sCOD and phosphorus removal in serially passaged wastewater reactors

To assess the performance and efficiency of serially passaged wastewater bioreactors and electro-bioreactors, sCOD and PO 4 3−-P concentrations were measured in the effluent (Fig. 1a and b). As shown, sCOD removal efficiency ranged from 91–98% in serially passaged control bioreactors and electro-bioreactors operated at CD of 3 and 7 Am−2. It was observed that passaging days 9, 12 and 13 had the highest sCOD removal of 97% in control bioreactor then slightly decreased to 91% by day 15, while the highest sCOD removal was 97–98% in electro-bioreactors operated at CD of 3 and 7 Am−2 on days 12 and 15. The high removal efficiency of sCOD in control bioreactors is due to the short hydraulic control of sludge age in terms of organic loading to recycled sludge across the passaging days. However, the decline observed on day 15 could be due to the dispersion of the activated sludge flocs which causes turbidity resulting in high sCOD concentration in the effluent; these results are in agreement with previous findings which confirms that sludge settling is an important factor affecting organic removal2. Nutrient removal was also investigated by reporting the removal efficiency of PO 4 3−-P in all serially passaged wastewater reactors tested. PO 4 3−-P was efficiently removed reaching 99% in all reactors during the whole serial passaging period. The high phosphorus removal in the control bioreactor could be due to biodegradation process such as the presence of polyphosphate accumulating organisms (PAOs) which are a relatively fast growing heterotrophs which can remove PO 4 3−-P. Overall, the removal efficiency of sCOD and PO 4 3−-P in electro-bioreactors is attributed to the effects of electrochemical processes (i.e. electrocoagulation, electro-osmosis and electrophoresis) in addition to biodegradation which was previously explored by4,20. It should be noted that no significant evolution of applied voltage was reported. Results revealed an average of 5.1 ± 1.1 V/cm over 15 days of experimental investigations in the electro-bioreactors operated at CD of 3 and 7 Am−2.

Figure 1 Removal efficiencies of (a) COD, (b) PO 4 3−-P in serially passaged control bioreactor and electro-bioreactors E3 and E7 operated at current densities 3 and 7 Am−2 respectively. Overall, the removal efficiency of sCOD and PO 4 3−-P in electro-bioreactors is attributed to the effects of electrochemical processes in addition to biodegradation. (c) pH variations in serially passaged control bioreactor and electro-bioreactors E3 and E7 operated at current densities 3 and 7 Am−2 respectively. Results confirmed that serially passaged electro-bioreactors maintained the pH which in turn stabilized the medium for the bacteria to be activated. Full size image

pH variations in serially passaged wastewater reactors

One of the physiochemical factors affecting the bioreactors and electro-bioreactors performance is the pH of the solution21. Also, studies on activated sludge systems show that most of biological systems and bacteria are activate in pH ranges of 4 to 922. As shown in Fig. 1c, pH was initially 9.2 ± 0.2 in all reactors tested during the serial passaging period. In control bioreactor pH declined by 36% to 5.9 ± 0.2 after 5 days of passaging then decreased to 3.5 ± 0.1 by day 9 then stabilized at 2.9 ± 0.1 from day 12 to 15 end of serial passaging. In electro-bioreactors operated at CDs of 3 and 7 Am−2, pH decreased by 11–14% to 8.2 ± 0.3 and 7.9 ± 0.2 after 5 days of passaging then by approximately 6% to 7.8 ± 0.1 by day 9 then stabilized in the range (6.9 ± 0.1–7.0 ± 0.1) from day 12 to 15 at CDs of 3 and 7 Am−2. Therefore, pH of the solution in control bioreactors and electro-bioreactors declined by 69 and 23%, respectively. This means that serially passaged electro-bioreactors maintained the pH which in turn stabilized the medium for the bacteria to be activated. Additionally, this confirms the high removal efficiency of sCOD and PO 4 3−-P as previously reported that depending on the pH of the solution, various metal species are produced during electrocoagulation which react with the pollutants leading to the destabilization and aggregation of suspended particles and the precipitation and adsorption of dissolved contaminants21. Studies aimed at understanding bacterial community flocculation determined that one aspect of flocculation is dictated by ionic interactions between organic polymers that are secreted into the environment by various bacteria. These organic polymers, mostly made up of protein and carbohydrates, vary in size and this is affected by enzymatic activity23. It is widely accepted that pH affects enzymatic activity, therefore fluctuations in pH can result in different organic polymer lengths and ionic charges, and ultimately affects biological flocculation. This change in pH could be one reason why the activated sludge particles in the control bioreactor did not settle and were kept in suspension.

Sludge volume index (SVI) and settling velocity (Vo) variations in serially passaged wastewater reactors

Sludge volume index (SVI) is often used to characterize the settleability of a specific sludge in treatment plants. It is the volume in milliliters occupied by 1 g of a suspension after 30 min settling. SVI was measured at the end of each experiment after 15 days in serially passaged control bioreactor and electro-bioreactors operated at CD of 3 and 7 Am−2. Results showed that SVI was reduced from 325 to 236.6, 141.5, and 54.7 mLg−1 in serially passaged control bioreactor and electro-bioreactors operated at CD of 3 and 7 Am−2, respectively reflecting the importance of applying electric field in the sludge cohesion and density. Sludge settling velocity (V o ) was calculated based on the mathematical formula by Akca et al.24:

$${V}_{o}=28.1\,{(SVI)}^{-0.2667}$$ (1)

according to the aforementioned formula, V o of 6, 6.5, 7.5 and 9.7 mh−1 was reported in the control bioreactor, and electro-bioreactors operated at CD of 3 and 7 Am−2, respectively. Those results show a significant improvement on the settling velocity (up to 161.7%) via creating denser flocs which tend to settle at a faster rate.

Effect of serial passaging on microbial diversity in wastewater bioreactors

To assess the microbial diversity in the serially passaged bioreactor and electro-bioreactors subjected to CDs of 3 and 7 Am−2, we performed alpha (α) diversity analysis on each sample and beta (β) diversity analysis across the samples. α diversity expresses the diversity of a population within a system; a community will have a higher α diversity when there is a higher number of unrelated species within the same sample25. α diversity was assessed by using the Chao1 index and Phylogenetic Diversity (PD) whole tree index26. The results of these measurements indicated differences in α diversity depending on passaging days. Specifically, control bioreactors and electro-bioreactors operated at 3 Am−2 started with low α diversity and continued to increase reaching the highest richness (peak) by day 12 (Fig. 2a). α diversity then lowered by day 13 and 15 in both control and electro-bioreactors as shown in Fig. 2a. Additionally, communities present in electro-bioreactors operated at 7 Am−2 had the highest α diversity by day 9 then lowered by day 12, 13 and 15 as shown in Fig. 2a. Furthermore, control bioreactors and electro-bioreactors operated at CD of 3 Am−2 had a higher α diversity than electro-bioreactors operated at 7 Am−2.

Figure 2 (a) Alpha diversity richness calculations using Chao1 and PD whole tree methods, (b) three-dimensional principal coordinate analysis (PCoA) plot showing the bacterial community variations present in serially passaged control bioreactors (red data points) and electro-bioreactors E3 (blue data points) and E7 (orange data points). It is noted that bacterial communities in electro-bioreactors clustered together when compared to control bioreactor. (S-D1: Fresh sludge sample without feeding with synthetic wastewater; C-Di: Control sample in day i; E-Dj: Electro-bioreactor sample in day j). Full size image

To more clearly compare the bacterial communities, a phylogeny-based weighted UniFrac distance analysis was used16. The bacterial communities prior to applying electric field taking into consideration the serial passaging time effect were highly distinct from each other as shown in the Principle Coordinate Analysis (PCoA) plot (Fig. 2b). As can be seen from the PCoA plot, fresh sludge sample without feeding with synthetic wastewater (S-D1) and control bioreactor sample fed with synthetic wastewater (C-D1) clustered together and were very distant from the rest of the reactors tested. Electro-bioreactors operated at CD of 3 (blue data points) and 7 Am−2 (orange data points) clustered together, indicating that the bacterial communities that developed in those reactors were more similar than any of the control bioreactors. Control bioreactor samples at days 6, 9 and 13 (C-D6, C-D9 and C-D13) clustered together on the extreme right side of the plot, while control bioreactors at days 12 and 15 (C-D12 and C-D15) clustered together in closer proximity to the electro-bioreactor samples. Taken together, this data indicates that applying an electric current differentiated the microbial community present in the electro-bioreactors and this caused early stabilization in the community since all data points clustered together. In contrast, the bacterial community in the control bioreactors seemed to fluctuate as the sludge was serially passaged.

A BIO-ENV trend and Spearman’s rank correlation analysis (using QIIME v 1.9.0) was performed (β-diversity) to investigate for electric field and operational parameters that is correlated with bacterial community diversity. This type of analysis calculates a bacterial community difference distance matrix and compares to Euclidian distance matrixes for each of the other measured variables (such as temperature, pH, ammonia content etc.). The variables that best explained the weighted Unifrac distance between all samples were best correlated with incorporation of current density, PO 4 3−-P, sCOD and pH (correlation = 0.412, 0.197, 0.111 and 0.496, respectively).

Clustering and phylogenetic analysis of microbial community structure

Raw Illumina MiSeq sequencing data was analyzed using QIIME for the 12 serially passaged reactor samples discussed here. Assigning sequences to different OTUs resulted in a total of 7019 OTUs with a minimum sequence depth of 24647 per sample and a table density of 0.404. Furthermore, after trimming and filtering according to the criteria mentioned in the methods section, the OTU biom table resulted in 6863 observed OTUs with a minimum sequencing depth of 24567. Rarefaction analysis of our sequencing data indicated that the sequencing depth we achieved per sample was sufficient to uncover low abundance species as rarefaction plots of reads vs OTU’s leveled off.

β-diversity analysis using UPGMA clustering revealed that the bacterial communities in the 12 samples could be clustered into two main groups containing: (i) fresh sludge sample and control bioreactor containing sludge fed with synthetic wastewater on day 1. (ii) serially passaged control bioreactors (C-D6 to D15) and electro-bioreactors operated at CD of 3 (E3-D6 to D15) and 7 Am−2 (E7-D6 to D15) (Fig. 3a). The second main branch is divided into 2 sub-branches according to the absence and presence of electric current. As can be seen in Fig. 3a, bacterial communities in control bioreactors clustered together on days 6, 9 and 13 except from days 12 and 15 which clustered with communities from the electro-bioreactor operated at CD of 7 Am−2. Whereas microbial communities from the electro-bioreactors operated at 3 Am−2 clustered together on all passaging days except from day 6 which clustered with communities from the electro-bioreactor operated at 7 Am−2 from the same day. Additionally, communities from the serially passaged electro-bioreactor operated at 7 Am−2 were separated because of serial passaging. For example, communities from days 6, 9 and 12 clustered together on one branch separated from another branch containing communities from days 12, 13 and 15. This means that the serial passaged reactors had an impact on microbial community structure and composition in the presence and absence of electric field. That could be explained referring to the consequences of serial passage in terms of evolutionary impact of environment on passaged microorganisms. In other words, the adaptation of microorganisms to the utilization of new substrates. An ability to utilize a new substrate has the potential to increase growth rates as well as organism end point-densities27.

Figure 3 (a) Beta-diversity using UPGMA clustering analysis using weighted Unifrac. Phylogeny of the bacterial communities in the control bioreactor and electro-bioreactors at (b) phylum level, and (c) family level. It is concluded that serial passaged reactors had an impact on microbial community structure and composition in the presence and absence of electric field. (S-D1: Fresh sludge sample without feeding with synthetic wastewater; C-Di: Control sample in day i; E-Dj: Electro-bioreactor sample in day j). Full size image

Phylogenetic analysis of the microbial communities present in the serially passaged reactors showed that Proteobacteria accounted for the largest fraction (59.7% average abundance across all samples) which is consistent with previous work28,29, followed by Bacteroidetes (16.7%), Chloroflexi (6.5%), Planctomycetes (5.3%), Firmicutes (3.3%), Acidobacteria (1.9%), Chlorobi (1.7%), Verrucomicrobia (1.6%) and Nitrospirae (1.2%) (Fig. 3b). In municipal WWTPs, the phylum Proteobacteria predominates, of which Betaproteobacteria (25.2% average abundance across all samples) is the most abundant class, largely responsible for organic and nutrient removal. The Proteobacteria encompass enormous morphological, physiological and metabolic diversity, and are of great importance to global carbon, nitrogen and sulphur cycles11. The results showed that the highest relative abundance of Proteobacteria members was in the serially passaged electro-bioreactor operated at CD of 3 Am−2 (63.3%) higher than the control bioreactor and electro-bioreactor operated at CD of 7 Am−2 (56.7 and 53.9%, respectively). A sub-dominant phyla Bacteroidetes correlated with serially passaged electro-bioreactor operated at CD of 7 Am−2 comprising a relative abundance of (20.2%) slightly higher than that of CD 3 Am−2 and control bioreactor (17.7 and 13.1%, respectively). This means that the application of electric field could have a positive effect on serially passaged microbial communities in wastewater reactors. It is noticed that when comparing the relative abundances of bacterial communities present in serially passaged electro-bioreactors and control bioreactor, there was an inflection point at certain passaging days where it reached its highest abundance then slightly decreased which could be due to serial passaging effect. For example, the relative abundance of Proteobacteria had the largest abundance on day 12 (70.4%) in control bioreactor, day 9 (77.2%) in electro-bioreactor operated at CD of 3 Am−2 and day 6 (70%) in electro-bioreactor operated at CD of 7 Am−2. Additionally, the average relative abundances of Chloroflexi in serially passaged electro-bioreactor operated at 7 Am−2 was higher than in control bioreactor and very low abundance in electro-bioreactor at 3 Am−2 (9.2, 5.9 and 0.4%, respectively). Previous studies reported that Chloroflexi as predominating filaments in municipal and domestic wastewater treatment plants30,31. Identification of filaments responsible for bulking enables to select optimal solution for their removal from a particular wastewater treatment plants. Furthermore, members of Firmicutes phyla known to occur in wastewater treatment plants treating penicillin-containing wastewater had the highest relative abundance in serially passaged control bioreactors and was depleted in both electro-bioreactors of CD 3 and 7 Am−2 with average relative abundance (8.6, 0.4 and 0.5%, respectively).

To more closely analyze the microbial community abundance and dynamics in the serially passaged reactors, a phylogenetic analysis at family level was constructed. After trimming and filtration, there are 27 families present with higher than 0.3% average abundance across all samples as shown in (Fig. 3c). Comamonadaceae (14.1%), Enterobacteriaceae (13.1%), Oxalobacteraceae (12.7%), Sphingomonadaceae (10%), weeksellaceae (9.7%), Pseudomonadaceae (7%), Cytophagaceae (5%) and Aeromonadaceae (4.8%) were the top abundant families across all samples. Our results showed that Enterobacteriaceae, Pseudomonadaceae, Streptococcaceae, Acetobacteraceae and Rhodocyclaceae had the highest average relative abundance across serially passaged control bioreactor samples than electro-bioreactors operated at CD of 3 and 7 Am−2 (18.8, 4.6 and 11.7%; 15, 1.2 and 2.7%; 8, 0.1 and 0.1%; 4.5, 0 and 0%; 2.1, 1.6 and 1.5%, respectively). This explains the high removal of sCOD and PO 4 3−-P (Fig. 2a,b) in control bioreactors on passaging days (6, 9 and 12) due to the high relative abundance of heterotrophic bacteria affiliated with organic and nutrient removal on these days such as Pseudomonadaceae; hydrocarbon utilizing organisms32, Streptococcaceae; glucose fermenting bacteria33 and Rhodocyclaceae; Phosphate accumulating organisms PAOs34. These families survived over time in serially passaged control bioreactors compared to both electro-bioreactors. Sphingomonadaceae (known to metabolize and remove micro-pollutants, such as polycyclic aromatic hydrocarbons (PAHs) and bisphenol A (BPA) degradation from wastewater35, Cytophagaceae; are Epiphetic protein hydrolyzers36, Aeromonadaceae; has been explored previously to treat sewage containing pharmaceuticals and personal care products (PPCPs)37, Neisseriaceae; involved in sCOD reduction in dairy wastewater38 and Flavobacteriaceae; heterotrophic bacteria associated with nutrient and micro-pollutant removal39 had the highest average relative abundance in serially passaged electro-bioreactors operated at CD of 3 Am−2 than in control bioreactor and electro-bioreactors operated at 7 Am−2 (17.3, 7.4 and 2.3%; 11.3, 1.9 and 2.8%; 7.6, 1.4 and 4.2%; 5.4, 1.4 and 1.8%; 2, 0.8 and 0.8%, respectively). These families continued to increase and survived through the serial passaging in the electro-bioreactor operated at CD of 3 Am−2 except for Flavobacteriaceae which started to decrease in abundance after day 9. Oxalobacteraceae; members of the family are heterotrophic and some genera fix nitrogen which are applied as plant growth-promoting bacterial inoculants in agriculture40, Comamonadaceae; is reported to be capable of performing a biological nutrient removal process with a reduced increase in cell mass when the oxygen supply is limited “see Table 1”41 and Weeksellaceae; present in hydrocarbon-polluted soil microbial communities or natural asphalts32 had the highest average relative abundance in serially passaged electro-bioreactors operated at CD of 7 Am−2 than in control bioreactor and electro-bioreactors operated at 3 Am−2 (17.2, 4.0 and 12.8%; 17.1, 7.3 and 14.6%; 17, 6.9 and 2.4%, respectively).

Table 1 OTU counts of bacterial families in serially passaged control bioreactor. Full size table

Linking functional microbial community function to sludge settling in serially passaged reactors

Bacterial communities that are responsible for activated sludge bulking and foaming play a role in the degradation of substrates such as fatty acids, amino acids and other bioorganic compounds2. These are Comamonadaceae, Pseudomonadaceae, Verrucomicrobiaceae and Flavobacteriaceae. These families are the major components of activated sludge of most wastewater treatment plants worldwide, playing crucial roles in degradation of organic compounds and forming floc structure of activated sludge. Moreover, many inductors of activated sludge bulking and foaming efficiently degrade stable organic compounds and their increase in content and biodiversity is reasonable42,43,44,45. Filamentous bacteria are usually present in wastewater treatment plants in a low number supporting the formation of microbial structures. The excessive growth of these bacteria causes sludge bulking11.

Therefore, it is important to assess the microbial subpopulations of functionally important bacteria in our serially passaged reactors, a heat map was constructed illustrating these differences (Fig. 4). This heat map, which is only based on functionally interesting bacterial families affiliated with sludge bulking and foaming (Comamonadaceae, Flavobacteriaceae, Verrucomicrobiaceaea and Pseudomonadaceae) and pathogenic microflora (Neisseriaceae, Enterobacteriaceae and Streptococcaceae)46, clearly differentiates the behavior of these bacteria when it is serially passaged in control bioreactor and electro-bioreactors. In serially passaged control bioreactors, the abundance of Comamonadaceae increased reaching the maximum OTU count by day 12 then slightly decreased and stabilized (see Table 1). The behavior was similar for Pseudomonadaceae, Streptococcaceae and Enterobacteriaceae which tend to an initial increase with highest abundance by day 9. Enterobacteriaceae was high in abundance till day 6 then continued to slowly decrease till the end of serial passaging while Pseudomonadaceae and Streptococcaceae dramatically decreased by day 12 then slightly increased on day 13 then decreased again by day 15. Neisseriaceae and Verrucomicrobiaceae members initially decreased then reached the maximum OTU count by day 12 then decreased and stabilized by day 15. Flavobacteriaceae had an inverse correlation by days as it tends to decrease from day 1 to day 15 which means that this bacterial family could not survive and depleted in the serially passaged control bioreactor.

Figure 4 Heat map of functional bacterial families in serially passaged control bioreactor and electro-bioreactors E3 and E7. It was observed that microbial subpopulations associated with sludge bulking and foaming are different in electro-bioreactors when compared to control bioreactor. (C-Di: Control sample in day i; E-Dj: Electro-bioreactor sample in day j). Full size image

In serially passaged electro-bioreactors operated at CD of 3 and 7 Am−2 (Tables 2 and 3), Comamonadaceae and Flavobacteriaceae was initially increased in abundance with highest OTU counts by day 9 at CD of 3 Am−2 and day 12 at CD of 7 Am−2 then deceased and stabilized. Verrucomicrobiaceae and Streptococcaceae were depleted through the 15-day while Pseudomonadaceae had the highest OTU count by day 6 then depleted by the end of serial passaging in both electro-bioreactors operated at 3 and 7 Am−2. Additionally, Enterobacteriaceae were in low abundance till day 9 then started to occur with high OTU counts by day 12 till day 15 of the serial passaging. Bacterial family Neisseriaceae had a different behavior through the serial passaging days which tended to increase in abundance through the 15-day of serial passaging in electro-bioreactor operated at CD of 3 Am−2 while dramatically decreased by day 12 in electro-bioreactor operated at CD of 7 Am−2.

Table 2 OTU counts of bacterial families in serially passaged electro-bioreactor operated at CD of 3 Am−2. Full size table

Table 3 OTU counts of bacterial families in serially passaged electro-bioreactor operated at CD of 7 Am−2. Full size table

Our results revealed that microbial communities structure changed in serially passaged sludge in all reactors tested. This could be due to the two phenomena involved in serial passaging experiments which are population growth of the microorganisms and the movement that occurs during passage (i.e., the dilution/transmission step). There can be differences in both the quality and intensity of selection imposed at each of these steps taking into consideration the duration of growth in the course of a single passage (difference between passaging days)28,47. Moreover, microbial communities structure, diversity and response changed in the serially passaged reactors which could be beneficial by optimizing the operating conditions and adjusting the duration and gap between the passaging days in bioreactor and electro-bioreactors treating wastewater.

This study indicates that the introduction of serial passaging, as a new approach to electro-bioreactors treating wastewater, is associated with greater sludge bulking and microbial community stability. Indeed, this approach represents a strategy that can be employed to solve sludge bulking and foaming problems, leading to improvement in sludge settleability and a more efficient waste water treatment process. The results reported here are the first to describe the effects of current density on microbial community structures in serially passaged sludge.