The average CODtotal, TN, and TP in the concentrated BW were 17.0, 2.53 and 0.20 g.L− 1, respectively. The concentration of CODtotal in the BW in the present study is higher than that reported by Zeeman et al. [5] and De Graaff et al. [42] for BW collected from vacuum toilets, but the concentrations of TN and TP are similar. The pH of the UASB reactor effluent was 7.70, within the recommended range for methanogenesis according to Chernicharo [14]. Alkalinity was higher in the effluent of the UASB reactor than in the influent, which is desirable because high concentrations of volatile acids can be buffered without causing a substantial drop in pH [14]. Appropriate total alkalinity can maintain the buffering capacity of the reactor at favorable levels [15]. The average CODtotal removal in the UASB reactor was 60.6%, lower than the value reported by De Graaff et al. (2010) due to some periods of instability in the influent flow of the reactor. The average removal of TN and TP in the UASB reactor was 30.4 and 30.0%, respectively; the majority of TN and TP remained in the liquid phase for subsequent removal/recovery in the tubular PBR. The TSS concentration in the sludge of the UASB reactor was 45.8 g.L− 1, which is classified as dense and flocculent according to Chernicharo (2017). The CODtotal in the effluent of the tubular PBR increased because of microalgal growth. The removal/recovery of TN and TP was 44.0% and 65.0%, respectively, from the influent to the tubular PBR.
Our results showed that the BW module increased microbial richness and diversity. From a microbial perspective, BW offers a wider niche for the UASB reactor, which likely promoted increased microbial community diversity via organic matter concentration. However, along the different modules of the bioreactor, the abundance of DNA sequences decreased dramatically. Also, the network analysis showed a decrease in the microbial community complexity from the beginning to the final steps of the bioreactor (SP1 to SP3), concomitant to a reduced diversity. Such a decrease is reasonable and reflects both changes in substrate composition and wash conditions. In the initial stage, the BW promoted increases in Bacteroidetes, Firmicutes, and Proteobacteria, which accounted for more than 60% of all DNA sequences affiliated to bacteria. Previous studies have reported the presence and high abundance of Bacteroidetes in human feces and water environments [16–18]. Bacteroidetes play an important role in acidification in UASB sludge [16, 18]. Moreover, Bacteroidetes are a group of proteolytic bacteria with a robust capacity to decompose proteins, amino acids, and high-molecular-weight compounds in water environments, explaining the dominance of this phylum in the initial phase [19, 20].
The high abundance and strong shifts in the phyla Bacteroidetes, Firmicutes, and Proteobacteria dominated the composition of the common microbiome in the BW module [21]. This is because Bacteroidetes and Firmicutes are capable of degrade various types of cellulose, mainly from toilet paper [18]. In the initial phase at BW there is a large amount of cellulose biomass which contributed to select cellulose-degrading bacteria. The high abundance of cellulose-degrading bacteria found in our study play an important role on fiber decomposition. The fiber decomposition compound can accumulate and hazardous impact the other steps such as sludge production and biogas production during the anaerobic processes [22]. Moreover, the predominance of Firmicutes in the SP1 tanks indicate a dependence on molecules produced by other groups of microorganisms once these syntrophic bacteria are capable of degrading cellulosic compounds and producing volatile fatty acids (VFAs) and H2, which are then consumed by hydrogenotrophic methanogens in metabolic reactions [23]. The third most abundant phylum, Proteobacteria, predominated in the SP3 tanks. This phylum comprises sulfate-reducing groups of bacteria that, like Firmicutes, are syntrophic and depends on symbioses (associated with methanogens) [24].
We also found significantly higher abundances of Acidobacteria and Cyanobacteria in the SP3 tanks. Members of the phylum Cyanobacteria are able to thrive in several critical conditions in bioreactor tanks. According to Stal [25], Cyanobacteria oxidize sulfides under anoxic conditions and perform anoxygenic photosynthesis utilizing a hydrogen sulfide-like electron donor. In a study of biofilms containing Cyanobacteria in scum from a bioreactor, [26] observed spatial proximity between Cyanobacteria and hydrogen sulfide bacteria.
The microbial community also showed several different functions along the bioreactor. The SP1 tank promoted functions related to the metabolism of carbohydrates, chemicals, drugs, and nitrogen. Human waste sewage is composed mainly of high concentrations of nitrogen and carbohydrates [27, 28]. Organic nitrogen is the major nitrogen form in human feces. Therefore, the transformation of organic nitrogen to its inorganic forms (ammonia/ammonium) requires an association with bacterial groups such as Bacteroidetes and Firmicutes [29][30]. Those groups likely transformed the nitrogen in the bioreactor system since they were the most abundant groups in the first stage of water decontamination. Bacteroidetes and Firmicutes also degrade carbohydrates in the human gut [31][32], their original habitat before transitioning to the bioreactor. In SP1, we also identified other functions related to cellular components such as stimulus, molecule transport, and pathogenesis. All these functions are likely associated with the high diversity of fecal components, which stimulates cell activity in the initial phase of the bioreactor.
In SP-S, we detected nitrogen metabolic processes resulting from initial nitrogen transformation. We also detected several other functions related to the exchange of electron acceptors and anaerobic transformation of waste. The synthrophic groups Synergistetes, Parvachaeota, and Chloroflexi play important roles in anaerobic processes [10, 33, 34]. According to [33], the predominance of these methanogenic groups indicates a syntrophic relationship with other bacterial groups. In the present study we also found high correlation with methanogenesis process in the SP-S. This function is directly related to the methanogenic degradation of toilet paper [35]. The water insoluble cellulose from toilet paper are a significant fraction of sewage and although cellulose is a type of carbohydrate its decomposition is very slow [36]. We here found that the partial degraded cellulose by Bacteroidetes and Firmicutes from SP1 activated the methanogenesis process from methanogenic Chloroflexi and Synergistetes at the SP-S which converted cellulose in methane.
In SP3, we found a high abundance of functions related to regulation of nitrogen, antibiotic biosynthetic and metal ion binding. Those functions are related to microalgal activity, which has shown efficient performance in decontaminating water [37–39]. In addition, these same microalgae have been shown to be used as rich nutrient in barley production [40]. Our system of wastewater treatment also decreased microbial functions related to pathogenesis, and the analyses confirmed that the microalgae-containing SP3 reduced bacterial richness and diversity. Consistent with our findings, [41] reported high performance of an algae system in reducing pathogenic bacteria.