, a short pre-aeration step was investigated as pre-treatment for thermophilic anaerobic digestion of the organic fraction of municipal solid waste (OFMSW). It was found that pre-aeration of 48 h generated enough biological heat to increase the temperature of bulk OFMSW to 60 °C. This was sufficient self-heating of the bulk OFMSW for the start-up of thermophilic anaerobic digestion without the need for an external heat source. Pre-aeration also reduced excess easily degradable organic compounds in OFMSW, which were the common cause of acidification during the start-up of the batch system. Careful consideration however must be taken to avoid over aeration as this consumes substrate, which would otherwise be available to methanogens to produce biogas. To accelerate methane production and volatile solids destruction, the anaerobic digestion in this study was operated as a wet process with the anaerobic liquid recycled through the OFMSW. Appropriate anaerobic liquid inoculum was found to be particularly beneficial. It provided high buffer capacity as well as suitable microbial inoculum. As a result, acidification during start-up was kept to a minimum. With volatile fatty acids (VFAs-acetate in particular) and H₂ accumulation typical of hydrolysis and fermentation of the easily degradable substrates during start-up, inoculum with high numbers of hydrogenotrophic methanogens was critical to not only maximise CH₄ production but also reduce H₂ partial pressure in the system to allow VFAs degradation. In a lab-scale bioreactor, the combined pre-aeration and wet thermophilic anaerobic digestion was able to stabilise the OFMSW within a period of only 12 days. The stabilised inert residual material can be used as a soil amendment product.     

Survival of bacterial pathogens during the thermophilic anaerobic digestion of biowaste: laboratory experiments and in situ validation

Anaerobic digestion is continually gaining importance for the processing of the organic fraction of municipal solid wastes. Although methods for studying the survival of pathogen exist, these methods often need adaptations, are expensive, time consuming or generally not well suited for the harsh conditions within an anaerobic digestion system. In the present study we investigated the applicability of commercially available, mechanically stable and inexpensive pathogen carriers to validate in situ pathogen inhibition within a 750,000l thermophilic, bio-waste treating anaerobic digester. None of the pathogens investigated (Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni) was capable of survival under the conditions of the biogas reactor for more than 24 h indicating that the temperature and physico-chemical properties of the sludge of the fermenter were effective in inhibiting the survival of these microorganisms.