Enhancement of Modeling Phased Anaerobic Digestion Systems through Investigation of Their Microbial Ecology and Biological Activity

Anaerobic digestion (AD) is widely used in wastewater treatment plants for stabilisation of primary and waste activated sludges. Increasingly energy prices as well as stringent environmental and public health regulations ensure the ongoing popularity of anaerobic digestion. Reduction of volatile solids, methane production and pathogen reduction are the major objectives of anaerobic digestion. Phased anaerobic digestion is a promising technology that may allow improved volatile solids destruction and methane gas production. In AD models, microbially-mediated processes are described by functionally-grouped microorganisms. Ignoring the presence of functionally-different species in the separate phases may influence the output of AD modeling. The objective of this research was to thoroughly investigate the kinetics of hydrolysis, acetogenesis (i.e., propionate oxidation) and methanogenesis (i.e., acetoclastic) in phased anaerobic digestion systems. Using a denaturing gradient gel electrophoresis (DGGE) technique, bacterial and archaeal communities were compared to complement kinetics studies. Four phased digesters including Mesophilic-Mesophilic, Thermophilic-Mesophilic, Thermophilic-Thermophilic and Mesophilic-Thermophilic were employed to investigate the influence of phase separation and temperature on the microbial activity of the digestion systems. Two more digesters were used as control, one at mesophilic 35 0C (C1) and one at thermophilic 55 0C (C2) temperatures. The HRTs in the first-phase, second-phase and single-phase digesters were approximately 3.5, 14, and 17 days, respectively. All the digesters were fed a mixture of primary and secondary sludges. Following achievement of steady-state in the digesters, a series of batch experiments were conducted off-line to study the impact of the digester conditions on the kinetics of above-mentioned processes. A Monod-type equation was used to study the kinetics of acetoclastic methanogens and POB in the digesters, while a first-order model was used for the investigation of hydrolysis kinetics. Application of an elevated temperature (55 0C) in the first-phase was found to be effective in enhancing solubilisation of particulate organics. This improvement was more significant for nitrogen-containing material (28%) as compared to the PCOD removal (5%) when the M1 and T1 digesters were compared. Among all the configurations, the highest PCOD removal was achieved in the T1T2 system (pvalue<0.05). In contrast to the solubilisation efficiencies, the mesophilic digesters (C1, M1M2 and T1M3) outperformed the thermophilic digesters (C2, T1T2 and M1T3) in COD removal. The highest COD removal was obtained in the T1M3 digestion system, indicating a COD removal efficiency of 50.7±2.1%. The DGGE fingerprints from digesters demonstrated that digester parameters (i.e., phase separation and temperature) influenced the structure of the bacterial and archaeal communities. This resulted in distinct clustering of DGGE profiles from the 1st-phase digesters as compared to the 2nd-phase digesters and from the mesophilic digesters as compared to the thermophilic ones. Based on the bio-kinetic parameters estimated for the various digesters and analysis of the confidence regions of the kinetic sets (kmax and Ks), the batch experiment studies revealed that the kinetic characteristics of the acetoclastic methanogens and POB developed in the heavily loaded digesters (M1 and T1) were different from those species developed in the remaining mesophilic digesters (M2, M3 and C1). As with the results from the mesophilic digesters, a similar observation was made for the thermophilic digesters. The species of acetoclastic methanogens and POB within the T1 digester had greater kmax and Ks values in comparison to the values of the T3 and C2 digesters. However, the bio-kinetic parameters of the T2 digester showed a confidence region that overlapped with both the T1 and T3 digesters. The acetate and propionate concentrations in the digesters supported these results. The acetate and propionate concentrations in the M1 digesters were, respectively, 338±48 and 219±17 mgCOD/L, while those of the M2, M3 and C1 digesters were less than 60 mg/L as COD. The acetate and propionate concentrations were, respectively, 872±38 and 1220±66 in T1 digester, whereas their concentrations ranged 140-184 and 209-309 mg/L as COD in the T2, T3 and C2 digesters. In addition, the DGGE results displayed further evidence on the differing microbial community in the 1st- and 2nd-phase digesters. Two first-order hydrolysis models (single- and dual-pathway) were employed to study the hydrolysis process in the phased and single-stage digesters. The results demonstrated that the dual-pathway hydrolysis model better fit the particulate COD solubilisation as compared to the single-pathway model. The slowly (F0,s) and rapidly (F0,r) hydrolysable fractions of the raw sludge were 36% and 25%, respectively. A comparison of the estimated coefficients for the mesophilic digesters revealed that the hydrolysis coefficients (both Khyd,s and Khyd,r) of the M1 digester were greater than those of the M2 and M3 digesters. In the thermophilic digesters it was observed that the Khyd,r value of the T1 digester differed from those of the T2, T3 and C2 digesters; whereas, the hydrolysis rate of slowly hydrolysable matter (i.e., Khyd,s) did not differ significantly among these digesters. The influence of the facultative bacteria, that originated from the WAS fraction of the raw sludge, and/or the presence of hydrolytic biomass with different enzymatic systems may have contributed to the different hydrolysis rates in the M1 and T1 digesters from the corresponding mesophilic (i.e, M2 and M3) and thermophilic (i.e., T2 and T3) 2nd-phase digesters

Biogas production from food waste via co-digestion and digestion-effects on performance and microbial ecology

In this work, performance and microbial structure of a digestion (food waste-only) and a co-digestion process (mixture of cow manure and food waste) were studied at mesophilic (37°C) and thermophilic (55°C) temperatures. The highest methane yield (480mL/g VS) was observed in the mesophilic digester (MDi) fed with food waste alone. The mesophilic co-digestion of food waste and manure (McoDi) yielded 26% more methane than the sum of individual digestions of manure and food waste. The main volatile fatty acid (VFA) in the mesophilic systems was acetate, averaging 93 and 172mg/L for McoDi and MDi, respectively. Acetate (2150mg/L) and propionate (833mg/L) were the main VFAs in the thermophilic digester (TDi), while propionate (163mg/L) was the major VFA in the thermophilic co-digester (TcoDi). The dominant bacteria in MDi was Chlorofexi (54%), while Firmicutes was dominant in McoDi (60%). For the mesophilic reactors, the dominant archaea was Methanosaeta in MDi, while Methanobacterium and Methanosaeta had similar abundance in McoDi. In the thermophilic systems, the dominant bacteria were Thermotogae, Firmicutes and Synergistetes in both digesters, however, the relative abundance of these phyla were diferent. For archaea, the genus Methanothermobacter were entirely dominant in both TDi and TcoDi.publishedVersio

Rumen and Cecum Microbiomes in Reindeer (Rangifer tarandus tarandus) Are Changed in Response to a Lichen Diet and May Affect Enteric Methane Emissions

Reindeer (Rangifer tarandus tarandus) are large Holarctic herbivores whose heterogeneous diet has led to the development of a unique gastrointestinal microbiota, essential for the digestion of arctic flora, which may include a large proportion of lichens during winter. Lichens are rich in plant secondary metabolites, which may affect members of the gut microbial consortium, such as the methane-producing methanogenic archaea. Little is known about the effect of lichen consumption on the rumen and cecum microbiotas and how this may affect methanogenesis in reindeer. Here, we examined the effects of dietary lichens on the reindeer gut microbiota, especially methanogens. Samples from the rumen and cecum were collected from two groups of reindeer, fed either lichens (Ld: n = 4), or a standard pelleted feed (Pd: n = 3). Microbial densities (methanogens, bacteria and protozoa) were quantified using quantitative real-time PCR and methanogen and bacterial diversities were determined by 454 pyrosequencing of the 16S rRNA genes. In general, the density of methanogens were not significantly affected (p>0.05) by the intake of lichens. Methanobrevibacter constituted the main archaeal genus (>95% of reads), with Mbr. thaueri CW as the dominant species in both groups of reindeer. Bacteria belonging to the uncharacterized Ruminococcaceae and the genus Prevotella were the dominant phylotypes in the rumen and cecum, in both diets (ranging between 16-38% total sequences). Bacteria belonging to the genus Ruminococcus (3.5% to 0.6%; p = 0.001) and uncharacterized phylotypes within the order Bacteroidales (8.4% to 1.3%; p = 0.027), were significantly decreased in the rumen of lichen-fed reindeer, but not in the cecum (p = 0.2 and p = 0.087, respectively). UniFrac-based analyses showed archaeal and bacterial libraries were significantly different between diets, in both the cecum and the rumen (vegan::Adonis: pseudo-F<0.05). Based upon previous literature, we suggest that the altered methanogen and bacterial profiles may account for expected lower methane emissions from lichen-fed reindeer

Fluctuations on the rumen and cecum archaeal microbiota in lichen-fed Norwegian reindeer.

<p>Mean values for the total 16S rRNA sequences assigned to each phylotype are displayed, with taxonomical classification at species/strain level. Standard error (black lines) and statistical significance (asterisk symbol; p<0.05) with permutation Welch’s <i>t</i>-test (9999 permutations) analysis were also included. Only samples from the same sampling site were used for statistical comparisons (i.e. rumen or cecum). Rumen sample from reindeer fed pellets (Rumen pellets) (red); Rumen-lichen (blue); Cecum pellets (green); Cecum lichen (orange).</p

Concentration of methanogens, bacteria and protozoa in the cecum of Norwegian reindeer.

<p>Concentration of methanogens, bacteria and protozoa in the cecum of Norwegian reindeer.</p

Box-plots within-sample community diversity comparisons from rumen and cecum samples in Norwegian reindeer.

<p>(A) Mean alpha diversity values for total unique OTUs (observed_species) calculated for the bacterial fraction of the microbiome. (B) Mean alpha diversity values for total unique OTUs (observed_species) calculated for the archaeal fraction of the microbiome. Box-plots were calculated using average values obtained from randomly subsampled datasets for each sample with a sample depth of 2000 sequences and 10 iterations at each subsampling step. Pairwise comparisons were performed only between samples from the same sample site (rumen or cecum) and statistical significance (asterisk symbol; p<0.05) was calculated with non-parametric t-test with Monte Carlo permutations (n = 999).</p

Concentration of methanogens, bacteria and protozoa in the rumen of Norwegian reindeer.

<p>Concentration of methanogens, bacteria and protozoa in the rumen of Norwegian reindeer.</p

Anatomical and physiological conditions in the rumen and cecum of Norwegian reindeer at sampling.

<p>Anatomical and physiological conditions in the rumen and cecum of Norwegian reindeer at sampling.</p

Comparisons of imputed metagenome prediction of the bacterial and archaeal metagenomes in Norwegian reindeer.

<p>Relative abundances for each KEGG metabolic pathway present in each metagenome were calculated and plotted with STAMP. KEGG pathways that were significantly different (IC: 95%. <i>p</i>-value <0.05) between diets for (A) bacterial and (B) archaeal predicted gene functions are showed. Color pattern is set based on diet composition: pellets concentrate (red); lichens (blue).</p