Characteristic change of WAS fermentation using different pretreatment
The particle size primarily changed through different pretreatment methods, which substantially influenced the subsequent release and conversion of various organic matter in the WAS. Ultrasonic treatment played the most noticeable effect on sludge structure break and scatter, leading to an average particle size distribution of 29.5 μm (see Additional file 1: Figure S1). Alkaline treatment slightly improved the sludge particle scatter, with an average of 56.3 μm in comparison to 60.8 μm of the control sludge without pretreatment. On the other hand, an obvious increase of particle size up to 387.5 μm was obtained by the freeze/thaw treatment, because flocks were produced after freezing. Consequently, the lysis ratio of increased SCOD to TCOD was 21, 6, and 11 % after alkaline, freeze/thaw, and ultrasonic pretreatment, respectively (see Additional file 1: Figure S2). The freeze/thaw pretreatment was not as effective as other methods on SCOD release, indicating that the flocks of larger particle size were not broken into smaller fragments in a short reaction time [40]. In another report on the effect of sludge pretreatment on sludge characteristics, the disruption of sludge flocks led to the release of intracellular and extracellular materials [41]. Moreover, alkaline (pH 10–12) treatment is known to further enhance the organic release during the pretreatment [42] and favor VFA production in the subsequent fermentation [20, 43]. In our study, an increased amount of soluble organics was released after pretreatment, mainly in the form of carbohydrates, proteins, and volatile fatty acids (VFAs) (see Additional file 1: Tables S1, S2). After 3 d fermentation, SCOD increased from 147 mg/L of the raw sludge to 452 mg/L of the control, 7690 mg/L of the alkaline pretreatment, 1760 mg/L of the freeze/thaw pretreatment, and 3461 mg/L of the ultrasonic pretreatment (see Additional file 1: Table S1). The VFAs were mostly produced by the alkaline pretreatment, which accumulated up to 5300 mg COD/L, accounting for 69 % of total SCOD. The same occurred with proteins, which reached 1749 mg/L, with a 24-fold increase compared to the control sludge without any pretreatment.
In our view, WAS pretreatment initially changed the particle characteristics, which played a major role in the studied process, influencing organic release and fermentative communities during pretreatment, and subsequently affecting the fermentation and VFAs production. Although all sludge pretreatments successfully improved hydrolysis and organic release, the subsequently generated short-chain fatty acids differed in terms of content and concentration, which are known to be important factors affecting the conversion rate and efficiency in bioelectrochemical systems [38]. Clearly, soluble organics play an important role, reducing the accessibility of substrates to bacterial disintegration, or stated differently, the initial particle size can affect the contact surface area, for subsequent bacterial action [44]. Our results showed that ultrasound led to the smallest particle size, followed by a higher acetate production (>80 % of total fermentative products). Alkaline pretreatment could increase the total production of short-chain fatty acids with a high conductivity fermentative liquid. Therefore, it seems to be one of the substantial factors to interact with electrode biofilm communities.
Furthermore, organics and conductivity of WAS fermentative liquid were the two key factors to the bioelectrochemical communities. To evaluate the influence of pretreatment on COD contribution in different sludge structures, COD was divided into four parts: soluble SCOD, loosely bound extracellular polymeric substances (LB-EPS), tightly bound extracellular polymeric substances (TB-EPS), and residual particles (Fig. 1). The alkaline pretreatment effectively released ~25 % particle organics (compared to the control) into SCOD (~21 %). A small part of particles (4 %) with reduced TB-EPS (~3 %) were converted into LB COD (~7 %). However, COD contribution was reduced to less than half of SCOD (6–11 %) from particles, when using the freeze/thaw or ultrasonic pretreatment. The SCOD of VFAs reached the peak accumulation during fermentation before methane production started, under the conditions of this study [45, 46]. The release of soluble matter also increased conductivity of fermentation solution. Even during fermentation without any pretreatment, there was a slight increase from 1.2 to 1.4 mS/cm in sludge fermentation liquid (SFL) (see Additional file 1: Table S1). Conductivity was further increased to 1.96–2.63 mS/cm by the freeze/thaw and ultrasonic treatment respectively, which matched the increasing trend of SCOD and inorganic ion release. The alkaline addition, using NaOH, highly enhanced the conductivity, reaching up to 6.23 mS/cm, which was almost close to 50 mM PBS (Phosphate buffer solution, pH 7.0) used for MEC reactor setup [35]. A high conductivity is to be considered potentially beneficial to electron transport in the following bioelectrochemical process [47]. Besides the additional alkaline contribution, organics and ion release from WAS improves during the pretreatment and is further enhanced during the fermentation. A previous study showed that the limiting factors, at the anodic biofilm, change from potential limitations at low conductivity, to dual potential and carbon source transfer limitations at a moderate conductivity, and to only mass transfer limitations at high conductivity [48]. A low conductivity (<1 mS/cm) was observed in common AD effluent after organic removal and biological treatment, moreover, a higher external voltage was required when connecting BES after AD to achieve biofuels [49]. In this respect, pretreatment is an important and flexible tool to regulate the performance of BES and AD integrated process, which would determine the total efficiency on waste treatment and biofuel recovery.
Pretreated SFL utilization and hydrogen production in MECs
The setup performance of the 15 MEC replicate reactors, before fueling SFL (see Additional file 1: Figure S3). The average coulombic efficiency was steadily around 92.2 ± 6.5 % in all replicates, with an average peak current of 3.75 ± 0.22 mA. The COD removal efficiency of acetate reached 86.8 ± 2.1 %. The 15 MEC reactors showed similar conversion efficiencies to hydrogen, with 3.3 ± 0.5 mol H2/mol acetate and a hydrogen production rate of 1.36 ± 0.26 mL/mg COD (1.46 ± 0.28 mL H2/mL reactor/d). Twelve reactors were randomly divided into four groups (three replicates each), to be fed with SFL obtained from the different pretreatment methods.
The pretreated sludge properties determined the subsequent fermentation process, leading to various levels of acidification, organic contents, and production rates (see Additional file 1: Figures S4, S5). The highest amount of VFAs was produced during the 3rd day fermentation of alkaline pretreated WAS, containing 2225.81 mgCOD/L and accounting for 42 % in total VFAs (see Additional file 1: Figure S4). There was 1077.25 mgCOD/L acetate produced in ultrasonic pretreated WAS, while still accounting for 41 % of total VFAs. The lowest amount of VFAs was observed with the freeze/thaw-pretreated WAS, though still showing a 3.8-fold increase compared to WAS without pretreatment. In all pretreated SFL, more VFAs were firstly utilized in MECs, showing a similar removal of around 70 % (see Additional file 1: Figure S5). Over 95 % of acetate and butyrate were utilized in alkaline SFL and ultrasonic SFL, while only ~85 % acetate and butyrate were removed in freeze/thaw SFL. Differently, a much higher percentage of propionate (removal amount was really low) was removed in freeze/thaw SFL than others at the same time. As a result, hydrogen production rate differed in MEC reactors, based on acid types and concentrations that were produced [38]. Previous results showed that pretreatment methods are very important to release organics and enhance degradation of various carbon sources from WAS [46, 50]. Probably, the cascade utilization of SFL could be regulated according to composition in VFAs, proteins, and polysaccharide [22, 51], while the energy recovery changed when the suitable organic compounds were degraded. It is therefore likely that the alkaline treatment performed best energy gains (Fig. 2) thanks to the high conductivity (increased from 2.1 ± 0.2 mS/cm for raw sludge to 3.5 ± 0.3 mS/cm for alkaline pretreatment) [51, 52] and SCOD, as well as high COD removal.
The current generation varied among different pretreated and fermented sludge (see Additional file 1: Figure S6). The highest peak current reached ~3.7 mA, when feeding with the alkaline pretreated SFL, which showed the highest acetate production of 2200 mg COD/L, as well as an enhanced conductivity. The peak current dropped to 2.5 mA for ultrasonic and 1.8 mA for freeze/thaw condition. The lowest current was only 1.0 mA, using SFL produced from raw WAS without pretreatment. The SCOD removal was slightly different in different SFL, with 61 ± 2 % for alkaline, 66 ± 5 % for freeze/thaw, and 69 ± 3 % for ultrasonic pretreatment in MECs (Fig. 2). However, the hydrogen production rate was quite different. The alkaline pretreated SFL achieved the highest hydrogen production of 1.22 ± 0.03 mL H2/mL reactor/d (compared to 1.46 ± 0.28 mL H2/mL reactor/d before fueling with SFL). The ultrasonic pretreated SFL was converted into hydrogen with a rate of 0.60 ± 0.15 mL H2/mL reactor/d. The freeze/thaw pretreatment, instead, was not able to effectively improve the cascade utilization of WAS, compared to raw sludge.
Clearly, organic products were the result of metabolic activities of the microbial community, which was characterized by different composition and structure during fermentation. Previous studies showed that with the proper enrichment of microbial communities, anaerobic processes can be improved and perform more efficiently [53]. In this study, the solid granular sludge changed based on different pretreatment methods. Although SCOD was increased and consequently converted to more hydrogen in MECs, the hydrogen yield was reduced by ~16 % when influent was changed from artificial wastewater (acetate, ~1140 mg COD/L) to SFL (acetate, ~2225 mg COD/L accounting for 29 % of SCOD in the alkaline pretreatment) with fermentative communities. Moreover, hydrogen yield was reduced by ~59 % when feeding the ultrasonic pretreatment SFL (acetate, ~1080 mg COD/L accounting for 31 % of SCOD). It has been pointed out that further increases in organic loading do not vary hydrogen production significantly [54, 55]. Therefore, it is likely that MEC performances changed in relation to anodic community structure, which interacted with dominant fermentative communities and organic compounds produced [56].
Methane production and archaea community change in integrated system
Methane production was detected in all MEC reactors after 2 weeks feeding SFL, however, the methane production rate was fluctuating, not being comparable among different pretreatments over all batch operations (data not shown). Although methane production was not substantially increased over 1 month (as we evaluated previously [57]), the MECs feeding SFL without pretreatment presented the highest methane production over all other conditions, together with the highest amount of acetotrophic methanogens (Methanosaeta), both in control SFL and MEC biofilms fed with control SFL (Fig. 3). It was interesting that the lowest amount of archaea were detected under ultrasonic pretreatment, leading to the lowest growth in MECs as a result. Compared to methanogens in initial biofilms fed with acetate (MEC sample), it seem that acetotrophic methanogens were substantially enriched to anode biofilm in all conditions. But hydrogenotrophic methanogens were further enriched with higher amounts than in SFL feeding, as shown in freeze/thaw and ultrasonic pretreatment, including Methanocorpusculum, Methanosphaerula, Methanoregula, Methanospirillum, Methanobacterium, and Methanobrevibacter. The extra hydrogen generation from MECs can favor hydrogenotrophic methanogens in anaerobic condition [57]. The H2 produced in a single chamber MEC can be lost through methanogenesis, which causes energy loss in the system [22, 51, 52].
Microbial community structure and anodic biofilm community shift in integrated process
A total of 244,761 raw sequences were analyzed over all community samples (Additional file 1: Table S2). Operational taxonomic units (OTUs) at 3 % distance were the most detected ones in raw WAS (5002), with the highest diversity (Shannon index 6.96), while being the least detected in the startup anode biofilm using acetate (2341), showing a reduced diversity (Shannon index 5.39) (see Additional file 1: Figure S7). Similar results were observed from ACE (abundance-based coverage estimator) and Chao1 indices (see Additional file 1: Table S3). Interestingly, microbial community diversity in the SFL decreased, indicating that specific fermentation bacteria were enriched and became dominant. On the other hand, an increase of diversity was detected in anodic biofilm communities, after initial MECs were connected to sludge fermentation (with or without pretreatment) for several days, showing an interactive effect of fermentative communities on initial anodic communities, thus leading to subsequent changes in MEC reactor performances.
After fermentation, the unpretreated sludge showed similar community structure to raw sludge (see Additional file 1: Figure S8). The most abundant phylum in SFL was Proteobacteria, accounting for 36.7 % in the control, 40.0 % in the alkaline pretreatment, 28.7 % in the freeze/thaw pretreatment, and 54.8 % in the ultrasonic pretreatment, over all microbial communities. Seemingly, sludge fermentation after ultrasonic pretreatment mostly increased Gammaproteobacteria. In comparison to the control sludge, Firmicutes (Bacilli sp. and Clostridia sp.) were all increased in SFL of the pretreated sludge. Bacteroidetes was the third most abundant community in the SFL.
When MECs were connected to the fermentation process, anodic biofilm composition obviously changed, compared to the original communities established using acetate (Fig. 4). Desulfovibrio [58] and Geobacter [14] (Deltaproteobacteria), responsible for electron transfer between bacteria and electrode, represented the key functional community. Geobacter was the most detected genus of the anode biofilm, in the case of reactors fed with acetate (startup MECs), and further increased after feeding with alkaline pretreated SFL, as well as the ultrasonic pretreated SFL (with a corresponding high energy conversion achieved in these reactors). The MEC fed with the freeze/thaw-pretreated SFL, instead, showed low abundances of Desulfovibrio and Geobacter, which was similar to the control SFL. On the other hand, compared to other treatments, freezing-thaw SFL led to an increased abundance of Pseudomonas in the anodic community. Moreover, large particles of organics in SFL led to enrichment of fermentative communities in the anode biofilm, including Anaerolinea (Levilinea and Longlinea), Bacteroida (Paludibacter and Parabacteroides), and Clostridia (Proteinilclasticum, Proteocatella, and Sedimentibacter). Compared to the original anode biofilm, four genera of the class Clostridia (namely Acetoanaerobium, Acetobacterium, Anaerovorax, and Fusibacter) decreased in all SFL-fed MECs.
Moreover, hierarchical cluster analysis clearly showed that SFL communities varied, depending on the different treatment method (Fig. 5, F-samples). The bacterial community structure of the control SFL (F-control) without pretreatment changed less, compared to the raw sludge (Raw), while the ultrasonic pretreatment led to the greatest difference in community structure in the SFL (F-f). Figure 5 (M-samples) showed how various SFL communities had different impacts on the change of the anodic biofilm communities, after combining fermentation and microbial electrolysis process. Interestingly, anodic biofilm communities were similarly grouped among the original MEC (Mec), MECs fed with the alkaline pretreated SFL (M-a), as well as MECs fed with ultrasonic pretreated SFL (M-u). Regarding the gas production, they performed much higher hydrogen yield than the SFL-fed control or the freeze/thaw pretreatment, which had a low hydrogen conversion, below 0.2 mL/mg COD, and peak current below 1.0 mA.
Bioelectrochemical communities were highly enriched in dominant functional groups related to Proteobacteria (63 %) and Firmicutes (25 %) when feeding acetate during reactor setup, inoculated with activated sludge (Fig. 6). They played the primary function of electron transfer and substrate degradation, with great potential on complex carbon utilization, as already suggested from a functional genes’ perspective [35]. The most abundant genera for extracellular electron transfer were Geobacter, Desulfovibrio, and Acinetobacter, belonging to Proteobacteria. Geobacter species are considered as the most efficient exoelectrogens in bioelectrochemical systems [59]. Desulfovibrio and Acinetobacter species are dissimilatory metal-reducing bacteria involved in contaminant degradation and metal reduction, outside the cell membrane [60, 61]. In Firmicutes, three genera of Clostridia were detected, namely Acetoanaerobium (5.9 %), Acetobacterium (8.5 %), and Fusibacter (5.0 %). They are supposed to play an important role in carbon recycling for anode respiring bacteria, as previous studies have shown that some Firmicutes may closely live with anode respiring bacteria, when fed with fermentative substrates [62].
Microbial community network on exoelectrogenic and fermentative communities
Based on the discussion above, a network representing the community change and linkage was constructed (Fig. 7), taking alkaline treatment samples as example. MEC biofilm was inoculated from raw sludge (Raw), then raw sludge was pretreated to produce fermentation liquid as feedstock for the MECs. During the cascade process, MEC biofilm interacted with fermentative communities to form a new MEC biofilm. The SFL primarily led to an increase in abundance of Bacteroidetes and Chloroflexi in anodic communities, thus reducing the abundance of Proteobacteria. Even though the phylum Bacteroidetes was commonly detected in bioelectrochemical system communities, few studies pointed out their negative impacts on electron transfer efficiency [33]. On the other hand, Bacteroidetes can be further enriched (over Proteobacteria) in an open-circuit BES to convert substrates, thus competing with anode respiring bacteria for power output [63]. Lately, it was highlighted that Bacteroidetes can be easily enriched in BES when supplemented with other electron acceptors (NO3
−) [64], thus potentially enhancing an electron flow that is separated from the energy yield in MECs. Chloroflexi also represented an enriched phylum in the open circuit, and it is usually predominant in anaerobic digester sludge. Bacteroidetes increased to 25.9 %, compared to 5.6 % in the initial setup MECs, with high coulombic efficiency and H2 yield. Proteobacteria, on the contrary, decreased to 17.8 % in SFL control MECs, although they increased to 26.8 % by feeding with freeze/thaw-pretreated SFL. More specifically, Gammaproteobacteria and Deltaproteobacteria were partially decreased, while Betaproteobacteria partially increased, thus leading, as a result, to a lower efficiency of electron transfer and hydrogen recovery.
MECs fed with alkaline pretreated SFL showed the best energy yield and shared the largest community of Geobacter, Desulfovibrio, Pseudomonas, and Clostridium, with initial MECs (Fig. 7). Simultaneously, Firmicutes substantially decreased, including Acetoanaerobium (1.1 %), Acetobacterium (0.2 %), and Fusibacter (0.3 %). Some of the anaerobes (>20 %), i.e., the class Bacilli (Pasteuria and Lactococcus) and Clostridia (Fusibacter, Anaerovorax, and Proteiniclasticum), were clearly enriched on electrode biofilm by SFL feeding, suggesting a synergistic effect with exoelectrogens to degrade complex organic matter [35] (like Lactococcus producing soluble electron shuttles to promote electron transfer between cells and the electrode surface [65]). Some genera belonging to Clostridia (Acetobacterium and Acetoanaerobium) (<10 %) were probably enriched in MECs by the availability of products such as hydrogen (electron). However, their function on carbon/electron recycling seems to be very limited in various BES systems [33], which would suggest only a limited hydrogen loss (or over 100 % coulombic efficiency) in MECs fed with acetate [66], carbohydrate [67], or fermentation liquid [51]. It is worth noting that a high protein or polysaccharide but low fatty acid content would lead to the dominance of Proteiniclasticum and Parabacteroides (increased by >10 %), which are able to produce VFAs as end products from fermentation [68, 69]. Thus, part of the microbial communities did not function on extracellular electron transfer; however, they were maintained in the fermentation niche of electrode biofilm, where they could provide labile products for electrode respiring bacteria. A substantial reduction in current and hydrogen recovery (Alkaline vs. Ultrasonic) was observed when introducing SFL with an increased abundance in Proteiniclasticum (Alkaline vs. Ultrasonic genus level in Additional file 1: Figure S8). Proteiniclasticum reached 11.4 % in the ultrasonic pretreated SFL, but only 0.4 % in the alkaline pretreated SFL. In fact, MECs presented a similar COD removal (61 and 69 %) but coulombs were reduced by 31 % and hydrogen production by 50 %.
Alkaline pretreatment could provide more short-chain fatty acids and higher conductivities than other pretreatments. These two aspects are known to favor mass transfer in anodic biofilm [48]. Geobacter increased in both, alkaline and ultrasonic pretreated SFL, in which accumulated VFAs were higher than 2500 mgCOD/L (with acetate reaching >1000 mgCOD/L). It appeared that some species, belonging to Parabacteroides, Clostridium, and Pseudomonas, were potentially enriched more in alkaline SFL with higher SCOD and conductivity, compared to ultrasonic SFL. But a delayed fermentation process on raw WAS, as well as freeze/thaw-pretreated WAS, substantially led to different fermentative communities in the anode biofilm (such as Parabacteroides and Proteinilclasticum), which produced little VFAs for anode respiring bacteria. In this situation, long time would be required to WAS cascade utilization, with a slow fermentation and inefficient electron generation in BES.