Isolation and growth kinetics of ammonia tolerant consortia (NH3)-mMPA biofilm
Ammonia tolerant consortia, (NH3)-mMPA, were isolated from the reactor enriched with mMPA communities. Consortia showed a Methanosarcinaceae MPA family prevalence. Consortia were able to grow in a planktonic phase at 848.8 mg NH3/L with methane representing a mean of approximately 70% of the produced biogas. (NH3)-mMPA consortia were inoculated into vials with ceramic spheres as support for 20 days, allowing biofilm formation. Growth kinetics determined by the Gompertz model showed a λ of 3.7 d (days) and µm of 0.67 d− 1, with r2 = 0.95 (Fig. 1A). In addition, methane production was monitored during biofilm formation. Methane peaked at 80% of total biogas after 20 days of incubation. A time lag of 5–6 days was observed in methane production. By performing SEM analysis, (NH3)-mMPA consortia were detected on the support surfaces after 4 days of incubation, consistent with the kinetic growth data that predicted micro-colony formation after 20 days of incubation (Fig. 1B).
Design of an anaerobic biofilm enriched with (NH3)-mMPA under NH3 shock
After a full year of operation, the control bioreactor (CR) showed a decrease of approximately 80% in TOC (Fig. 2A), with 85% methane content in the produced biogas. The methane production curve obtained for the treatment bioreactor (TR) gradually increased, approaching 70% of the biogas produced in the first 50 days (Fig. 2B). The alkalinity alpha factor remained below 0.3 in both reactors, TOC consumption varied between 80 ± 20% in CR and 62 ± 17% in TR. Methane content in the biogas ranged from 86 ± 8% in CR, and 81 ± 18% in TR (Fig. 2A and 2B).
Morphological and genotyping characterization of (NH3)-mMPA consortia and ammonia Tolerant biofilm
To determine the microbial morphologies present in (NH3)-mMPA consortia, concentrated planktonic cultures and ceramic support fragments were observed by SEM. Sarcina morphotypes characteristic of Methanosarcinaceae were found (Fig. 1B). Analysis of the 16S rDNA from (NH3)-mMPA consortia revealed that gene sequences were grouped with Methanosarcinaceae members (Methanosarcinaceae, Methanosarcinales). Phylogenetic relationships of archaeal 16S rDNA partial sequences (EU544306; EU544307), archaeal mMPA ammonium tolerant strain 16S rDNA partial sequence (EU544305) and methanogenic species sequences from the Gene bank show that mMPA sequence EU544305 (868 bp), was grouped in the Methanomethylovorans genus. This is because it showed higher similarities with uncultured archaeon (99%) and Methanomethylovorans hollandica (Lomans 1999) (98%), and two different species of Methanosarcina genera which were predominant components of the consortia, and similar to cloned sequences. EU544306 (547 bp), uncultured Methanosarcina sp. clone HUB A5, and EU544307 (521 bp), were grouped into the Methanosarcina genus, showing high similarity with Methanosarcina sp. (95%) and Methanosarcina baltica (von Klein 2002) (95%). When a BLAST analysis compared the sequences with stored species Types of uncultured Methanosarcina sp. clone HUB A5, this is located near Methanosarcina baltica in a phylogenetic tree at a Neighbor Joining distance of 0.05 (not shown). M. baltica is an archaeon isolated from anoxic layers of sediment in the Baltic Sea that grows fast in marine culture, in methylamines, methanol, and acetate (vonKlein et al. 2002). Uncultured Methanosarcina sp. clone HUB A6 sequence EU544307 was also grouped in the Methanosarcina genus, showing high similarity to Methanosarcina acetivorans (Sowers 1984) (93 %) and uncultured Methanosarcina sp. (92%). Uncultured Methanosarcina sp. clone HUB A6 is located near Methanosarcina acetivorans in a phylogenetic tree at a Neighbor Joining distance of 0.2 (not shown), also described in anoxic marine environments and found as a single cell in marine cultures (Maeder et al. 2006).
Microbiological and molecular description of biofilm in bioreactors: Ammonia concentration in both bioreactor influents ranged between 37.7 and 128 mg NH3-N/L until day 152, when both bioreactors were subjected to ammonia shock, increasing ammonia concentrations to 755 mg NH3-N/L. Between day 180 and 208, ammonia concentrations returned to average levels (37.2–41.1 mg NH3-N/L). Significant differences in ammonia concentration were observed in phases C and D in both TR and CR effluent outputs (Fig. 2A and 2B). The experimental bioreactor showed a lower concentration of ammonia residual (137–184 mg NH3 L− 1) when compared with the control bioreactor (327 − 128 mg NH3 L− 1). Shock NH3-N effects in CR and TR are shown in Fig. 2A and 2B, respectively. Methanation in CR began at 90%, dropping to 70% during ammonia shock (Fig. 2A). A different response was observed in TR, where no significant variations were observed during the ammonia shock, reaching methanation levels above 90% throughout the experiment (t-test, p 0.0005-0.01) (Fig. 2B). In both cases (CR and TR), correlations between the percentage of TOC consumed and the concentration of methane produced were significant (r2: 0.72 p ≤ 0.05 for CR, and r2 = 0.77 p ≤ 0.05 for TR) (Fig. 2). Production of methane specific was determined based on biomass as volatile suspended solids (VSS) were registered in both reactors. In CR, a production of 0.32 ± 0.04 mL CH4/g (VSS * d) was registered, while TR showed a production of 0.65 ± 0.08 mL CH4/g (VSS * d), this activity was re-controlled post ammonia shock where it was found a production of 0.30 ± 0.05 mL CH4/g (VSS * d) in CR and 0.38 ± 0.04 mL CH4/g (VSS * d) in TR. Increased methanogenic activity per biomass unit from day 152 to 166 was 20.5-fold higher in TR when compared to CR, which indicates that methanogenic archaea maintained their activity under ammonia shock conditions.
FIGURE 2. Total Organic Carbon consumption, methanization and ammonia concentration in an anaerobic filter type reactor enriched with mMPA. 2A. CR. 2B. TR. Phase A: CR anaerobic biofilm development with ammonia operating range of 27.45–75 mg NH3-N/L; Phase B. Reactor acclimatization- Initial phase bioreactor functioning. Acclimatization at 37.7–128 mg NH3-N/ L. Phase C: Ammonia shock at 755 mg NH3-N/L, Phase D: Final phase bioreactor functioning, reactor recovery from 37.1 to 41.2 mg NH3-N/L. ■- % Total Organic Carbon (TOC) consumption; - □ -% methane; --- ○ --- NH3 input concentration; --●-- NH3 output concentration.
When comparing the first 100 days of both reactors, the control bioreactor consumed significantly more TOC than the experimental bioreactor (t-test, p < 0.0001–0.007). Methane production measure by gas chromatography was also significantly higher in the CR (89.48% − 95%) when compared to the TR (29% -75%) (p < 0.0001–0.029). These differences diminished from day 54 to 82 (Fig. 2A and 2B). During and after the ammonia shock, (phase C - day 152 to 208), a higher consumption of TOC was observed in both bioreactors, although it was significantly higher in the TR until day 180, after which it decreased significantly to 65.78% on day 208. Methane production in the TR remained above 90%, significantly higher than in the CR (p < 0.0005–0.0417), which showed a methane production that fluctuated between 69% − 86% (Fig. 2A and 2B). Significant differences (t-test, α ≤ 0.05) were observed for both methane production and TOC concentrations when comparing the CR and TR during the ammonia shock step. Figures 2A and 2B show how the ammonia shock caused a significant decline (t-test, α = 0.05) in methane production and TOC consumption in the CR. These results clearly indicate that the TR was resistant to the deleterious effect of ammonia, as the mMPA biofilm community was able to maintain its methanogen activity even under high ammonia concentrations.
Bands in the gel were conspicuous in phase B, the acclimatization phase (Fig. 3A, lane B), but tended to diminish in phase C, the ammonia shock, (Fig. 3A, lane C). However, the abundance of archaea increased between phase B, the recovery phase, and phase D, the final phase (Fig. 3A, lane D). Besides, lower archaea biodiversity was observed in the CR throughout the profile when compared with TR biofilm (Fig. 3C). Moreover, it was observed that (NH3)-mMPA enriched biofilm produced fewer bands in the bacteria domain in the CR than biofilm enriched with mMPA tolerant to ammonia (Fig. 3B, lanes B, C and D). However, 16S rDNA bacterial profile similarities were higher in the CR than in the TR. Similarities were lower for archaea than bacteria, as shown in Fig. 3C and 3D.
Figure. 3.A. DGGE profiles of archaea. Gel bands recovered from 16S rDNA gels were sequenced and identified as EU544305 Methanomethylovorans sp., EU544306 and EU544307; two different species belonging to Methanosarcina sp. B. DGGE profiles of bacteria. C. Cluster analysis (WPGMA) using data from DGGE profiles of archaea in anaerobic biofilm to assess the similarity of taxonomic composition of consortia assigned to treatment of industrial waste rich in proteins, obtained from the control reactor (CR). D. Treatment Reactor (TR) lanes. B lanes- phase B, initial acclimatization at 37.7–128 mg NH3-N/L; C lanes- phase C, ammonia shock at 755 mg NH3-N/L; D lanes- phase D, final reactor recovery from 37.1 to 41.2 mg NH3-N/L.
This study demonstrates that efficient wastewater treatment and stable methane production can be achieved under high ammonia concentrations by using anaerobic biofilms formed in fixed bed reactors containing (NH3)-mMPA (ammonia tolerant archaea biofilms).
Zheng et al. (2015) demonstrate that the selection of a suitable support for fixed-bed reactors is crucial for the anaerobic digestion of ammonium-rich livestock wastes, since they found that using zeolites as support they achieve a methane production over 80%, compared to other types of support, which is comparable to that shown in this research. NH3-tolerant microbial consortium, defined as a microbial community composed of archaea and bacteria, give rise to long-term stable communities whose tolerance to ammonia stress can be increased with an acclimated consortium. The main components (archaea) found in this study, of selected tolerant consortia shared general characteristics with the Methanosarcinaceae family, with the mMPA EU544305 sequence related to the Methanomethylovorans genus, and the other two sequences (EU544306 and EU544307) related to the Methanosarcina genus, the first one of which is considered an obligate methylotrophic methanogen (Supplementary Material SI 1. to SI 3.). Members of Methanosarcinaceae have been found in a wide variety of anaerobic environments where methane is produced, can grow on a diversity of substrates, and represent the most versatile group of Archaea (Oren 2014). The predominant archaea throughout the experiment were Methanosarcina and Methanomethylovorans (He et al. 2017; Cadavid-Rodríguez et al. 2019). Some researchers consider these species to be keystone species. For example, Methanosarcina acetivorans (Galagan et al. 2002), is a unique species which may have a wide range of metabolisms that support the reactor’s operation under ammonia shock.
In a study by Wu and Song (2021), adding inoculum through anaerobic co-digestion of waste activated sludge (WAS) and fish waste (FW), high methane production rates were obtained with mixtures of 1.5% and 3% of FW, but methanogenesis was inhibited with an addition of 6% of FW or higher, finding a high accumulation of ammonia and fatty acid. In a similar trial, Cadavid-Rodríguez et al. (2019) found that when using from 1.5% of FW in anaerobic digestion, methanogenesis is inhibited. Thus, the alternative of enriching the inoculum with ammonia-tolerant mMPA is attractive and feasible to avoid the inhibition of methanogenesis in the treatment of protein-rich fishery waste.
Furthermore, the analyses of the microbial community carried out by Wu & Song (2021) showed that methane production was dominated by hydrogenotrophic and methylotrophic methanogenic archaea, which is to be expected since fishery residues are rich in methylated amines, which would favour the growth and maintenance of the Ammonia tolerant mMPA consortia.
Physiological studies of this strain have reported that it tolerates NaCl concentrations of 100 mM. In this study, the NH3 tolerant consortia were able to grow and maintain constant methanogenic activity at 400 mM NaCl, a characteristic that favours their use for fishery waste treatment at high salt concentrations (Aloui et al. 2009, Chen et al. 2018, He et al. 2017). Because salinity has been reported to deteriorate removal performance, activated sludge characteristics, and change microbial community in anaerobic reactors (Chen et al., 2017; He et al., 2017).
We identified a strong relationship between reactor performance and abundance of Methanosarcina cells. Until day 398 (750 FAN mg/L; 6000 TAN mg/L), highly active Methanosarcina cells were identified in large multicellular structures (clusters). Calli et al. (2005) found an abundance of Methanosarcina species at high ammonia concentrations (160–747 mg).
The experimental bioreactor showed constant low values of ammonia in the output effluent after an ammonia shock when compared to the control bioreactor. Similar results have been previously reported (Dai et al. 2016). The 16S rDNA profile of archaea and bacteria obtained by PCR-DGGE shifted at OTU (Operational Taxonomic Units) composition, confirming that an acetoclastic methanogenesis dominated acetate utilization in the bioreactor. Galagan et al. (2002) described the metabolic and physiological diversity of Methanosarcina acetivorans, suggesting that the formation of multicellular structures (cell envelope and extracellular matrix) is an adaptation to stress and likely plays an important role in the ability of Methanosarcina to colonise diverse environments. This species metabolises a broad spectrum of carbon compounds into methane, a process controlled by different methanogenesis genes. Individual copies of the duplicated genes may display differential regulation and kinetic properties, allowing the species to change between substrates, consistent with the reported plasticity that Methanosarcina species show in nature (Angelidaki et al. 2011), an important characteristic that allows the community to tolerate stress. High bacterial biodiversity was observed in the communities, but we did not find significant differences between these communities. This idea has been expressed before in studies on stability in an anaerobic bioreactor (Fernández et al. 2000), where bacteria were found in the biofilm, even under stress. Nevertheless, according to our results, when the communities were exposed to changing conditions, the bacterial activity was modified, and archaea did not exhibit significant differences in abundance.
Our coastal zone is characterized by high primary productivity favoured by wind predominance and marine oceanographic current system in the South Eastern Pacific Ocean, that produce constant upwelling and for decades or centuries had been registered major fish strandings (Hernandez-Miranda et al. 2010, 2012, 2017). It is probable that an unknown endemic rich microbial ammonia methanogenic (bacteria and archaea) community potentially had evolved associated both sea water column and sediments because high protein content product of strandings. This potential microbial community sampled from effluent industrial fishery was used in the bioreactor and bioaugmented in the treatment bioreactor and our objective was characterized archaea components.