Effect of bioaugmentation on digestate metal concentrations in anaerobic digestion of sewage sludge

This study examined the influence of bioaugmentation on metal concentrations (aluminum, cadmium, chromium, cobalt, copper, iron, lead, manganese, molybdenum, nickel and zinc) in anaerobically digested sewage sludge. To improve the digestion efficiency, bioaugmentation with a mixture of wild-living Archaea and Bacteria (MAB) from Yellowstone National Park, USA, was used. The total concentration of all metals was higher in the digestate than in the feedstock. During anaerobic digestion, the percent increase in the concentration of most of metals was slightly higher in the bioaugmented runs than in the un-augmented runs, but these differences were not statistically significant. However, the percent increase in cadmium and cobalt concentration was significantly higher in the bioaugmented runs than in the un-augmented runs. At MAB doses of 9 and 13% v/v, cadmium concentration in the digestate was 211 and 308% higher than in the feedstock, respectively, and cobalt concentration was 138 and 165%, respectively. Bioaugmentation increased over 4 times the percentage of Pseudomonas sp. in the biomass that are able to efficiently accumulate metals by both extracellular adsorption and intracellular uptake. Biogas production was not affected by the increased metal concentrations. In conclusion, bioaugmentation increased the concentration of metals in dry sludge, which means that it could potentially have negative effects on the environment.


Introduction
Currently, a major part of heavy metal emissions resulting from human activity enters into wastewater. The physical-chemical processes involved in wastewater treatment are responsible for accumulation of these metals in suspended-growth biomass (activated sludge). A typical metal content in sludge is about 0.5-2% on a dry weight basis [1] and may reach 4% [2]. However, this depends on many diverse factors, such as local conditions (e.g. type of industries in a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Bioaugmentation has also been applied to the degradation of specific organic substances [27], lipid-rich substrates [28,29], keratin-rich waste [30], lignocellulosic matter [25,[31][32][33][34], as well as seed biomass [35]. Schauer-Gimenez et al. [36] and Li et al. [37] proposed this method to reduce the recovery time of digesters exposed to toxic events. Fotidis et al. [38,39] suggested bioaugmentation for efficient anaerobic digestion of ammonia-rich waste through alleviation of ammonia toxicity effect. Li et al. [40] found it helpful for improving the digestion performance in the systems with feedstock of high C/N ratio. Other authors have applied bioaugmentation to accelerate digester start-up [41] as well as for odor reduction [26,42]. The beneficial effects mentioned above indicate that the influence of bioaugmentation on anaerobic digestion of sewage sludge is worth investigating. The question is how to get the helpful microorganisms. The microorganisms strains for bioaugmentation may be obtained by culturing in the laboratory combined with the strains selection, growing in situ using indigenous consortia or buying commercial products designed for specific applications [26,36,43]. Commercial products are easy to obtain, store and dose and thus are the most useful in practice. Some of them, in addition to selected strains of microorganisms, contain enzymes (lipase, protease, cellulase, hydrolase) needed to accelerate the decomposition of biopolymers as well as micronutrients essential for anaerobic digestion processes (Fe, Ni, Cu, Mn, Zn, Se, Mo). However, the composition of most commercial products is usually not known because of patent protection. There are also bioaugmentative products based on the wild-living microorganisms. Among them, the mixture of Bacteria and Archaea (MAB) from Yellowstone National Park, USA, is worth considering. Application of the microorganism from archaeal domain seems to be promising because of their adaptations to extreme habitats (assisted by the possession of unique cell wall types), including environments of high salt content, high temperature, low pH, and acute anoxia. The enzymes provided by Archaea result in higher reaction rates and reduce the contamination problems [44]. This was confirmed by the recent study of Lebiocka et al. [45] on the anaerobic digestion of sewage sludge. The authors reported a significant increase in the rate constant of biogas production along with the MAB dose despite the decrease in hydraulic retention time. The advantages of bioaugmentation contributed to more frequent application of this method in a technical scale. However, the environmental effect of its implementation to the existing digesters should also be examined. Thus far, the influence of bioaugmentation on digestate quality has not been evaluated. This subject is worth investigating, especially in the aspect of heavy metal concentration and the possible environmental risk related to the agricultural application of digestate. Importantly, the researchers involved in bioaugmentation studies did not consider all the possible effects of using this strategy, focusing more on biogas production and organic removal. Considering all the aspects of bioaugmentation (both benefits and hazards) is of great importance in the context of its implementation in full-scale systems. Thus, the proposed holistic approach constitutes a novel concept of bioaugmentation research. In the present study, the influence of bioaugmentation on total metal concentrations in digestate was examined for anaerobic digestion of sewage sludge. The presented results are the continuation of previously mentioned research [45]. The novel aspect of the work involved evaluating the effect of adding a mixture of wild the bacteria and Archaea from Yellowstone National Park, USA, on the metal contents in the digestate, and determining how it may influence the environment.

Material characteristics
Sewage sludge that included residues from primary and secondary treatment was obtained from the Puławy municipal wastewater treatment plant (WWTP), Poland, operating at the flow rate of 13 000 m 3 d -1 . Primary sludge from the gravity thickener and waste sludge from the mechanical belt thickener were sampled once a week and transported to the laboratory in separate containers. Therein, sludge was mixed at the volume ratio of 60:40 (primary:waste sludge), recommended as beneficial for obtaining maximal biogas production, then homogenized, manually screened through a 3-mm screen and partitioned. The sludge samples were stored at 4˚C in a laboratory fridge for a week at the longest. Sludge prepared in this manner (designated as SS) was used as material for the study. The main characteristics of SS during experiments was as follows (the average value ± standard deviation are given): chemical oxygen demand (COD) of 46 ± 6.3 g L -1 , volatile fatty acids (VFA) of 838 ± 251 mg L -1 , total solids (TS) of 40.1 ± 2.5 g kg -1 , volatile solids (VS) of 31.0 ± 1.9 g kg -1 , pH 6.8 ± 0.3 and alkalinity of 812 ± 100 mg L -1 .
The mixture of Bacteria and Archaea used for bioaugmentation was provided by Soltech Co. the main representative of ArchaeaSolutions Inc. (Evansville, IN, USA) in Poland. The MAB liquor was prepared in continuous mode using a powdery substrate (interchangeable after 30 days), generator and the procedure given by ArchaeaSolutions Inc. (Evansville, IN, USA). The associated scheme is presented in Fig 1. The release of an appropriate microbial content of 1.08 g L -1 h -1 was achieved ensuring the flow rate of dechlorinated water through generator of about 0.5 L min -1 (Fig 1).The average TS and VS of the liquor were 0.48 and 0.042 g kg -1 , respectively, and the COD value reached 22 g m -3 . The VFA concentration of 21 g m -3 , the alkalinity of 330 g CaCO 3 m -3 , and pH value of 7.16 were obtained. The microbial composition of the powdery substrate (ArcheaSolutions Inc.) after 1 day of cultivation at 37˚C under constant mixing conditions in distilled water was investigated by next-generation sequencing (NGS), and sequencing results were deposited in the Sequence Read Archive (SRA, BioProject PRJNA431048). The sequencing was performed as described below (section Analytical methods). In the bioaugmenting mixture the most numerous genera were: Methanosaeta (32.8%), Methanomethylovorans (1.7%), Methanobacterium (1.7%), Exiguobacterium (16.9%), Janthinobacterium (12.1%), Acinetobacter (11.7%), Stenotrophomonas (10.6%), Flavobacterium (2.1%), Brevundimonas (1.9%) and Herbaspirillum (1.1%).

Laboratory installation and operational set-up
The study was performed in anaerobic reactors operating at a temperature of 35˚C in semiflow mode. The laboratory installation consisted of three completely mixed digesters (each with an active volume of 40 L) working in parallel, equipped with a gaseous installation, an influent peristaltic pump and storage vessels. Feedstock was supplied to the upper part of the digester, and digestate was wasted through the bottom by gravity. The gas was taken up using a system consisting of pipelines linked with the pressure equalization unit and a mass flow meter. The scheme of the installation is shown in Fig 2. Inoculum for the laboratory reactors was taken from the WWTP as a collected digestate from a mesophilic anaerobic digester. The adaptation of the digester biomass was achieved after 30 d.
Three runs were conducted, each lasting 90 days (d): 30 d for acclimatization and 60 d for measurements. The digesters were supplied once a day with an applied volume of SS or mixture of SS and MAB. In the first run (R1 -control) the reactor was fed with mixed sludge only. The second run (R2) and the third run (R3) were conducted to evaluate the influence of bioaugmentation on anaerobic digestion efficiency and digestate quality. The SS:MAB volumetric ratios were assumed on the basis of the results of batch experiments conducted previously by the authors. The main criterion was the enhanced biogas production efficiency as compared to the sole of SS anaerobic digestion, however retaining a suitable hydraulic loading rate of approximately 5% was also considered as in for semi-continuous systems. The detailed experimental settings are presented in Table 1.
It is widely known that in municipal wastewater treatment plants the HRT of 20 days is recommended for anaerobic digesters to ensure a highly efficient biogas production and organics removal. While introducing the MAB solution at the assumed doses, hydraulic loading rate was enhanced from 0.05 (R1) to 0.055 and 0.058 (for R2 and R3, respectively), because of an increase in the daily feedstock volume from 2 L to 2.2 and 2.3 L. Thus, the related HRT shortened from 20 days to 18.2 and 17.4 days.

Analytical methods
TS and VS were determined according to the Standard Methods for the Examination of Water and Wastewater [46]. Analogously, the distinctive parameters (COD, VFA, alkalinity, and pH) were measured. The analysis was made once weekly in the feedstock and twice weekly in the digestate (for each reactor) during the entire cycle of measurements (60 d). VS removal (η VS ) was evaluated based on organic matter decomposition. The biogas production was measured using Aalborg (Orangeburg, NY, USA) digital mass flow meter. The biogas composition was determined using Trace GC-Ultra (Thermo Fisher Scientific, Milan, Italy) gas chromatograph coupled with a thermal conductivity detector (TCD) fitted with divinylbenzene (DVB) packed columns. The Rt-Q-Bond column was used for determination of CH 4 and CO 2 concentrations. The analysis was made using the following parameters: injector 50˚C and detector 100˚C. The carrier gas was helium with a flux rate of 1.5 cm 3 min -1 . Peak areas were determined by the computer integration program (CHROM_CARD).
Determination of total metal content, both in the feedstock that was supplied to the reactors and in the digestate was carried out using ICP-OES method (inductively coupled plasma optical emission spectrometry). Quantitative analysis was performed on a JY238 Ultrace (Jobin Yvon-Horriba, France) using an external calibration method following microwave digestion. The homogenized samples of 1 g were digested in an acid mixture of HNO 3 :HCl (5:2). The digestion was carried out for 45 min at 180 � C and at a pressure of 1. The feedstock samples for metal content analysis were taken three times during the entire cycle of measurements. This was performed once weekly, on the same day for all reactors. The digestate was also sampled three times, considering that the hydraulic retention time (HRT) was adequate for specified runs. The measurements were made in triplicate for each sample and the results were presented as the mean of the nine measurements for each reactor.

Statistical analysis
The statistical analysis of biogas production was accomplished via ANOVA involving Shapiro-Wilk's, Levene's and Tukey's tests by the means of Statsoft Statistica software (version 10, TIBCO Software Inc., Palo Alto, CA, USA). The differences were considered as statistically significant at p < 0.05.

Molecular analysis
Microbial structure of biomass from digesters was analyzed after 3 months of digester operation. The samples of biomass were stored at -20˚C. Isolation of DNA was performed using GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich, Germany). Integrity of DNA was confirmed by agarose electrophoresis while purity and concentration was measured using a Biophotometer (Eppendorf, Germany). Archaeal and bacterial 16S rDNA gene sequences were amplified using a universal 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGG GTWTCTAAT) primer set and then sequenced in Research and Testing Laboratory (USA) using the MiSeq Illumina platform. The obtained sequences were analyzed bioinformatically

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The metal contents in bioaugmented digestate as described in Świątczak et al. [47]. Because samples had a similar number of reads normalization was not performed to avoid data loss. For rarefaction analysis a module of the RDPipeline was applied. The sequences have been deposited in SRA as BioProjectPRJNA380917 entitled 'Metagenome of bioaugmented anaerobic digesters'.

Results and discussion
The environmental impact analysis of the digested medium was performed by evaluating the changes of TS, VS and metal concentrations before and after digestion in bioaugmented and non-bioaugmented systems. In order to assess the possible toxic impact of metals on the anaerobic digestion process, the biogas yields, VS removal and methane concentration were determined ( Table 2). More detailed information in the field of biogas production, kinetics and process stability is presented in the study performed by Lebiocka et al. [45].
The average TS were greatest in R1 (sewage sludge) both in feedstock and digestate: 40.1 and 24.0 g kg -1 , respectively. In contrast, when feedstock was enriched by MAB there were correspondingly lower levels in R2 (36.4 and 21.9 g kg -1 ) and R3 (34.2 and 20.1 g kg -1 ). The same trend was observed for VS. The average values in feedstock were as follows: 31.0 (R1), 28.1 and 26.4 g kg -1 (R2 and R3) and in digestate VS reached 16.5, 15.0 and 13.5 g kg -1 , respectively. Thus, the greater the MAB dose, the lower the average TS and VS content. This could probably be explained by dilution of the reactor's influent with MAB as well as an effective decomposition of organic and inorganic matter to gaseous end products. Good decomposition of organic substrates may have resulted from the presence in all experimental reactors microorganisms belonging to Bacteroidetes (18.9-12.3%, Fig 3). Sewage sludge is rich in hard-to-degrade lignins, cellulose and hemicelluloses [48]. It was observed that bioaugmentation with an acetatetype fermentation bacterium belonging to Bacteroidetes increased the methane yield by 19-23% during corn straw digestion mostly due to increased removal rates of cellulose and hemicelluloses [21].
The bioaugmented anaerobic digestion of sewage sludge was more efficient (R2 and R3) compared to the control (R1). Despite the progressive decrease in HRT from 20 d (R1) through 18.2 d (R2) to 17.4 d (R3), the biogas yields were still similar (Table 2) (the differences of means were not statistically significant). Moreover, methane content was similar, independently of the MAB dose. The process was stable and the pH was maintained in the range typical for methanogenesis 7.72-7.99 (lower value for R3, upper for R1 and middle value of 7.86 for R2). The VS removal was higher despite the shortened HRT (Table 2) indicating that MAB improved the sludge digestion due to a better decomposition of organic substrates. This explanation is consistent with research by Duran et al. [26] regarding selected strains of the Baccillus, Pseudomonas and Actinomycetes species used for bioaugmentation.
As mentioned above, TS decreased with the amount of MAB added, both in feedstock and digestate. Considering weight loss during anaerobic digestion, an increment of total metal content in digestate could be expected-with the maximum for the bioaugmented run with the greatest dose of MAB (i.e. R3).
Determination of the average total concentration of metals both in the feedstock and digestate was performed for specified runs ( Table 3).
The average total metal content on a dry weight basis (Table 3) was used for environmental impact analysis. This parameter allowed normalization and comparison of data from different runs.
On this basis, it can be estimated that the total concentration expressed in mg L -1 was lower for most metals (Al, Cr, Cu, Fe, Mn and Zn) in bioaugmented feedstock than for non-bioaugmented sewage sludge (Table 3); moreover, concentrations decreased along with the increase of MAB dose (9 and 13% v/v, respectively). However, for some metals there were similar levels in all comparisons (Cd, Co and Pb) or no clear trend was observed (Mo and Ni). It was assumed that the addition of MAB probably diluted the sewage sludge (the lower TS value in bioaugmented runs seems to confirm this), thus the metal concentrations in feedstock decreased in most cases with the exception of Cd, Co and Pb. In contrast, there was an opposite trend on a dry weight basis. An increase of the total metal content in bioaugmented feedstock occurred (mostly related to a dose of MAB) due to the lower TS content.
The relative abundance of metals in sewage sludge samples before digestion followed the general order: Cd < Mo < Co < Ni < Cr < Pb < Cu < Mn < Zn < Fe < Al (Tables 1 and 2). Table 2. Biogas production, methane concentration, and VS removal for runs [45]. (the mean value and standard deviation are given).

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The metal contents in bioaugmented digestate A similar sequence was observed in samples enriched by MAB, except that there was less Co than Mo in R2.These results are consistent with other research focused on sewage sludge (11,3); however, in these studies a small group of metals was investigated and their concentrations were one order of magnitude higher than in the present study (except for Cd and Pb). This may be due to the lack of high industrial loads in the supply to the Puławy WWTP, which was reflected in low levels of metals in sewage sludge. In contrast, Dong et al. [49] noted a different order in dewatered sewage sludge (with the use of a high-molecular weight flocculant based on polyacrylamide): Pb < Ni < Cu < Cr < Zn. The digested medium had an increased total content of all metals compared to the feedstock (Table 3). This observation is consistent with research on sewage sludge by Chipasa [11], who found that Cd, Cu, Pb and Zn concentrations in digestate exceeded the corresponding values in undigested sludge by 50-99%. In the present study, the corresponding increases were approximately 70-103% in R1, with the exception of 111 and 116% increases in the concentrations of Cd and Co, respectively (Fig 4). The pattern of increases was similar in the bioaugmented runs (i.e. R2 and R3), although the percentage increases for most metals (74-112%) were a little higher. Only the changes in Co and Cd concentrations differed significantly between reactors (one-way ANOVA); their concentrations exceeded the corresponding values in feedstock by 211 and 308% (Cd) and 138 and 165% (Co) in R2 and R3, respectively, and the size of the increase depended on the MAB dose (Fig 4).
The mass balance for heavy metals showed their bioaccumulation throughout anaerobic digestion both in bioaugmented and un-augmented reactors (Fig 5). However, different levels of bioaccumulation were found with a spectacular difference for Cd and Co corresponding to the above-mentioned increases. It should be noticed that in bioaugmented runs there was no association between the increases in metal concentration and the biogas yields, methane concentrations, and VS removal; thus the metals did not seem to have a toxic effect. This was in contrast to the results of the study by Abdel-Shafy and Mansour [50], who reported a significant decrease in both biogas production and VS removal, as well as an accumulation of organic acid intermediates resulted from Hg, Cd and Cr(III) toxicity in non-bioaugmented anaerobic digestion of sewage sludge. The above might indicate an immobilization of heavy metals in bioaugmented systems, resulting both from the differences in microbial selectivity and ability to take up metals. Metal remediation potential is strictly related to the selection of species in the bioaugmentation mixture. A study on removal of extractable metals such as Pb, Cu, and Al through bioaugmentation conducted with inocula of bacterial species isolated from leachate-contaminated soil has shown that the efficiency of metal removal was significantly lower when all isolated microbes were used than when only three species were used (Lysinibacillus sp., Bacillus sp., and Rhodococcus sp.) [51].

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The metal contents in bioaugmented digestate Bioaugmentation with even one group of bacteria involved in methane fermentation affects all steps of the process. Bioaugmentation of methane fermentation with hydrogen-producing acetogens (HPAs) obtained through enrichment and subculturing of anaerobic sludge with the use of butyric acid improved not only hydrogen-producing acetogenesis but also hydrolysis/acidogenesis and methanogenesis. At a bacterial number-to-activated sludge ratio of 1:9, inoculation with HPAs improved the yield and rate of methane production by a factor of 2.1 and 2.0, respectively [52]. In our study, bioaugmentation favored growth of Pseudomonas sp. that were not present in the MBA. The percentage of Pseudomonas sp. increased from 0.8% in the control reactor to over 3.5% in the bioaugmented digesters (Fig 3). In the same time increased Cd and Co accumulation was observed in the bioaugmented digesters. Previous studies indicated that Pseudomonas sp. may improve the uptake of heavy metals from the environment. During their active growth phase, Pseudomonas aeruginosa strain efficiently accumulated more than 75% of soluble cadmium (8 mM) from growth medium, and more than 89% from Cd-amended industrial wastewater; cadmium accumulated mostly in the membrane and periplasm of cells [53]. Pseudomonas sp. were also involved, along with Brevundimonas sp. and Stenotrophomonas sp., in nickel, cobalt and copper uptake from contaminated systems [54].
The primary mechanism of metal accumulation by microbes is biosorption followed by bioaccumulation into the cells, controlled siderophore production, enhanced respiration and modified protein profile [55,56] Pseudomonas halodenitrificans efficiently biosorbed Co on its cell walls and the biosorption was significantly inhibited by the presence of divalent ions such as Ca [57]. Pseudomonas sp. exposed to Al shifts its metabolism toward the production of organic acids and lipids that play a vital role in chelating and immobilizing Al [58]. Low molecular weight ligands that chelate iron and other metals produced by microorganisms are called siderophores [59,60]. The major siderophore produced by Pseudomonas aeruginosa is pyoverdine (PVD). Hannauer et al. [60] found that PVD-metal complexes could accumulate in the periplasm in different concentrations depending on the presence or absence of a specific efflux pump (PvdRT-OpmQ). Its absence resulted in higher levels of metals in the periplasm since the complexes were not expelled into the extracellular medium. With an inactive efflux pump, metal content increased as much as 1050% for Co 2+ and 1005% for Zn 2+ , or as little as 45% for Cd 2+ and 32% for Fe 3+ . It is worth noting that, in addition to changes in transport mechanisms, differences between microbial consortia may also lead to differences in metal accumulation. Investigation of protein response to Cd presence in Pseudomonas aeruginosa san ai cells indicated that almost a third of the total numbers of 60 proteins that were up-regulated in Cd-amended culture were metalloproteins. In the presence of Cd denitrification proteins were over expressed but not active, suggesting their protective role in conditions of heavy metal exposure [56].
Metals can also be accumulated by adsorption on extracellular polymeric substances that are excreted by bacteria. EPS have an important influence on metal binding capacity because of their high concentration of active sites, e.g. carboxyl groups [61]. Pseudomonas sp. can produce adhesive EPS, and this production increases as the length of incubation in starvation conditions is extended [62]. Moreover, it depends on the available source of organics. Production of EPS by Pseudomonas aeruginosa G1 and Pseudomonas putida G12 in media containing various amounts of glucose, mannose, fructose, and xylose was highest in the medium containing xylose. Maximum EPS yield was 368 mg L -1 with strain G1 cultivated in 3% (w/v) xylose [63]. P. aeruginosa are also able to produce biosurfactants such as rhamnolipids that complex heavy metals. Neilson et al. [64] evaluated rhamnolipid synthesis in the presence of heavy metals. Cd-induced rhlB expression was observed in mid-stationary phase (53 h) and sustained production of rhamnolipid was completed in 96-h late stationary growth phase. Presence of Cd also increased the ratio of dirhamnolipids to monorhamnolipids, which was favorable because the complexation constant for dirhamnolipid-Cd is several orders of magnitude larger than that of monorhamnolipid-Cd.
In our study, bioaugmentation increased the number of sequences that have only been identified at the kingdom level, indicating that many unknown species were present in the biomass and may have been involved in metal up-take.
The increase in content of a metal during digestion was not proportional to its initial content in the feedstock. This was consistent with research by Chipasa [11]. Although the relative abundance of metals after digestion was similar to that in undigested samples (only the relative abundance of Mo and Cd differed between bioaugmented and non-bioaugmented runs). Although bioaugmented and non-bioaugmented systems differed in terms of the percent increase of individual metals, these percent increases followed the same order in both systems (Fig 4). The results suggested that for the lowest total metal content in sludge before digestion there was the highest increase following the process. It was especially so regarding Cd and Co. However, introducing MAB led to much greater increases and the metals were much more concentrated in dry bioaugmented compared to non-bioaugmented sludge.
In principle, the contents of metals in bioaugmented and non-bioaugmented sludge after anaerobic digestion were in accordance with the compliance limits for agriculture land application [65]. However, since bioaugmentation increased concentration of metals on a dry weight basis, the use of bioaugmentation should be carefully considered for situations in which sewage sludge is rich in metals. In such an event, bioaugmentation could exclude the digestate from safe use in agricultural application or reclamation.
To sum up, the total metal content observed in the digestate tended to increase more in bioaugmented runs; moreover, the response to MAB was dose-dependent for most metals (the higher the dose, the more concentrated metals in the dry digested sludge). Thus, the addition of MAB could unfavorably affect the bioaugmented digestate quality.