Sequential recovery of protein and ammonium from waste sludge and functional metabolism in a combined process of nutrient recovery electro- fermentation (NREF)

doi:10.21203/rs.3.rs-3321199/v1

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Research Article

Sequential recovery of protein and ammonium from waste sludge and functional metabolism in a combined process of nutrient recovery electro- fermentation (NREF)

https://doi.org/10.21203/rs.3.rs-3321199/v1

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published 31 Mar, 2024

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Background: Waste sludge, as an inevitable by-product during wastewater treatment, is a valuable resource for nitrogen nutrient recovery (e.g. protein and ammonium). Electro-fermentation system (EFS) has a unique advantage in boosting sludge fermentation while recovering ammonium. However, the simultaneous recovery of protein and ammonium during sludge treatment has not yet been achieved. And it remains obscure how microbes cooperate regarding their molecular metabolic mechanisms during EFS treating sludge.

Results: We proposed a novel approach for sequential recovery of protein and ammonium and simultaneous sludge treatment by pretreatment-EF cascading system. The results showed that 72.23% of protein was recovered by alkaline-thermal pretreatment of dewatered sludge, which was recycled as yeast extract and peptone substitute for bacterial growth. The cascading two-chamber EFS facilitated the ammonium recovery of 71−80% and 45−50% in cathode chamber of residual pretreated dewatered sludge (EFS-TS) and raw waste sludge (EFS-RS), respectively. Additionally, the EFS significantly enhanced the COD removal, where EFS-TS obtained the highest TCOD removal which increased by 6.39−14.53% over anaerobic digestion. Microbiome analysis demonstrated that EFS attributed to the enrichment and syntrophic interaction of electroactive bacteria (Geobacter), fermentative bacteria (Rikenella, Lentimicrobium, and Petrimonas), and nitrogen-fixing bacteria(Geobacter and Azonexus). Metagenomics analysis uncovered that this syntrophic interaction facilitated the organic degradation pathways, intracellular and extracellular electron transfer, and nitrogen transformation.

Conclusions: Our study provides insights into syntrophic interaction between biofilm and suspension in the nutrient recovery electro-fermentation, and presents a promising approach for processing sludge with full form nitrogen recovery.

Electro-fermentation

Protein recovery

Ammonium recovery

Waste sludge

Metagenomics

Electroactive bacteria

The increasing waste activated sludge (WAS) amount and its potential environmental concerns severely impedes the sustainable industrial development and arouses researcher’s attention ^{1, 2}. In 2020, Europe Union generated 13.5 million tons of dry solids of sludge ³, while in China the WAS (80% moisture) production was estimated to over 90 million tons in 2025 ⁴. Recently, instead of a social burden WAS has been considered as a renewable resource due to its high contents of organics (59–88% w/v) and nutrients (N and P), which can be converted to value-added products, such as biomaterials, biochemicals, and biofuels ⁵. Therefore, it’s vital to develop novel strategies of organic waste treatment toward low-carbon, energy-saving, and sustainable development goals.

Protein, as the major component of WAS (32–41% of TS), has drawn great interest in its recovery from sludge ⁶. Instead of treating all sludge components as a hazardous waste, the direct extraction of protein from sludge presents solutions to overcome excess energy and resource input in sludge management. To extract protein, different technologies and processes have been employed to lyse cells and release intracellular compounds in WAS, including physical, thermal, chemical, biological and combined pretreatment methods ^7–9. While the combined pretreatments, such as alkaline-thermal, acid-thermal, and enzyme lysis-thermal pretreatments, were widely used and proved more efficient ^{6, 10}. After sludge disintegration, isoelectric precipitation has been widely applied to precipitate and recover protein from sludge hydrolysates because of its economically viable for scale-up ^{11, 12}. While various of protein recovery methods were evaluated, the research of protein recovery from dewatered sludge (DS) was limited. Effective techniques are needed to integrate with sludge residual treatment after protein recovery.

Electro-fermentation system (EFS), as one type of bioelectrochemical system (BES), can steer metabolic product profile via coordinating the extracellular electron transfer (EET) or internal electron transfer, the interspecies interaction of electroactive bacteria (EAB) and fermenters, the ratio of NADH/NAD⁺, extracellular oxidation − reduction potential (ORP), and substrate transmembrane transport ^13–15. Anodic electro-fermentation system (AEFS) use a working electrode as an anode, allowing microorganisms to transfer excess electrons to oxidize organic compounds. In contrast, cathodic electro-fermentation system (CEFS) use a working electrode as a cathode, allowing microbes to consume the electrons to synthesize more reduced products ¹⁶. Moreover, CEFS has potential to utilize organic waste or CO₂ to produce high-value products by capturing electrons from renewable energy supplements such as solar, wind, and biomass, which helps to mitigate carbon emissions ^{13, 17}. A recent study demonstrated that an AEFS improved H₂ and ethanol production by Ethanoligenens harbinense from glucose with different anode potentials ¹⁴. In addition, a CEFS boosted methane production yield by 54.3% through improved electron transport to methanogens ¹⁸, while another CEFS achieved 28% increased caproate production compared with open circuit ¹⁹. Notably, EFS is more effective at degrading WAS than anaerobic digestion (AD). It has been reported that combining the AD with microbial electrolysis cells (MEC) can further enhance organics degradation and H₂ recovery ^{20, 21}. However, a large amount of ammonium (NH₄⁺) will release during sludge hydrolysis and acidogenesis, which will cause operational instability and inhibit microbial activity, thereby limiting their potential conversion into value-added products ^{22, 23}. Hence, ammonium extraction from WAS not only recovers nutrients but also reduces subsequent microbial inhibition, which can be used for fertilizer and nitrogen source additives. Nowadays, compared to conventional biological nitrification and denitrification processes, the ammonium recovery from wastes is expected to reduce energy and carbon emission and while achieving resource recovery ^24–26. BESs, as a low-cost and eco-friendly technology, have been proven to recovery NH₄⁺ by electromigration across the cation exchange membrane (CEM) to cathode, meanwhile realizing its removal from sludge ²⁷. For example, the two-chamber EFS fed with Fe-sludge resulted in over 75% of NH₄⁺ recovery in catholyte ²⁸. The alkaline cathodic circumstance further stripped ammonium to NH₃ without chemical dosing or heating, resulting in 93% NH₄⁺ recovery from real urine in BES ²⁹. However, the understanding of the molecular mechanisms involved in carbon- and nitrogen-transforming metabolisms, and electron transfer pathways in EFS for treating WAS is still limited.

Therefore, the aim of this study is to develop the nutrient recovery electro-fermentation (NREF) approach for simultaneous recovery of protein and ammonium and dewatered sludge (DS) treatment based on pretreatment-EF cascading system. As acid-thermal pretreatment was usually reported to be less effective than AT pretreatment for hydrolyzing biomass cells in sludge ¹⁰. The synchronous protein extraction and disintegration by enzyme lysis and alkaline-thermal pretreatment were compared, then the reduction of residual treated DS and raw WAS in EFS were investigated. Furthermore, microbiome and metabolic function high-throughput sequencing and metagenomics analysis were performed to evaluate the mechanisms underlying enhanced sludge degradation at a molecular level.

2.1. Sludge characteristics

The waste activated sludge (WAS) and dewatered sludge (DS) were obtained from the Wenchang Municipal WWTP (Harbin, China). The WAS was filtrated and thickened by gravity and stored at 4 ℃ prior to the experiment. The main characteristics of the concentrated WAS and DS are shown in Table 1.

Table 1

Main characteristics of the WAS and DS.
Parameters	WAS	DS
Moisture content	97.48 ± 0.04%	79.38 ± 0.34%
Total suspended solids (TSS) (g/L)	30.27 ± 0.27	0.227 ± 0.037 (g/g)
Volatile suspended solids (VSS) (g/L)	15.31 ± 0.02	0.085 ± 0.006 (g/g)
Total chemical oxygen demand (TCOD) (g/L)	24.54 ± 0.11	108.54 ± 4.95
Soluble chemical oxygen demand (SCOD) (g/L)	1.02 ± 0.02	/

2.2. Protein recovery and disintegration methods

Enzyme lysis (EL) and alkaline-thermal (AT) pretreatment were carried out to extract crude protein of DS. The DS (330 g) was mixed twice the volume of distilled water to achieve a moisture content of approximately 93%. Then, for the EL pretreatment, alkaline protease (2%, w/w) was added to the diluted DS suspension, following by adjusting the pH of DS suspension to 8. After that, this suspension was treated at 55 ℃ for 4 hours then heated at 100 ℃ for 15 min in a magnetic stirring hotplate (DF-101S, Qiuzuo technology Co., China). For AT pretreatment, the pH of diluted DS suspension was adjusted to 12.5, then stirred at 100 ℃ for 4.5 h in a magnetic stirring hotplate. After hydrolysis, two kinds of supernatant containing soluble protein were obtained by centrifuging the sludge hydrolysate for 30 min (10000 rpm, 4°C), and the hydrolytic precipitate (HP) was saved for later EF treatment. Then the isoelectric precipitation was carried by adjusting the pH of the protein supernatant to 3 using dilute H₂SO₄ solution. After stirring for 15 min and centrifuging at 12000 rpm for 30 min at 4°C, the crude protein was recovered. The crude protein pellet was lyophilized for heavy mental analysis using inductively coupled plasma atomic emission spectrometry (ICP-AES Optima8300, PerkinElmer Inc. USA). In addition, the recovered protein pellet was tested as yeast extract and/or peptone substitute in Luria–Bertani (LB) medium for Pseudomonas aeruginosa PAO1 growth. Further details were given in the Supporting Information (SI). The residual of treated DS (supernatant and HP) was mixed together and referred to as TS.

2.3. Construction and operation of electro-fermentation reactors

The reactors used in this study were two-chamber EFS reactors consisting of two 120 mL chambers separated by a cation exchange membrane (Ultrex CMI-7000, Membranes International Inc., USA) (Fig. 1). Graphite felts (25 × 45 mm, 3 mm thick) were used as anode and cathode, connected by titanium wire, while a KCl saturated Ag/AgCl reference electrode (+ 199 mV vs. the standard hydrogen electrode, SHE) (Ida Co., Tianjin, China) was placed in the anode chamber. The EFS reactors were pre-acclimated to enrich electroactive biofilm on the anode. The EFS reactors generated the higher current (4.16 mA) and had a larger double-layer area than those of the unenriched reactors (Con), thus proving the effective electroactive biofilm formation. Moreover, the CV showed two redox systems at approximately − 0.376 V and − 0.295 V vs. Ag/AgCl which were the characteristic peak of Geobacter (Figure S1) ³⁰. The detailed start-up procedures were shown in SI.

The mixed solution (120 mL, pH 7) of the TS or raw waste activated sludge (RS) with a 100 mM nutrient phosphate buffer solution was used as substrate of EFS (Table S1). The cathode chamber was loaded with 100 mM NaCl solution, while the anode chambers were purged with N₂ (99.999%) for 30 minutes to removal oxygen. For EFS condition, the anode potentials of TS and RS loaded reactors were poised at 0.2 V (EFS-TS and EFS-RS), respectively, with a multichannel potentiostat (WMPG1000, WonATech, Korea) as previously described ¹⁴.

Open-circuit EF reactors of TS and RS (AD-TS and AD-RS) were operated as AD control, respectively. The reactors were refilled with half of fresh anodic and cathodic solution when the current decreased below 0.5 mA and all reactors were operated with magnetic stir bars mixing in for two batch cycles at 35 ℃. All groups were tested in triplicate. The cathode and anode chamber of each reactor were connected to a gas bag for gas collection. Liquid samples from each chamber were periodically taken for chemical analysis.

2.4. Analyses and Calculations

Liquid samples collected daily were centrifuged and filtered through 0.45 mm syringe filters, after which were analyzed for VFAs, SCOD, nitrogen (NH₄⁺-N, NO₂^--N, and NO₃^--N), pH, soluble protein, and polysaccharide. Gas composition (H₂, CO₂, and CH₄) from gas bag and reactor headspace, and VFAs concentrations were quantified using the GC (GC7890A, Agilent Technologies Co. Ltd., America) as previously described ³¹. The total suspended solid (TSS), volatile suspended solid (VSS), total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD), NH₄⁺-N, NO₂^--N, NO₃-N, and pH were determined according to the Standard Methods ³². Soluble protein concentrations were measured by a BCA Protein Assay Kit (Tiangen Biotech Co., Beijing, China), while polysaccharide was tested by the phenolesulfuric method ³³. The cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were conducted before and after enriching the electroactive biofilm using a VMP3 electrochemical workstation (Bio-Logic Inc., France). The scan rate of CV was 1 mV s^− 1 and 5 mV s^− 1 in the potential range from − 0.8 V to 0.2 V versus Ag/AgCl reference electrode, while EIS was conducted over a frequency range of 10⁵ to 10^− 2 Hz.

The protein recovery efficiency was calculated as Eq. (1)

$${\eta }=\frac{\text{C}\text{t}-\text{C}0}{\text{C}\text{t}}$$

where η is the protein recovery efficiency, c_t is the protein concentration in supernatant after pretreatment, c₀ is the protein concentration in supernatant after isoelectric precipitation.

2.5. Microbiome analysis based on 16S rRNA gene amplicon sequencing

To compare microbiome composition, anode biofilm and suspension samples from EFS-TS (EFS-TS-bio and EFS-TS-ss) and EFS-RS (EFS-RS-bio and EFS-RS-ss), and suspension samples from AD-TS and AD-RS were harvested at day 35, and were immediately stored at -20°C for further DNA extraction. The various region V3–V4 of 16S rDNA was amplified using a bacterial universal primer pair 338F and 806R. The qualified libraries were sequenced on a NovaSeq6000 platform (Illumina, San Diego, CA, USA) with paired-end sequencing. The amplicon sequence variants (ASVs) were clustered using the Silva138.1 database. Species classification, phylogenetic relationship construction, alpha and beta diversity assessment were conducted by Quantitative Insights Into Microbial Ecology 2 (QIIME2) software (Version QIIME2-202006). Over 94,000 qualified reads and a total of 2,424 amplicon sequence variants (ASVs) were identified (Table S2). Further details of analysis are provided in the SI. All raw reads associated with 16S rRNA sequencing have been submitted to the NCBI Sequence Read Archive under the project ID PRJNA1010495.

2.6. Metagenomics analysis

To identify microbial metabolic functions of four groups, DNA from the EFS-TS-bio, EFS-TS-ss, EFS-RS-bio, EFS-RS-ss, AD-TS, and AD-RS were extracted at day 35 for metagenomics sequencing. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s instructions. The library preparations were sequenced on an Illumina PE150 platform with a paired-end 150 bp sequencing kit (Illumina, San Diego, CA, USA). The assembled Scaftigs (> 500 bp) were used to predict the open reading frames (ORF) using the MetaGeneMark (V2.10, http://topaz.gatech.edu/GeneMark/) software. Metagenomic sequencing generated over 10,000 Mb of clean sequences and assembled over 185,900 contigs with an N50 over 1,091 bp from each group (Table S3). Taxonomic affiliation was determined by blasting non-redundant gene catalogue from the NR database (Version: 2018-01-02, https://www.ncbi.nlm.nih.gov/) of NCBI with e-value cutoff of 1e^-5 using DIAMOND software (V0.9.9, https://github.com/bbuchfink/diamond/). Gene functions have been annotated by KEGG (Version 2018-01-01, http://www.kegg.jp/kegg/) and eggNOG database (Version 4.5, http://eggnogdb.embl.de/#/app/home) to investigate the metabolic activity of the community. The detailed methods for metagenomics sequencing and data analysis are provided in SI. All raw reads associated with metagenomic sequencing have been deposited in the NCBI Sequence Read Archive under the project ID PRJNA1007408.

3.1. Synchronous dewatered sludge disintegration and protein recovery

The protein concentrations in supernatant after alkaline-thermal (AT) pretreatment and enzyme lysis (EL) were 4171.25 ± 23.75 mg/L (148.71 mg/ g VSS) and 3400.71 ± 43.15 mg/L (121.23 mg/ g VSS), respectively (Fig. S2), This was significantly higher than the concentration of 108.24 ± 0.67 mg/L (3.86 mg/ g VSS) in untreated DS, indicating that these two methods fulfilled effectively synchronous sludge disintegration and protein extraction. AT pretreatment led to a higher release in the protein concentration compared with EL. When the pH adjusted to 3, the protein precipitated and the concentrations decreased to 1158.12 ± 16.87 mg/L (41.29 mg/ g VSS) in the AT-treated sludge, which was lower than that in the EL (2054.28 ± 78.3 mg/L). AT pretreatment presented a higher protein recovery (72.23%) compared to EL (39.59%), thus it was chosen to recover soluble proteins from the DS followed by electro-fermentation of residual component after protein recovery.

The concentrations of heavy metals in DS and recovered protein by AT pretreatment were further measured (Table S4). Through the protein recovery process, 59.77 − 63.77% of Al, Ca, and Pb, and 88.65 − 90.12% of Cu, Cr and Ni were removed. While Fe and Zn were not accumulated in recovered pellet. The concentrations of some toxic heavy metals (Pb, AS, Cd, Hg, and Se) were below the limit of the hygienic standard for feeds (GB 13078 − 2001) (Table S4). In addition, the recovered protein was proved to support Pseudomonas aeruginosa PAO1 growth as yeast extract and peptone substitute, with a comparable cell growth amount in RP-1000, RPY-3700, and RPP-1800 compared to those in LB medium (Figure S3).

3.2. EFS facilitates degradation of organic matter

For the TS treatment, EFS fed with TS (EFS-TS) achieved a 28.80% increase in the SCOD degradation efficiency during the first batch compared to the open-circuit EFS fed with TS (AD-TS) (51.44 ± 2.59%) (Fig. 2A). Interestingly, the SCOD degradation rate of EFS-TS in the first 7 days (246.04 mg COD/L-day) was 7.36-fold higher than that in AD-TS (one-way ANOVA, p < 0.05, n = 3), suggesting that EFS accelerated organics degradation. The EFS-TS obtained higher TCOD removal of 75.91 ± 1.15% compared with 71.35 ± 1.11% in the AD-TS (Fig. 2B). Polysaccharide and protein degradation were faster in the EFS-TS than in the AD-TS during the first 5 − 9 days (Fig. 2C and D). Polysaccharide removal efficiency accounted for 51.41 − 61.24% in the EFS-TS and AD-TS (Figure S5A), while protein concentration showed a slight increase and ended up no significantly difference in the first batch (one-way ANOVA, p < 0.05, n = 3). In the second batch, the tendency of COD removal was similar to that of the first batch (Figure S4A and B), while the protein and polysaccharide concentrations dropped by 48.15 ± 0.34% and 35.66 ± 3.44% in the EFS-TS, respectively, which was significantly enhanced compared with those in the AD-TS (39.9 ± 3.15% and 5.97 ± 2.73%) (one-way ANOVA, p < 0.05, n = 3) (Figure S5B).

For the RS treatment, the SCOD of EFS-RS and AD-RS fluctuated around about 800 mg/L to 1300 mg/L during the first batch and remained relatively constant, indicating that RS released more soluble organics. Furthermore, the TCOD removal of EFS-RS (21.33 ± 1.99%) was higher than that of the AD-RS. The EFS-RS showed 1.35 times higher polysaccharide degradation efficiency (31.51 ± 6.65%) than the AD-RS (Fig. 2C and D). Although the protein concentration increased, the increment of EFS-RS (74.14 ± 4.26%) was lower than that in AD-RS (91.29 ± 5.94%), suggesting more protein degradation in EFS-RS (Figure S5A). The tendency of organics removal in the second batch was similar to that of the first batch (Figure S4A and B). All EFSs exhibited significantly enhanced TCOD removal, faster protein and polysaccharide degradation compared to AD (one-way ANOVA, p < 0.05, n = 3). These results demonstrated that EFS accelerated disintegration and hydrolysis of WAS.

The VFAs concentration of TS increased after AT pretreatment, while acetate was the predominant SCFA detected in all reactors (Fig. 3A and B). During the EFS fed with TS, the concentration of acetate decreased sharply in the first 7 days and then gradually reduced to 109.60 ± 6.81 mg/L, while that of AD-TS accumulated in the first 11 days and slightly decreased to 973.60 ± 20.64 mg/L afterwards. The VFAs concentration of EFS/AD-RS was much lower than EFS/AD-TS, but the variation exhibited a similar trend in that the acetate degraded faster in the EFS compared with the AD (Fig. 3C and D).

3.3. EFS enhanced ammonium recovery

The hydrolysis and acidogenesis of protein-rich sludge released a large amount of ammonium into the supernatant (Fig. 4A). With the current flow in two-chamber of EFS, cations migrated from the anode chamber to the cathode chamber, resulting in ammonium enriching in the catholyte. The NH₄⁺-N concentration in the anode chamber of EFS-TS decreased to 100.00 ± 5.56 mg-N/L after 17 days of operation in the first batch, while it increased to 248.80 ± 28.11 mg-N/L in the catholyte (Fig. 4A and B) achieving a 71.33% ammonium recovery from sludge digestate. By contrast, the NH₄⁺-N concentration in sludge suspension in the AD-TS increased to 326.85 ± 6.25 mg-N/L after 17 days (Fig. 4A). Meanwhile, there was still 115.74 ± 8.64 mg-N/L of NH₄⁺-N in the catholyte of AD-TS owing to the diffusion of concentration gradient. The second batch of operation showed similar results, with 299.07 ± 10.98 mg-N/L of NH₄⁺-N migrating to the catholyte of EFS-TS, leading to 79.63% ammonium recovery efficiency (Figure S7).

Less NH₄⁺-N migrated to the cathode chamber of the EFS-RS due to lower current and NH₄⁺-N release (Figure S7D). NH₄⁺-N of 44.97% in the EFS-RS was migrated to the catholyte during the first batch, higher than that in the AD-RS (26.89%) (Fig. 4). During the second batch, the NH₄⁺-N concentrations of the EFS-RS and AD-RS increased to 260.65 ± 6.69 mg-N/L and 194.72 ± 19.78 mg-N/L in the cathode chamber, respectively. Hence, the EFS-RS achieved 49.88% ammonium recovery from the fermented sludge liquor (Figure S7). In addition, proton migration in the EFS led to a decrease of pH at the anode (Figure S8A), while proton consumption and hydroxyl ions (OH⁻) production at the cathode led to an increase in pH (Figure S8B).

3.4. EFS shaped microbiome of biofilm and suspension

The Pielou evenness, Shannon, and Simpson indices of the suspension samples were higher, indicating higher community diversity and more evenness distribution. The Chao1 estimator showed that the low abundance species in the suspension samples was higher than in the anode biofilms (Table S3). Principal coordinates analysis (PCoA) revealed a distinct difference in microbial community structure between the RS and TS, and electrode biofilm and suspension. However, the suspension of the EFS and AD had similar bacterial composition (Fig. 5Aand B). This suggests that EFS shaped microbial community structure.

Bacteroidota, Desulfobacterota, Firmicutes, and Proteobacteria were the foremost predominant phyla in all samples (Fig. 5C). The relative abundance of Bacteroidota decreased from 35.75 − 54.83% in suspension samples (AD-RS, AD-TS, EFS-RS-ss, and EFS-TS-ss) to 24.09 − 26.25% in the anode biofilm of EFS-TS (EFS-TS-bio) and EFS-RS (EFS-RS-bio). Conversely, the poised anode potential promoted the enrichment of Desulfobacterota which relative abundance accounted for 54.86% in the EFS-RS-bio and 44.20% in the EFS-TS-bio. In the EFS-TS-bio, Geobacter (38.80%), Hydrogenophaga (3.62%), and Petrimonas (9.32%) were enriched, while the EFS-TS-ss and AD-TS shared the same predominant genera (Rikenella, Lentimicrobium, Petrimonas and Azonexus). The relative abundance of Lentimicrobium, Petrimonas, and Azonexus increased by 31.70%, 43.55%, and 673.89%, respectively, in the EFS-TS-ss compared to AD-TS (Fig. 5D). In the EFS-RS-bio, Geobacter (51.27%), Lentimicrobium (12.00%), and Leptolinea (1.54%) were enriched compared with AD-RS, which had Clostridium (11.76%), Smithella (8.81%), Rikenella (5.18%), and Petrimonas (4.95%) as its predominant genera. The relative abundance of Lentimicrobium (20.96%), Rikenella (11.82%), and Leptolinea (2.55%) increased in the EFS-RS-ss compared with AD-RS.

The clustering analysis heatmap showed a clear distinction in archaeal communities between biofilm and suspension samples (Fig. 5E, Figure S9). The predominant phyla in all reactors belonged to Euryarchaeota, Candidatus Bathyarchaeota, and Candidatus Lokiarchaeota. Methanosarcina and Methanothrix, the acetoclastic and methylotrophic methanogens, respectively, dominated all communities. While the relative abundance of most methanogens reduced in the EFS-TS-bio and EFS-RS-bio compared to that in the AD-TS/RS, which is consistent with therir lower methane production (Figure S6). For example, the relative abundance of Methanosarcina decreased by 71.23% in the EFS-TS-bio compared with the AD-TS, while that of Methanothrix decreased by 62.02% in the EFS-RS-bio compared with the AD-RS.

3.5. Difference in functional gene enrichment between EFS and AD

The metagenomic analysis showed that Geobacter (46.94 − 48.51%) was the most predominant genera in the biofilm of the EFS (Figure S10). COG categories illustrated that the genes involved in signal transduction mechanisms (T) had 1.28 and 1.07 times higher relative abundances in the EFS-TS-bio and EFS-RS-bio, respectively, than those of AD (Figure S11). Clustering heatmap of KEGG also showed the shift of metabolic pathways between the EFS and AD reactors (Figure S12A), such as the increased abundances of fatty acids biosynthesis, TCA cycle, protein export, and two-component system encoding genes in the EFS biofilm. Furthermore, the abundance of putative transposase encoding gene related to replication and repair increased 5.62-fold in the EFS-TS-bio than that in the AD-TS (Figure S12B). Besides, genes encoding methyl-accepting chemotaxis protein, putative ABC transport system permease protein, and branched-chain amino acid transport system substrate-binding protein were identified to be increased in the EFS-TS-bio and EFS-RS-bio compared to the AD.

3.6. EFS enriched genes involved in carbon and nitrogen transformation

The critical pathways for sludge degradation include polysaccharides degradation, monosaccharide metabolism, and protein hydrolysis. Compared with the AD, the abundant genes involved in starch and sucrose metabolism pathways enriched in the EFS-TS and the EFS-RS, including alpha-amylase (treS), glucoamylase (gla1), and 4-alpha-glucanotransferase (malQ), especially for treS which abundance increased by 1.31 times in the EFS-TS-bio vs the AD-TS (Fig. 6A and B). Additionally, gene encoding fructose-bisphosphate aldolase (fbaA) exhibited 47.46% higher abundance in the EFS-TS-bio than the AD-TS. The genes involved in protein hydrolysis enzymes, including serine endopeptidases (gluP and degQ) and cysteine peptidases (pfpI) showed 1.67 − 5.17-fold higher abundance in the biofilms of the EFS-TS/RS vs the AD (Fig. 6C and D). The results that EFS-TS and EFS-RS increased the abundance of the genes involved in polysaccharides and protein metabolism were consistent with their higher polysaccharides and protein degradation efficiency, which provided a reasonable explanation for the enhancement of COD removal (Fig. 2 and S4).

Additionally, key genes involved in glycolysis pathway were identified to be more abundant in the EFS than AD, especially in the EFS-TS-bio, including hk, tal-pgi, pfkABC, fbaAB, tpiA, gapAN, gapor, pgk, apgM, eno, korAB, and porABDG, which catalyzed the hydrolysis of D-glucose to α-D-glucose-6P, followed by β-D-fructcose-6P oxidation to glyceraldehyde-3P and further to phosphoenol-pyruvate, and pyruvate conversion to acetyl-CoA (Fig. 7A). Among them, the gapor exhibited the highest increased abundance of 4.98-fold in the EFS-TS-bio vs the AD-TS. The relative abundance of gene encoded acetate kinase (ackA) increased to over 0.04% in the biofilm of EFS-TS/RS, whereas its abundance in the AD accounted for about 0.02%. Additionally, the abundance of gene encoded acetyl phosphotransferase (pta) accounted to 0.02% in the EFS-TS-bio and EFS-RS-bio while decreased to 0.01% in the AD (Fig. 7B). Furthermore, the genes involved in acetate oxidation had higher abundance in the EFS than those in the AD, suggesting that the capacity for acetate degradation was enhanced in the EFS.

The electrode biofilm and suspension hosted various pathways for nitrogen transformation (Fig. 7C). The abundance of genes encoding glutamate dehydrogenase (gdhA), hydroxylamine reductase (hcp), nitrogenase delta subunit (anfG), nitrogenase molybdenum-iron protein (nifDHK), and nitrite reductase (nrfAH), which catalyzed ammonium formation from L-glutamate, hydroxylamine, nitrogen, and nitrite, were higher in the EFS-TS-bio and EFS-RS-bio than in the AD, consistent with high ammonium recovery in EFS. The nifDHK and nrfAH genes showed 2.46 − 3.90-fold higher abundances in the anode biofilms than those of AD. On the other side, the abundance of genes encoding nitrite reductase (nirABD), which involved in the generation of ammonium from nitrite, was higher in the suspension vs the anode biofilm. In addition, the microorganism in suspension samples possessed more genes encoding ammonia monooxygenase (amoABC), hydroxylamine dehydrogenase (hao), periplasmic dissimilatory (napA), assimilatory nitrate reductase (nasA), membrane-bound nitrate reductase (narGH), nitrite reductase (nirKS), and nitrate/nitrite transporter (nrtP).

3.7. Abundant genes associated electron transfer in EFS

Genes related to electron transfer were abundantly enriched in electrode biofilm and suspension of EFS, but rarely detected in AD samples (Fig. 7B). Genes encoded cytochrome c peroxidase (macA) and cytochrome c-type biogenesis protein (ccdA) more enriched in electrode biofilm than in suspensions, which functioned in EET of Geobacter. The relative abundance of macA increased to 0.21 − 0.29%, while the abundance of ccdA increased to 0.25 − 0.28% in the electrode biofilm of EFS. The fixABCX associated electron transfer flavoprotein (ETF): quinone oxidoreductase complexes, pyrDII encoded dihydroorotate dehydrogenase electron transfer subunit, along with nuoA-N, rnfA-EG, and hdrA-E associated ETF-independent redox complexes were more abundant in the EFS-TS-bio and EFS-RS-bio than those in suspensions. In addition, the electron transport complex protein associated genes (rnfA-EG) also showed higher abundance in the suspentions of EFS compared with the AD. These results confirmed the promotion of EET pathways in EFS.

4.1. Sequential resource recovery and disposal of waste sludge

The recovery of energy and resources is an important valorization strategy for sludge management, including production of methane, H₂, VFAs, and nutrients ^{34, 35}. Numerous works have been conducted to extract proteins from waste sludge, while ammonium recovery from the waste is another bonus in BESs, but the high energy consumption and carbon emission associated with further utilization of these resources become the primary bottleneck ^{6, 36}. Therefore, an effective sequential resource recovery strategy is needed to fulfill sufficient sludge management while achieving full form nitrogen recovery. This study for the first time proposed an approach that combined sequential recovery of protein and ammonium with DS degradation in a combined process of nutrient recovery electro-fermentation (NREF). AT pretreatment yielded synchronous protein recovery of 72.23% and sludge disintegration. The recovered protein, as a renewable source, could be used as yeast extract and peptone substitute to culture microorganism like P. aeruginosa, which achieved nitrogen source recycling. In addition, AT pretreatment was reported to enhance the sludge biodegradability ³⁷. The following EFS showed more advantages for COD removal efficiency compared with AD. A maximum of 5,196.08 mg/g-VSS and 776.52 mg/g-VSS of TCOD were removed in EFS-TS and EFS-RS, respectively, which increased about 6.39% in the EFS-TS and 2.67-fold in EFS-RS than those of AD (Fig. 2B, Figure S4B).

The current of EFS was lower than that in microbial electrosynthesis because its main purpose was not to maximize the electron transfer efficiency by generating electricity, therefore with a low power supply could achieve higher ammonium recovery efficiency of 71.33 − 79.63% in the EFS-TS and 44.97 − 49.89% in the EFS-RS (Fig. 4, Figure S7) ¹⁶. These results were comparable with the 70% ammonium recovery efficiency obtained from iron-based primary sedimentation sludge ²⁸. As there was no nitrate and nitrite accumulated, ammonium was considered as the terminal form in all reactors (Figure S7). Then the recovered ammonium can be further used for fertilizer and food production ³⁸. In general, the AT pretreatment achieved more protein recovery while the cascading EFS accomplished efficient sludge treatment and ammonium recovery. The NREF highlights the unique potential for sequentially recovering nitrogen nutrients while also removing COD from sludge. Moreover, the resulting cathodic ammonium concentrate minimizes the chemical dosing for further extraction, making it more energy efficient than conventional biological nitrification-denitrification and NH₃ stripping methods ³⁹. The economic analysis revealed that the NREF process was cost effective and the net profit was estimated at 2.539 × 10^− 4 $/g DS (Table S5). Although, the reuse of recovered protein and ammonium can offset the cost of NREF operation and make it more feasible. To reduce energy consumption in the future, upgrading the scale-up system, optimizing electrode material, biocatalysts, and continuous flow mode configuration need further investigation.

4.2. EFS shaped microbiome and metabolic functions

Microbiome analysis showed that the majority of dominant populations in the anode biofilm of EFS was Geobacter, which is a representative EAB that capable of donating or accepting extracellularly electron transfer ⁴⁰. In addition, fermentative bacteria (FB) such as Rikenella, Lentimicrobium, and Petrimonas also prevailed in the suspension solution of EFS than in AD, implying that cross-feeding occurred between EAB and FB (Fig. 5D and 8) ^{41, 42}. This indicated that the syntrophic interaction of EAB and FB facilitated COD removal of EFS by improving intercellular and extracellular electron transfer ^{43, 44}. The genes associated with carbon metabolism and electron transfer were enriched abundantly in the EFS, which further supported this community interaction at molecular level (Fig. 8).

It is worth noting that through the catalyzed intracellular proteolysis into smaller peptides by degQ, gluP, ftsH, ctpB, and sppA encoding enzymes ^{45, 46}, and the boosted endo-hydrolyzing the α-1,4 or α-1,6 linkages in polysaccharides (e.g. starch and glycogen) to glucose by treS and gla1 encoding enzymes ⁴⁷, the EFS accelerated protein degradation into amino acids and polysaccharide hydrolysis into monosaccharide (Figs. 2 and 6). Then FB degraded glucose into VFAs as more genes involved in glycolysis pathway (hk, tal-pgi, pfkABC, fbaAB, gapor, and apgM) were identified in the EFS than AD, thereby facilitating COD degradation (Figs. 7 and 8).

It was found that the biofilm and suspension of EFS displayed different acetate oxidation pathways. The increased abundances of ackA and pta in the anode biofilms of EFS compared to AD indicated the activization of the phosphotransacetylase-acetate kinase (Pta-Ack) pathway, which was identified as a representative acetate degradation way in Geobacter (Fig. 8) ⁴⁸. 76.04% and 71.36% of annotated pta were affiliated to Geobacter in the EFS-TS-bio and EFS-RS-bio, respectively, while 81.69% and 75.20% of ackA were assigned to Geobacter in the EFS-TS-bio and EFS-RS-bio, respectively (Figure S13). Meanwhile, the suspension of EFS also showed the increased abundance of genes encoded enzymes that catalyze one-step conversion of acetate to acetyl-CoA (aarC and pct) ⁴⁹. Geobacter harbors these genes associated with acetate oxidation and was considered to contribute to the faster acetate consumption, thereby accelerating organic removal through underlying syntrophic associations with other acetogens (e.g. Lentimicrobium and Petrimonas) ^{50, 51}.

In addition, macA and ccdA encoded key enzymes that transport electron from the inner membrane to the outer membrane were enriched in the EFS. The majority of macA and ccdA were affiliated to Geobacter in the EFS-TS-bio and EFS-RS-bio (77.87 − 85.88%) (Figure S13) ⁵². These results suggest that Geobacter performed acetate utilization and EET metabolic pathway with electrons transporting through the TCA cycle to the quinone pool, then directly transferred to outer membrane by c-type cytochromes (macA and ccdA) (Fig. 8) ⁵³. The electron transfer between (i) quinone/quinol and NAD(H) (nuoA-N), (ii) H⁺ and NAD(H) and Fd (rnfA-EG), and (iii) Fd and unknown electron carriers (hdr) were enhanced in the EFS ^{54, 55}. Through these components, syntrophic partners employed intracellular electron transfer and EET, accordingly accelerating the electron transfer capability which may be a key for the faster COD degradation in EFS. Overall, these results have shed light on the mechanisms of how EFS accelerated the organic degradation from microorganism and molecular level, which highlights the importance of syntrophic interactions of EAB and FB and electron transfer during sludge treatment.

4.3. The mechanism of boosting nitrogen recovery in NREF

AT pretreatment promoted the disruption of sludge cells and caused effective release of protein, carbohydrates, and VFAs (Figure S1, 2, and 3). Therefore, more protein (107.61 mg/ g VSS) was recovered through AT pretreatment. Moreover, the higher current observed in the EFS-TS than that in the EFS-RS implied that more released acetate provided more soluble substrates for EAB growth (Figure S7D), which contributed to more ammonium recovery in cathode chamber of cascading EFS-TS. Furthermore, the enhanced EET capability in EFS facilitated the ammonium production pathway (Fig. 8). This is likely due to two reasons, firstly is attributed to dissimilatory nitrate reduction to ammonium (DNRA) by Geobacter. Geobacter was identified as composed of the DNRA pathway of nitrate to nitrite catalyzed by nitrate reductase followed by nitrite rapidly converted to ammonium through nitrite reductase complex (nrfA/H) ⁵⁶. The nitrate may come from the oxidation of ammonium to nitrate, catalyzed by ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO) from comammox bacterium Nitrospira (Fig. 7and S10) ^{57, 58}. The nrfA/H involved in DNRA were significantly enriched by 3.03-fold and 2.46-fold in the EFS-TS-bio and EFS-RS-bio vs the AD, respectively, implying that the electroactive biofilms enhanced DNRA pathway. This resulted in ammonium accumulation and then recovery at the cathode by electromigration. Another reason for higher ammonium concentration is the promotion of nitrogen fixing pathway. Geobacter, Hydrogenophaga, and Azonexus, which were reported to be nitrogen-fixing bacteria (NFB), were detected more abundant in the EFS-TS and/or EFS-RS than in the AD ^{59, 60}. Nitrogenases encoding genes (anfG and nifDHK) and fixABCX from NFB catalyzed the fixation of nitrogen to ammonium, which were enriched greatly in the EFS-TS-bio and EFS-RS-bio vs the AD (Fig. 8) ⁶¹. These genes were related to electron transfer to nitrogenases, suggesting that the nitrogen fixing pathway required a high electron driving force to maintain the redox balance ⁶². Therefore, it was assumed that the enhanced EET capability in EFS provided more electrons for NFB, thereby facilitating the conversion of nitrogen gas to ammonium. In this regard, EFS, as a technology for enhancing the sludge treatment, could also become a promising resource recovery approach for simultaneous ammonium recovery.

This study offers an innovative approach for improving sludge degradation and simultaneously recovering protein and ammonium recovery through alkaline-thermal (AT) pretreatment combined NREF. AT pretreatment achieved higher sludge disintegration and protein recovery (72.23%) compared to enzyme lysis. Compared with processing RS and TS in open-circuit reactors, the NREF enabled faster SCOD degradation and maximum TCOD removal of EFS-TS and EFS-RS reached 75.91% and 46.51%, respectively. These were attributed to the enhanced protein, polysaccharide, and acetate degradation. The two-chamber EFS separated by CEM accomplished 71 − 80% ammonium recovery in the cathode chamber of EFS-TS, while 45 − 50% in EFS-RS driven by the electrical flow. The 16S rDNA sequencing showed that EAB (Geobacter) was enriched in electrode biofilm of EFS, while the FB (Rikenella, Lentimicrobium and Petrimonas) were enriched in suspensions of EFS, indicating the facilitated microbial syntrophic interaction and interspecies EET. Metagenomics revealed that polysaccharides and protein metabolisms were enhanced in EFS. The facilitated glycolysis, acetate oxidation, and nitrogen conversion pathways through EET between FB, EAB, and NFB in EFS were constructed. These findings improve our understanding of the conversion metabolisms of syntrophic microorganism in NREF and highlight the feasibility for accomplishing resource recovery while synchronously removing COD from sludge in this integrated NREF.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (No. U22A20443), Heilongjiang Key Research and Development Program (2022ZX02C19), State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology) (No. 2022TS06), Interdisciplinary Research Foundation of HIT, and Heilongjiang Touyan Innovation Team Program (HIT-SE-02-02).

AUTHOR INFORMATION

Authors' contributions

JG designed the experiments and DX provided general concept for experiments. JG conducted all the experiments and analyzed data. HZ, JW, KF guided the experiment. JG wrote the initial manuscript, while DX, GX, BL revised the manuscript.

Corresponding Author

Correspondence to Defeng Xing.

ETHICS DECLARATIONS

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interest.

AVAILABILITY OF DATA AND MATERIALS

All raw data and sequencing information can be requested by contacting the corresponding author Defeng Xing ([email protected])

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Sequential recovery of protein and ammonium from waste sludge and functional metabolism in a combined process of nutrient recovery electro- fermentation (NREF)

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Abstract

Figures

1. BACKGROUND

2. METHODS

2.1. Sludge characteristics

2.2. Protein recovery and disintegration methods

2.3. Construction and operation of electro-fermentation reactors

2.4. Analyses and Calculations

2.5. Microbiome analysis based on 16S rRNA gene amplicon sequencing

2.6. Metagenomics analysis

3. RESULTS

3.1. Synchronous dewatered sludge disintegration and protein recovery

3.2. EFS facilitates degradation of organic matter

3.3. EFS enhanced ammonium recovery

3.4. EFS shaped microbiome of biofilm and suspension

3.5. Difference in functional gene enrichment between EFS and AD

3.6. EFS enriched genes involved in carbon and nitrogen transformation

3.7. Abundant genes associated electron transfer in EFS

4. DISCUSSION

4.1. Sequential resource recovery and disposal of waste sludge

4.2. EFS shaped microbiome and metabolic functions

4.3. The mechanism of boosting nitrogen recovery in NREF

5. CONCLUSION

Declarations

References

Additional Declarations

Supplementary Files

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