3.1 Physicochemical conditions of Anaerobic digesters
The physicochemical conditions of the digester operation are given in Table 2. All three digesters were operated in almost similar physicochemical conditions, i.e., under mesophilic temperature (34 °C), neutral pH range of 7.13–7.55. At the same time, dry solids, volatile solids concentrations varied respectively, between 2.56 to 5.74% and 43.75 to 70.95 mg L− 1. Among the three digesters, however, AD3 had a relatively low organic loading rate of 5.84 kg. ODS/m3. d compared to AD1 (6.84 kg. ODS/m3. d) and AD5 (6.61 kg. ODS/m3. d). The electrical conductivity (EC) of all sludge digestors found to be towards the higher range. The highest conductivity of sludge samples was found for AD5 (12.33–15.46 mS cm− 1) followed by AD3 (11.71–14.44 mS cm− 1), and AD1(9.77–13.45 mS cm− 1). The EC value range found in this study was similar to a study conducted recently in Austria in which the effects of various co-substates were evaluated for the microbial community composition of seven full-scale anaerobic digesters fed with or without co-substrates [17]. Overall, three anaerobic digesters showed acceptable levels of main operational parameters and were performing stably during the sampling period.
Table 2
Physicochemical parameters of anaerobic digesters samples
Digester
|
Digester sample
|
Temperature
(°C)
|
pH
|
EC
(mS cm− 1)
|
Dry solids (% [wt/vol])
|
Volatile solids
(% [wt/wt] of TS)
|
Volatile fatty acids
(mg HAc/L)
|
Dissolved sulfide (mg/L)
|
Alkalinity
(mg/L)
|
AD1
|
AD1N
|
34.3
|
7.33
|
10.77
|
3.34
|
70.27
|
177
|
30.6
|
3249
|
AD1D
|
33.8
|
7.13
|
13.45
|
2.91
|
70.56
|
165
|
37.2
|
3014
|
AD1F
|
33
|
7.33
|
9.77
|
4.34
|
70.95
|
195
|
38
|
3451
|
AD3
|
AD3S
|
34.5
|
7.22
|
14.44
|
2.56
|
43.75
|
153
|
34.40
|
3390
|
AD3D
|
33.5
|
7.27
|
11.71
|
3.35
|
70.15
|
168
|
32.40
|
2992
|
AD3J
|
34.2
|
7.55
|
13.76
|
5.74
|
43.76
|
205
|
37.20
|
3512
|
AD5
|
AD5S
|
34.4
|
7.36
|
12.33
|
2.79
|
50.3
|
150
|
16.80
|
3190
|
AD5N
|
33.8
|
7.45
|
13.75
|
3.41
|
56.52
|
145.5
|
27.60
|
2893
|
AD5J
|
32.9
|
7.46
|
14.39
|
3.35
|
54.54
|
195
|
26.40
|
3857
|
AD5F
|
33.4
|
7.50
|
15.46
|
3.54
|
67.49
|
191
|
47.20
|
3498
|
3.2 Illumina sequencing of 16S rRNA genes
To check the quality of Illumina sequencing results, the number of raw reads, total data obtained, and the average base quality for each sample are shown in supplementary Fig. 2. It can be observed that all the samples, except two of them, have an average Phred score of more than 30. To retain only the high-quality data reads with a Phred score > Q30 is taken for down-stream analysis. The total number of raw reads of 16S rRNA genes identification, and CR (conserved region for V3-V4) trimmed reads are presented in supplementary Fig. 3. Overall, 3340 MB sequencing data were generated using Illumina sequencing, revealing 1.6 × 106 raw reads. Using quality filtering, 60% of the total reads were removed, resulting in an average of 0.6 × 106 reads per sample. A total of 419 bacterial community taxa found and classified at Phyla (33), Class (64), Order (99), Family (116), and Genera level (107). To find out the major taxa in each level, filtration is applied to retain taxa, which are present at least 1% in any of the samples, resulting in 84 OTUs based on 97% similarity. While 6.1 ± 1.24% reads were assigned to Archaea, which included five major phyla, 83 Genera, and 152 species. Bacteroidetes, Firmicutes, Synergistetes, and Theromotogate, OP8, and Chloroflexi were found to be dominant in all the digesters, accounting for 90.85% of all sequences (Fig. 1). In this study, Bacteroidetes were found to be the most abundant phylum (41.44%), followed by Firmicutes (23.42%), Synergistetes (7.22%), Thermotogae (6.70%), and Chloroflexi(6.69%) and OP8 (5.3%). Other phyla, detected at percentages > 1% were affiliated to Actinobacteria (3.1%), Proteobacteria (1.4%), and WWE1 (1.21%). The overall abundance of bacterial community phyla observed in this study is similar to closely related studies where a group of Phyla, namely Chloroflexi, Firmicutes, and Bacteroidetes were found to be dominant in full-scale anaerobic sludge digester samples [17, 23, 24]. However, unlike these studies, the percentages of Bacteroidetes (44%) detected in this study were significantly high and the population of proteobacteria was very low (1.4%). The higher population of Bacteroidetes phyla can be associated with high hydrolytic activity in full-scale anaerobic digesters[25].
3.3 Bacterial community diversity at genus level
The rarefaction curves show that among the three digesters, the highest diversity was present in AD5. At the same time, the least diverse microbial community was found in AD1 (Fig. 2A). Fifty-one genera were detected as core genera in all the digesters, including Acetobacteroides (Blvii28), Coprothermobacter, Fervidobacterium, Clostridium, Caldilinea, Allochromatium, Sediminibacter, and T78 (Fig. 2B). In addition to these genera, many of the bacteria were identified as unknown bacteria. Most of these genera are associated with an anaerobic digester, some have been reported from anaerobic digester earlier also[11]. Notably the highest population of genus Acetobacteroides (Blvii28) was present in all the digesters. The cultured representative member of the genus is known to produce acetate, H2, and CO2 as the product of fermentation [26]. Intra-group comparison of three AD3 samples collected between September 2016 and January 2017 showed that the concentration of Blvii28 increased significantly in samples from September 2016 to December 2016 and January 2017 (supplementary file 2). A similar trend was observed for intragroup comparison of AD5 samples collected between September 2016 and January 2017 (supplementary file 3). In contrast, T78 is possibly metabolizing carbohydrates and alcohol via syntrophic interactions [27]. T78 is the member of the phylum Chloroflexi often found to be abundant in full-scale mesophilic anaerobic digesters receiving excess sludge from wastewater treatment plants[28]. Intragroup comparison of AD5 (Jan-Feb 2017) samples found nine-fold increase in concentration of T78 in February 2017 sample compared to January 2017. Many genera found predominantly in the digesters were thermophilic or halophilic. Like, Coprothermobacter is a known proteolytic anaerobic thermophilic bacteria found in many thermophilic anaerobic digester [29]. This genus can also improve protein degradation by establishing a syntrophy with hydrogenotrophic archaea [29]. Other thermophilic bacteria found in the anaerobic digester include Fervidobacterium and Caldilinea, that ferment carbohydrates to lactate, acetate, hydrogen, and carbon dioxide [30, 31].
In comparison, genus Sediminibacter which was initially isolated from marine sediment, was also found as one of the dominant genera [32]. This can be due to the simple reason that most of the water used in Dubai is obtained from the sea and is being used after desalination [33, 34]. The incoming feed may contain elevated levels of salts, or it may be due to the fact that Sediminibacter has a unique light-driven sodium ion pump, which helps in their survival in marine habitats [35]. Several purple sulfur bacteria like Allochormatium and Thermotogales AUTHM297 were also part of the microbial community in these digesters. Many sulfate-reducing bacteria were also found, including Desulfomicrobium and Desulfobacter, as the two most dominant genera of SRB. In our previous work using FISH technique, we found that in all three digesters members of the genus Desulfobacter and Desulfobacterium were consistently present in large numbers[19]. It is interesting to note that Desulfomicrobium is also known to be associated with the marine habitat [36].
The process of anaerobic digestion involves the degradation of organic compounds to simple organic compounds, which is most likely carried out by the members of the phylum Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, and Thermotogae. Other dominant phyla in the fermenters are shown in Fig. 3. Previous studies also demonstrate the presence of these phyla in anaerobic digester as macromolecules degrading bacteria[11]. Mycobacterium, which is associated with the production of Lipases and Lipolytic activity, is also found as one of the predominant genera. Another critical process of anaerobic digestion is acidogenesis, wherein bacteria convert organic monomers into acids like acetic, propionic, and butyric acids. The population of Acidogenic bacteria like Acetobacteroides, Fervidobacterium, Clostridium, and Paludibacter producing acetate lactate or propionate was high in the digesters as reported above. The conversion of these organic molecules to CH4 is carried out mainly by Archaea. However, some bacteria may influence the production of methane. For example, acetate-utilizing uncultured bacteria of Synergistes group 4 and its competition with aceticlastic methanogen [37].
The boxplots of intergroup comparisons for differentially present genera among three anaerobic digestor groups (AD1 v/s AD3; AD3 v/s AD5 and AD1 v/s AD5) are shown in Fig. 4 and supplementary file. Three genera Rhodobacter, Allochromatium, and Bifidobacterium, are found to be differentially present across the two groups AD1 and AD3, but among them, only Bifidobacterium showed differential abundance between AD3 and AD5. For the third Comparison (AD1 v/s AD5), genera Propionicimonas, Clostridium, and Paracoccus showed differential abundance between the two groups AD1 and AD5. To find out if the samples showed an overall group-specific trend, a principal component analysis based on all the genera detected is performed. The PC1 vs PC2 PCA plot (Fig. 5) showed no group-specific signature. Overall, from the taxonomic classification at the genera level, it can be observed that different digesters alter the microbiome profile differently. For intergroup comparisons, no abundant genera (present at least 1% in any of the samples) showed significant differentiation.
3.4 Archaeal community diversity at genus level
When analyzed at the genus level, the highest diversity was observed in digester AD5, followed by AD1 and AD3 (Fig. 6A). This was evident from the highest Shannon index obtained for AD1, followed by AD3 and AD5. The predominant Archaea in the three digesters were the members of the phylum Euryarchaeota followed by Crenarchaeota. The population of hydrogenotrophic archaea was higher in the digesters. Methanocorpusculum alone constitutes more than 50% of the total population of Archaea. Other predominant genera of archaea include Metallosphaera, Methanocella, Methanococus, Acidianus, Natronobacterium, and others shown in Fig. 6B. Methanocorpusculum is one of the hydrogenotrophic methanogens, which was isolated for the first time from the biodigester of the wastewater treatment plant [38]. In contrast, the second most predominant genus was Metallosphaera, which is an extreme thermoacidophile with optimal growth at 74 °C and pH 2.0 [39]. Methanocella, which was the third most dominant genus, is a mesophilic, hydrogenotrophic methanogen [40]. While Methanococcus is also a thermophilic hydrogenotrophic methanogen and at least one species of the genus is also known to fix nitrogen[41].
It is clear from the microbial community analysis that a core microbial community exists in the anaerobic digesters studied. However, the degradation is carried out by the members of various phylum reported earlier also from the anaerobic digesters. These include the members of the phylum Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes, and Thermotogae [13]. While several genera mainly involved in the conversion of these organic substrates into acetate, butyric acid, propionate, and H2 were found to be the dominant genera in the anaerobic digesters. However, Euryarchaeota, mainly the hydrogenotrophic methanogens, were found as predominant members in the digesters (Fig. 6). The predominant hydrogenotrophic methanogens activities compared to aceticlastic ones, is in agreement with another study[25]. Overall, abundant microbial genera observed in this study, and their predicted role in anaerobic digestion associated with a specific process is shown in Fig. 7. Although the digesters were operated under mesophilic conditions, many thermophilic genera of bacteria and archaea were found to predominate in the digester. It may be because of the presence of these bacteria in the incoming feed. Whether or not these bacteria are initially present in the feed is a matter of further investigation. Furthermore, the bacteria of marine origin like Sediminibacter and Haloterrigena as one of the predominant genera maybe because most of the water used in Dubai is desalinated seawater. The high salinity range suggested indirectly by higher electrical conductivity values observed in this study may be the reason for the occurrence of halophilic bacteria found in anaerobic digester systems.