The microbial community structure in industrial biogas plants influences the degradation rate of straw and cellulose in batch tests

Diversity indices

Analysis of the bacterial communities in the ten industrial-scale biogas plants by 454 pyrosequencing resulted in 36,523 sequences after quality trim and chimera check, with a range from 2573 to 4915 sequences per sample. Unique barcode was assigned to each replicate of one sample. The triplicate sequencing analysis was evaluated with unweighted UniFrac principal coordinate analysis (PCoA), where no outlier was observed (data not shown). The triplicates were then pooled in silico and randomly subsampled according to the sample having the lowest number of sequences (2500 sequences per sample). The number of OTUs per sample ranged from 52 to 258, with a comparatively lower value for the CD plants (Table 3). The rarefaction curve revealed the same general trend, i.e. a lower number of OTUs in the co-digestion plants compared with the WWTP (Table 3; Fig. 1). Based on the observed species and the Chao1 index, the sequencing covered 64.7–89.7 % of the total bacterial community. The three co-digestion plants CD01, 02 and 04 had low diversity and evenness of the bacterial community, as indicated by low values of species richness, Shannon and Simpson index. However, WWTP04 had similarly low values in this regard (Table 3).

Sample	Chao 1	OTUs	Shannon	Simpson
CD01	58	52	1.937	0.456
CD02	94	69	3.111	0.763
CD03	147	120	5.042	0.947
CD04	109	96	3.619	0.767
WWTP01	294	227	5.851	0.956
WWTP02	215	187	4.737	0.861
WWTP03	304	258	5.823	0.930
WWTP04	209	135	3.088	0.609
WWTP05	354	244	5.899	0.955
WWTP06	280	242	5.857	0.930

CD 01-04 co-digestion plants, WWTP 01-06 wastewater treatment plants

The low diversity in CD01, 02 and 03 correlated with the comparably higher level of ammonia in these digesters (two-sample t test and nonparametric Monte Carlo permutations, n = 999, P < 0.01). In biogas plants processing protein-rich materials, the ammonia released during the AD process is known to have a strong impact on both the community structure and the diversity, most probably because of the inhibitory effects of ammonia [40–42]. High evenness and richness have been suggested to be associated with good conversion efficiency of fatty acids to methane [43]. High initial evenness has also been shown to be important for preserving functional stability in microbial communities in general [44]. In the present study, CD01, 02, 04 and WWTP04 had lower evenness and diversity than the other digesters, implying that these biogas plants might have a comparatively lower potential to reach high degradation efficiencies. Indeed in the BMP tests started with inoculum from CD01 to CD02 a comparably lower methane production rates was seen for both substrates (Table 2). However, both CD04 and WWTP04 showed higher degradation rates, similar to the rates obtained with inoculums having a higher bacterial diversity (CD03, WWTP01–3 and WWTP05–6).

Phylogenetic analysis across samples

The phylogenetic composition, as determined by PCoA of unweighted UniFrac matrices (Fig. 2a), clearly revealed a clustering of the WWTPs, which suggests close phylogenetic distance within these different plants, while the CDs were more separated from each other and from the WWTPs. Considering the relative abundance, as revealed by the weighted UniFrac matrices, the plant CD03 was more closely related to the WWTPs, while the other three CDs plants were still distantly separated from each other and from the WWTPs (Fig. 2b). This confirms findings by Sundberg et al. [45] of two separate clusters distinguishing co-digestion plants and plants processing sewage sludge. In general, Firmicutes and Bacteroidetes were identified as predominant phyla in the industrial-scale biogas plants studied here, but other phyla were also present (Fig. 3). For example, the phyla Chloroflexi, Proteobacteria and OP8 were more associated with plants processing sewage sludge (Welch’s t-test, P < 0.01), while the co-digesting CD01 and 02 biogas plants contained a large fraction of sequences belonging to a unclassified cluster at phylum level. Bacteroidetes and Firmicutes are the two dominant phyla commonly found in various AD processes [21, 28, 31, 35, 36, 46, 47].

The phylum Firmicutes was detected in all biogas plants, but at different relative abundance, from 5.2 to 67 % (Fig. 3). Within this phylum, the class Clostridia was dominant, with relative abundance of 4.4–64 %. Other classes were also present but at lower levels, such as Bacilli (<2.7 %) and Erysipelotrichi (<0.4 %) (Additional file 1: Figure S1). The order Clostridiales (3.8–56.3 %) was dominant within the clostridia, while Thermoanaerobacterales (<0.6 %) and unclassified orders such as MBA08 and SHA-98 comprised the rest of this class (Additional file 1: Figure S2). In anaerobic environments, Clostridiales has been reported as the main cellulose degrader [10]. This order has frequently been recovered from various digesters operating with mono- and co-digestion of lignocellulosic materials [31, 32, 46, 48–50]. However, the class Clostridia also contains proteolytic members and replacement of cellulolytic clostridia with proteolytic members has been observed when using protein-rich material as substrate with an inoculum originating from a biogas plant processing pig slurry and maize silage [51, 52]. A genus Caldicoprobacter (OTU 481, Additional file 1: Figures S4, S5) within Clostridiales was found at a higher relative abundance (0.68 %) in CD01, 02 and 04 compared to the rest samples (<0.04 %). This genus contains several xylanolytic bacteria [53–55]. The order MBA08 was detected in four samples in the present study CD01 (2.8 %), CD02 (1.5 %), CD04 (6.8 %) and WWTP06 (0.4 %) (Additional file 1: Figures S2, S5). This cluster was first identified in a thermophilic laboratory-scale digester [56] and later also in other thermophilic digesters [31, 57]. The presence of representatives from this cluster in the mesophilic biogas plants included in this study suggests that MBA08 contains organisms growing at wide range of temperatures. The order SHA-98 was observed at comparably higher levels in the high ammonia digesters CD01 and CD02, with relative abundance of 5.5 and 8.8 %, respectively. The levels in the rest of the digesters were 0.3–3.2 %. This order was represented by two dominant OTUs, OTU11 representing the main OTU in the high ammonia digesters CD01/02/04 (up to 7.9 %) and OTU 35, comparably more abundant in all other digesters (up to 2.6 %) (Additional file 1: Figure S5). In line with previous findings by Sundberg and co-workers [45], the family Clostridiaceae had higher relative abundance in WWTPs (2.2–5.4 %) than in CDs (0.2–0.9 %) (Welch’s t test, P < 0.01) (Additional file 1: Figure S3). Notably, the genus Gallicola, classified to the proposed family Tissierellaceae [58], was present at high relative abundance in plant CD04 (46.2 %) (Additional file 1: Figure S5). This family was not detected in any other biogas plant except WWTP06 (0.1 %) (Additional file 1: Figure S4). The first representative of the genus Gallicola was isolated from chicken manure and it has been shown to grow on purines, such as uric acid, xanthine, 6,8-dihydroxypurine, guanine and hypoxanthine [59].

The phylum Bacteroidetes had a relative abundance of 10.1–69.2 % (Fig. 3). Within this phylum, the order Bacteroidales (class Bacteroidia) was dominant in all samples (Additional file 1: Figure S2, S3). Members of the Bacteroidetes are able to degrade various polysaccharides [60, 61]. In a study with batch fermentation of straw and hay, the relative abundance of Bacteroidetes was higher at the end than at the beginning of the batch cultivation, indicating the importance of this phylum for degradation of lignocellulose [36]. An increase in relative abundance of this phylum has also been observed in response to straw addition in a laboratory-scale digester initially operating with cattle manure [31]. However, the Bacteroidetes have been shown to decrease in abundance at high levels of ammonia [41, 42, 51, 62]. In line with this, the lowest relative abundance of this phylum was seen for CD01 and CD04, both with relatively high ammonia levels (Fig. 3). However, CD02 also had a high ammonia level but had a similar relative abundance of Bacteroidetes as the low ammonia digesters. At family level, the Bacteroidaceae (up to 2.3 %), Marinilabiaceae (up to 0.2 %), Porphyromonadaceae (up to 9.2 %), Rikenellaceae (up to 0.7 %) and a few unclassified families were present (Additional file 1: Figure S3). One unclassified family, SB-1, was also observed, with higher abundance in CD03 (12.6 %) and WWTP01-06 (4.0–65.3 %) than in CD01, 02 and 04 (<0.2 %), possibly suggesting sensitivity to high ammonia levels (Table 1; Additional file 1: Figure S5). In addition, the Porphyromonadaceae were observed at relative abundance of 9.8 % in CD02 and 8.0 % in CD03, while other orders represented less than 2.0 %.

The candidate phylum Cloacimonetes (formerly WWE1) was observed at levels of less than 0.1 % in plants CD01 and CD02, but ranged from 1.0 to 14.2 % in the other eight biogas plants (Fig. 3; Additional file 1: Figure S5). Representatives of this phylum were first discovered in AD plants processing sewage sludge [63], but have since been found in a full-scale plant fed energy crop (mainly maize silage) [32] and a laboratory-scale digester fed cattle manure as the sole substrate or co-digested with wheat straw [31]. A study using stable isotopes suggested that members of this phylum are engaged in either cellulose hydrolysis or uptake of cellulose fermentation products [64]. The uncultured cluster at phylum level SAR406 was observed in CD03 with a relative abundance of 8.2 %, while the level in CD01-02 and in all WWTP was less than 1 % (Fig. 3; Additional file 1: Figure S5). The candidate name Marinimicrobia has been proposed for this cluster [65] and preliminary genome analysis suggests that members within this phylum are proteolytic and amino acid degraders [64, 66]. The phylum Proteobacteria was present at a level of 3.4–9.6 % in the WWTP, but less than 0.3 % in the CDs (Fig. 3; Additional file 1: Figure S5). This phylum has previously been found in various digesters processing sewage sludge [45, 67–69], but recently also in mono-digestion of fodder beet silage [46] and in a process co-digesting food waste with Chinese silvergrass [70]. Representatives of the uncultured phylum Hyd24-12 corresponded to 12–25 % in WWTP 01, 05 and 06, but were much less abundant in the other biogas plants (<2.3 %). This uncultured cluster has been found in other methanogenic digesters [71, 72], but its function in the methanogenic environment is still unknown [73]. The phylum Chloroflexi was represented with relative abundance of 6.5–11.5 % and 0.1–3.9 % in the WWTPs and CDs, respectively, and was dominated by an uncultured genus T78 (5.4–9.2 % in WWTPs and 0.1–3.0 % in CDs) (Fig. 3; Additional file 1: Figure S5). This phylum has been previously found in anaerobic digesters processing sewage sludge [74] and in co-digestion of whey permeate and cow manure [75], and members have been suggested to be carbohydrate utilisers [76, 77]. In addition, an uncultured cluster was found at high levels in CD01, 02 and 04, with a relative abundance of 73.7, 45.2 and 11.4 %, respectively (Fig. 3; Additional file 1: Figure S5). This large cluster was represented by one single OTU and had 84 % similarity based on 16S rRNA gene with an uncultured Clostridium (OTU1). The dominance of the community by such a large fraction represented by a single OTU is somewhat surprising and, to our knowledge, has not been reported previously for biogas digesters. Considering the feedstock for these biogas plants, this OTU might represent a protein-fermenting bacterium enriched by the protein-rich feedstock. Alternatively, this bacterium has a selective advantage at the high levels of the free ammonia in these digesters (0.218–0.408 g L⁻¹). Ammonia has in previous studies been shown to have a strong selective pressure on the microbial community [41, 42].

cel5

The cellulolytic community structures were investigated using T-RFLP combined with clone library analysis (Fig. 4a). Analysis of the inoculum samples revealed that T-RF 275 bp was present in all digesters except CD04, with a higher relative abundance in CD01–02 (51.1–52.1 %) than in the other plants (<21.1 %). T-RF 362 bp was also present at higher levels in CD01–02 (14.0–24.8 %) than in the other samples, with detectable levels only in samples WWTP02–04 (1.3–2.9 %). T-RF 85 bp had the highest relative abundance in CD03 (76.1 %), followed by WWTP01–04 (44.3–71.4 %), WWTP05–06 (14.5–15.4 %) and CD01–02 (2.8–9.2 %), while it was not detected at all in CD04. T-RF 396 bp dominated in CD04 (88.8 %) and WWTP05–06 (66.1–80.8 %), while T-RF 78 bp showed higher relative abundance in WWTP06 (13.1 %), WWTP04 (13.7 %) and WWTP03 (23.9 %), but was present at lower levels (<8 %) in the other plants.

Incubation with cellulose and straw in the batch test clearly changed the cel5 TRFLP profile compared to the one observed at the beginning of the experiment. However, the response varied both with inoculum and with substrate (Fig. 4a). In CD01, the relative abundances of the dominant T-RFs 275 and 362 bp were lower after the incubation with cellulose (28.3, 14.0 %) compared to levels at the beginning of the batch test. Instead the abundance of T-RFs 74, 78, 85, 156 and 396 bp were comparably higher (27.2, 6.0, 11.5, 10.6 and 7.4 %, respectively) than at the starting point (0, 1.8, 2.8, 1.2 and 2.6 %, respectively). For CD02, a similar decrease was seen for T-RF 362 bp, while with this inoculum T-RF 275 bp increased to 79.9 %. The other T-RFs (i.e. 74, 78, 85, 156 and 396 bp) that increased to a smaller extent in CD01 were not detected at the end point for this inoculum. Using straw as substrate resulted in a similar T-RFLP profile change as for cellulose for both CD01 and CD02. For CD03 and WWTP01–04, the relative abundance of the most dominant T-RF 85 bp decreased after incubation with both cellulose and straw. For WWTP03, the level of T-RFs 78 and 85 bp was reduced to non-detectable, while instead T-RFs 275 and 362 bp increased to 76.5 and 23.5 % for cellulose (83.6 and 16.4 % for straw). However, at the end point using both substrates, T-RFs 74, 78 and 156 bp increased their level for CD03, WWTP01–02 and WWTP04, except T-RFs 74 and 156 bp for WWTP04c. For WWTP04c, the level of T-RFs 275 and 362 bp increased. T-RF 122 bp increased in CD03c and WWTP01–02c, while T-RF 130 bp increased in WWTP01–02c. For CD04 and WWTP05–06, the most pronounced change was for the dominant T-RF 396 bp, which decreased to 7.9, 8.1 and 7.8 %, respectively. Instead, for CD04c, T-RFs 211, 85, 156 and 74 bp increased to 44.1, 26.5, 11.9 and 8.2 %, respectively. In addition, for CD04 s T-RF 78 bp increased to 15.8 %. For WWTP05, T-RFs 74, 78, 85 and 275 bp increased to 14.1, 10.5, 37.1 and 12.4 %, respectively, for cellulose, while T-RFs 74, 85 and 275 bp increased to 23.4, 33.7 and 9.5 %, respectively, for straw. The profile of WWTP06 was similar to that of WWTP05 but, in addition, T-RFs 156 and 78 bp increased to 16.1 and 30.5 % for WWTP06c and WWTP06 s, respectively.

cel48

The T-RFLP profile obtained by the cel48 primer set of the inoculum samples showed a high relative abundance of T-RF 62 in the majority of samples from the WWTPs, with WWTP01, 02, 03 and 05 having relative abundance of 28.6–81.5 % (Fig. 4b). The level in WWTP04 and WWTP06 was lower, 4.8 and 1.4 %, respectively. This T-RF was only detected in two CDs (CD02–03) and at low levels (1.5–2.3 %). For WWTP04, the dominant T-RFs were instead 141 bp (21.2 %) and 238 bp (38.6 %), while for WWTP06, the two most dominant T-RFs were 357 bp (48.2 %) and 328 bp (12.8 %). For CD01 and CD04, the T-RFLP profile was dominated by T-RFs 357 bp (35.4 and 17.4 %) and 328 bp, (30.1 and 48.0 %). For CD02, the level of T-RF 328 bp was similar (29.9 %), but T-RF 357 bp represented only 1.9 %. Instead, T-RFs 231, 224 and 73 bp were present at a level of 17.9, 11.5 and 10.2 %, respectively. For CD03, the two most dominant T-RFs were 247 bp (34.9 %) and 321 bp (33.0 %), which were present at a relatively low level in all other biogas plants.

In contrast to the cel5 community, the end point cel48 community in most cases was not the same when using cellulose and straw as substrate (Fig. 4b). For CD01, the dominant T-RFs 328 and 357 bp decreased in both cases, but T-RF 316 bp (47.9 %) increased in the cellulose incubations and T-RF 296 bp (53.6 %) increased after incubation with straw. For CD02, the dominant T-RF after incubation with straw and cellulose was 268 bp (30.5 %) and 357 bp (61.6 %), respectively. For CD03, the starting point T-RF 247 bp disappeared for both end points, while T-RF 321 bp increased in response to cellulose addition (67.0 %), but declined in abundance when straw was used as substrate (2.5 %). In addition, T-RF 62 bp increased to 89.0 % when straw was used. For CD04, T-RF 357 bp dominated after digestion with cellulose (90.9 %), while T-RFs 321 bp (38.4 %), 328 bp (16.2 %) and 357 bp (14.1 %) dominated in the straw cultures. For WWTPs, after digestion with cellulose, the major peak was T-RF 62 bp (13.0–94.6 %). In addition, T-RFs 296 bp (78.7 %) and 321 bp (45.1 %) were high in WWTP03 and 04, respectively. For the end point with straw, T-RF 62 bp remained as the major peak in WWTP02 (38.9) and 06 (74.1 %) and T-RF 296 bp was also high in WWTP02 (41.4 %). For WWTP01 s and 04–05 s, the dominant peak changed to T-RF 164 bp, with a relative abundance of 94.0, 49.3 and 93.6 %, respectively. For WWTP03 s, T-RF 357 bp, with a level of 36.3 %, was the major peak in this plant.

Clone libraries and phylogenetic analysis

cel5

T-RF 275 bp, represented by OTU07 and present in all digesters except CD04, and T-RF 362 bp (OTU08), highly abundant in CD01 and CD02, were both most closely related to an uncultured bacterium (AEV59723), with an identity of 77.0 and 76.0 %, respectively. Mahella australiensis (phylum Firmicutes) was the closest cultivated bacterium, with an identity of 57.4 and 58.4 %, respectively (Table 4, Fig. 5). M. australiensis is able to ferment different carbohydrates, including cellobiose [78]. It has previously been detected in thermophilic digesters operating with chicken manure, where it is suggested to be sensitive to ammonia [79, 80]. This species has also been observed in the cel5 community in a CSTR digester fed cow manure and steam-exploded straw operating at 44 °C [29]. T-RF 85 bp, present at high level in most digesters and represented by OTU02 and OTU03, was closely related to an uncultured bacterium (AGW24153), with an identity of 100 and 89.1 %, respectively. This OTU has previously been observed during anaerobic digestion of cow manure and steam-exploded straw at 37 °C [29]. The closest cultivated bacterium was Echinicola vietnamensis, with an identity of 58 and 61.4 %, respectively. E. vietnamensis was first isolated from seawater [81], and is able to hydrolyse starch and grow up to 44 °C with 15 % NaCl. T-RF 396 bp (OTU10), dominating in CD04, WWTP05 and WWTP06, was verified as C. cellulolyticum (identity 100 %). This is a well-studied non-ruminal mesophilic cellulolytic bacterium, initially isolated from decayed grass [82] that has the ability to degrade cellulose and hemicellulose to acetate, ethanol, lactate and H₂ [83]. Moreover, it has been shown that bioaugmentation with C. cellulolyticum can increase the cellulose degradation efficiency of wheat straw during batch cultivation [84]. T-RF 78 bp, which increased in most of the digesters in response to cellulose and straw, was unfortunately not found in the clone library in this study. However, in a previous study investigating the cel5 community in digesters operating with manure and steam-exploded straw, a T-RF of the same size was shown to correspond to a clone related to Eubacterium siraeum (49.5 % identity) [29]. The abundance of the organism in that study was shown to increase from 14  to 52 % with increasing temperature from 37 to 52 °C. This bacterium has been isolated from human faeces and belongs to a genus commonly existing in the human gut and involved in cellulose degradation [85] [86]. T-RF 211 bp, which increased in the CDs in response to both straw and cellulose, was represented by OTU06. This OTU was related to an uncultured bacterium (AGO64733; 75.0 % identity) previously identified in a laboratory-scale reactor operating with manure and straw [29] and grouped with P-OTU05 and 07, identified in the same study (Fig. 5). The closest cultured relative, Marinilabilia salmonicolor (phylum Bacteroidetes, 66.3 % identity) was isolated in seawater and has been shown to have the ability to degrade cellulose, monomeric sugars and starch [87].

cel48

T-RF 238 bp (OTU05), dominating in WWTP04, was most closely related to Ruminococcus sp. (identity 82.3 %). T-RFs 358 bp (OTU11) and 328 bp (OTU10), dominating in WWTP06, were most closely related to uncultured bacterium AGO64695 (99.0 % identity) and AGO64673 (99.1 % identity) as identified previously [29]. The closest cultivated relative was Clostridium straminisolvens JCM 21,531(78.8 % identity) and Clostridium termitidis CT1112 (73.8 % identity) for OTU11 and OTU10, respectively (Fig. 6). Both C. straminisolvens and C. termitidis have previously been shown to have the ability to degrade a variety of monomeric sugars, as well as cellobiose [88, 89]. Clostridium straminisolvens has been detected in various types of biogas digesters, such as an anaerobic thermophilic digester fed municipal waste [24] and an anaerobic mesophilic digester fed pig manure, rice straw [90, 91]. The T-RF with size 238 bp was also found in digester WWTP03 at an abundance of 11.9 %. In this case, the corresponding clone (OTU06) was closely related to Streptomyces griseorubens (61 % identity). This result highlights one drawback of T-RFLP analysis, with several different sequences resulting in the same T-RF size, and the importance of combining this method with a clone library. S. griseorubens has been isolated from soil in both India and China, and the ability to degrade lignocellulose has been demonstrated [92–94]. Clones OTU07 and OTU09 represented T-RFs 247 bp and 321 bp, respectively, which were present at comparatively high levels in CD03. For these, two uncultured bacteria (AGO64692; identity 75.2 % and AGO64682; identity 85.6 %), respectively, both retrieved from anaerobic digester sludge, had the highest similarity. The closest known bacteria were Acetivibrio cellulolyticus (71.2 % identity) and Ruminiclostridium thermocellum (75 % identity), respectively. A. cellulolyticus can utilise cellulose, cellobiose and salicin and has previously been found in a methanogenic enrichment culture from municipal sewage sludge [95]. R. thermocellum, synonym of Clostridium thermocellum, is a well-studied anaerobic cellulose-degrading thermophilic organism which has been suggested as a potential candidate for different biotechnological applications [96]. It has been isolated from plants and from cow and horse manure [97] and has been demonstrated to play a key role in cellulolytic degradation in different types of biomethane production digesters [98–100]. After incubation with straw, T-RFs 296 and 164 bp increased in abundance in CD01 and WWTP01, respectively. These two T-RFs could not be found in the clone libraries. However, in a previous study of laboratory-scale digesters operating with manure and straw at 44 °C [29], both T-RFs were found and suggested to represent species closely related to A. cellulolyticum (74.3 % identity) and Ruminococcus champanellensis (61.8 % identity), respectively. R. champanellensis is able to utilise cellulose, cellobiose and xylan, but not starch and pectin [101]. OTU02 (T-RF 68) and OTU08 (T-RF 290) grouped together in the phylogenetic tree (Fig. 6) and showed similarity to Ruminococcus, but with relatively low identity (50.0 and 50.5 %, respectively). OTU04, 09 and 11 grouped together with P-OTU03 and 08 from a previous study investigating potential cellulose-degrading bacteria in digesters operating with straw and cow manure [29].