Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: Microcosm and microbial diversity studies

Journal Pre-proof Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: Microcosm and microbial diversity studies Wei-Han Lin, Ssu-Ching Chen, Chih-Ching Chien, Daniel C.W. Tsang, Kai-Hung Lo, Chih-Ming Kao PII:

S0013-9351(20)30189-4

DOI:

https://doi.org/10.1016/j.envres.2020.109296

Reference:

YENRS 109296

To appear in:

Environmental Research

Received Date: 28 November 2019 Revised Date:

19 January 2020

Accepted Date: 23 February 2020

Please cite this article as: Lin, W.-H., Chen, S.-C., Chien, C.-C., Tsang, D.C.W., Lo, K.-H., Kao, C.M., Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: Microcosm and microbial diversity studies, Environmental Research (2020), doi: https://doi.org/10.1016/ j.envres.2020.109296. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.

Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: Microcosm and microbial diversity studies

Wei-Han Lina, Ssu-Ching Chenb, Chih-Ching Chienc, Daniel C.W. Tsangd, Kai-Hung Loa, Chih-Ming Kaoa,*

a

Institute of Environmental Engineering, National Sun Yat-Sen University, Kaohsiung, Taiwan

b

c

Department of Life Sciences, National Central University, Chung-Li City, Taoyuan, Taiwan

Graduate School of Biotechnology and Bioengineering, Yuan Ze University, Chung-Li City, Taoyuan, Taiwan d

Department of Civil and Environmental Engineering, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China

*Corresponding author: Prof. C. M. Kao Address: Institute of Environmental Engineering National Sun Yat-Sen University Kaohsiung 80424 Taiwan E-mail: [email protected]

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ABSTRACT Hexavalent chromium (Cr6+) is a commonly found heavy metal at polluted groundwater sites. In this study, the effectiveness of Cr6+ bioreduction by the chromium-reducing bacteria was evaluated to remediate Cr6+-contaminated groundwater. Microcosms were constructed using indigenous microbial consortia from a Cr6+-contaminated aquifer as the inocula, and slow-releasing emulsified polycolloid-substrate (ES), cane molasses (CM), and nutrient broth (NB) as the primary substrates. The genes responsible for the bioreduction of Cr6+ and variations in bacterial diversity were evaluated using metagenomics assay. Complete Cr6+ reduction via the biological mechanism was observed within 80 days using CM as the carbon source under anaerobic processes with the increased trivalent chromium (Cr3+) concentrations. Cr6+ removal efficiencies were 83% and 59% in microcosms using ES and NB as the substrates, respectively. Increased bacterial communities associated with Cr6+ bioreduction was observed in microcosms treated with CM and ES. Decreased bacterial communities were observed in NB microcosms. Compared to ES, CM was more applicable by indigenous Cr6+ reduction bacteria and resulted in effective Cr6+ bioreduction, which was possibly due to the growth of Cr6+-reduction related bacteria including Sporolactobacillus, Clostridium, and Ensifer. While NB was applied for specific bacterial selection, it might not be appropriate for electron donor application. These results revealed that substrate addition had significant impact on microbial diversities, which affected Cr6+ bioreduction processes. Results are useful for designing a green and sustainable bioreduction system for Cr6+-polluted groundwater remediation.

Keywords: bioreduction, hexavalent chromium, groundwater contamination, metagenomics assay, green and sustainable remediation.

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1. Introduction Chromium (Cr) is a commonly found metal at polluted groundwater sites (Wang et al., 2015). Chromium has been applied by different industries (e.g., dye industry, leather tanning, stainless steel plants), and thus, improper wastewater discharges or illegal waste dumping could cause the release of chromium to the environment (Marinho et al., 2019). Chromium compounds exist in the form of chromium metal, hexavalent chromium [Cr6+ or Cr(VI)], and trivalent chromium [Cr3+ or Cr(III)]. Cr(III) is a nutrient and can help human body to utilize protein, fat, and sugar (Demir and Arisoy, 2007; Zayed and Terry, 2003). However, Cr(VI) can result in carcinogenicity and mutagenicity for humans (Bagchi et al., 2002). Thus, Cr(III) and Cr(VI) have contrasting properties (Francisco et al., 2002) and environmental remediation of Cr(VI) is needed. Most of the Cr(VI) is in the form of chromate, which is a toxic, carcinogenic, and water soluble chemical. When the subsurface environment is contaminated by Cr(VI) or chromate, it needs to be removed to prevent the migration of chromate to farther downgradient area and endanger the ecosystem and human health (Rajapaksha et al., 2018; Wan et al., 2019; Zhong et al., 2018). The most applicable and cost-effective remediation goal is to reduce Cr(VI) to less toxic Cr(III), which usually exists in the subsurface as precipitates (Guertin, 2004; Han et al., 2010). Different remedial technologies (e.g., adsorption, bioreduction, electrolysis, membrane separation, chemical precipitation) have been applied for chromate removal for chromate-contaminated groundwater remediation (Barrera-Díaz et al., 2012; Jobby et al., 2018). Among these developed technologies, attention has been given to bioremediation (e.g., bioaccumulation, bioreduction, biosorption) because of its low operational and maintenance costs and environmental friendliness (Anastopoulos et al., 2017; Duan et al., 2017; Gu et al., 2019; Kongjan et al., 2019; Marinho et al., 2019; Martín-Domínguez et al., 2018). Although biosorption and bioaccumulation using specific microorganisms or plants

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could result in Cr(VI) removal, the following microbial separation and plant harvesting/treatment would cause operation, maintenance, and final disposal problems (Samuel et al., 2012). Application of the biostimulation method for in situ remediation of polluted groundwater sites has been considered to be a feasible remedial method because it is a costly and environmental friendly option (Adams et al., 2015). Thus, application of in situ biostimulation to enhance the bioreduction mechanism would be a potential treatment process to cleanup Cr(VI) (or chromate) polluted groundwater (Das et al., 2014; Samuel et al., 2012; Satarupa and Paul, 2013). Cr(VI) reduction can occur under either abiotic or biotic mechanisms. Primary carbon substrates can be supplied as the electron donors and injected into the Cr(VI)-contained groundwater to enhance the bioreduction of Cr(VI) to less toxic Cr(III) (Wen et al., 2017). Certain indigenous bacterial species are able to enzymatically reduce Cr(VI) to Cr(III) under different oxidation-reduction processes. Cr(VI) reduction can also occur chemically through natural reduction processes using Fe(II) or H2S (produced via anaerobic biodegradation processes) as the reducing agents (Fan et al., 2019; Mosher et al., 2012). Chromium-reducing bacteria (CRB) (Somasundaram et al., 2009) can be isolated from chromium-contained

wastewaters

(e.g.,

wastewaters

from

tannery,

electroplating

manufacturing, and textile industries), chromite mines (Dey and Paul, 2012; Dey and Paul, 2016; Irazusta et al., 2018; Wu et al., 2017), and soils contaminated with chromium (Joutey et al., 2016; Maqbool et al., 2015) for Cr(VI) reduction. Bacteria have the capabilities for Cr(VI) reduction including strains of Shewanella, Bacillus, Enterobacter, and Pseudomonas (Adams et al., 2015; DeFilippi, 2018; Xia et al., 2019). Under anaerobic conditions, Cr(VI) could be bioreduced to Cr(III) by CRB interceded via membrane-linked or dissolvable enzymes (Xia et al., 2019). Cytochromes b and c are responsible for the enzymatic enhanced electron transport respiratory pathway during Cr(VI) bioreduction within cells (Han et al., 2016;

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Pradhan et al., 2017). Bioreduction of Cr(VI) can also proceed under aerobic conditions (Pradhan et al., 2017). From an engineering point of view, it is more feasible and practical to enhance Cr(VI) under anaerobic conditions due to the lower operational and maintenance cost (Huang et al., 2016). Next generation sequencing (NGS) offers an useful technique for sequencing with high-throughput of target genomes and genes, which offer information to construct desirable microbial community for bioremediation of polluted sites (Gholami et al., 2019; Mustafa et al., 2018). Metagenomics can be applied for the construction of microbial diversity catalogue (Hao and Chen, 2012; Jobby et al., 2018). Different NGS platforms have been developed for the analyses of metagenomic datasets for microbial diversities (Johny et al., 2018). For environmental samples, Illumina and Roche 454 are two NGS platforms used in metagenomic analyses (Bokulich et al., 2013; Jobby et al., 2018; Werner et al., 2012). Comparable to full length 16S rDNA sequence, Illumina reads can offer accurate taxonomic information (Hao and Chen, 2012; Jobby et al., 2018). Researchers evaluated the application of soluble substrates for Cr(VI) bioreduction (Chen et al., 2015). However, frequent supplement of soluble substrates (e.g., maltose, sodium acetate, fructose, lactose) is required to maintain a higher carbon concentrations in the subsurface because they are more biodegradable and easily flushed to farther downgradient areas by groundwater flow (Molins et al., 2015). To minimize the expense of primary substrate supplements, slow-releasing and emulsified polycolloid-substrate (ES) can be explored as the substrates and electron donors (Lien et al., 2016). In our previous study, slow-releasing ES was produced and injected into the contaminated groundwater to remove chlorinated ethylenes via the reductive dechlorination (Lien et al., 2016; Sheu et al., 2016). The main components of slow-releasing ES are cane molasses (CM) (for the early-stage carbon supplement), vegetable oil (for the second stage

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carbon supplement), soya lecithin (Long Island Medicines, Japan), surfactant (Simple GreenTM) (Sunshine Makers, USA), and vitamins (Kuo et al., 2014; Lien et al., 2016; Sheu et al., 2016). The goals of this microcosm and bacterial diversity study were to (1) assess the effectiveness of using CM, ES, and NB (as the electron donor) for the bioreduction of Cr6+ (as the electron acceptor) to less toxic Cr3+ in a microcosm study using indigenous microbial consortia as the inocula, and (2) apply metagenomics assay to assess the diversification in microbial community and dominant bacterial species during Cr6+ bioreduction. In this study, the effectiveness of Cr6+ bioreduction by the chromium-reducing bacteria was evaluated. Complete Cr6+ reduction via the biological mechanism was observed using cane molasses and emulsified substrate as the carbon sources under anaerobic processes with the increased trivalent chromium production. Part of the produced Cr3+ was precipitated onto the soil particles after the bioreduction process. Substrates addition caused the increased bacterial communities for Cr6+ bioreduction. Sporolactobacillus, Clostridium, and Ensifer were the dominant species caused for Cr6+ bioreduction under anaerobic conditions. Results from this study would be helpful in understanding the main mechanisms causing Cr6+ removal under anaerobic bioreduction processes. Moreover, the dominant bacterial species and the optimal operational conditions of the Cr6+ bioreduction process could be determined. Results could be applied to design an in situ Cr6+ bioreduction process to cleanup the Cr6+-contaminated groundwater.

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2. Materials and methods 2.1 Microcosm experiment An electroplating factory site located in the southern Taiwan area was contaminated by Cr6+ due to the leakage of the Cr6+-contained wastewater into the subsurface. The Cr6+ concentrations in site groundwater varied from 14 to 19 mg/L. Soils and groundwater samples were collected from the electroplating factory site for the microcosm experiments. The collected soil and water samples were kept on ice in a cooler before they were transported to the laboratory for analyses. In the laboratory, all samples were kept refrigerated at 4 °C until analyzed (APHA, 2005). The characteristics of collected groundwater and soils are shown in Tables 1 and 2. The effectiveness of Cr6+ bioreduction under anaerobic conditions was evaluated in the microcosm experiments, and aquifer sediments were used as the inocula in microcosms. The collected soils and groundwater were sparged before use. CM, ES, and nutrient broth (NB) were used as the primary substrates. The ES was prepared following the procedures described in Kuo et al. (2014). In this study, five groups of microcosms were constructed. Table 3 presents the components of each group of anaerobic microcosm. The five groups of microcosms included Group A (intrinsic bioreduction or live control) (LC group), Group B (killed control) (KC group), Group C (NB addition) (NB group), Group D (CM addition) (CM group), and Group E (ES addition) (ES group). Each microcosm was prepared using 50 mL site groundwater containing Cr6+, amendments (substrates or groundwater), and soils (15 g) in a serum bottle (120 mL) sealed with a Teflon-lined rubber septa. NB (Difco 003-01, USA), CM (Taiwan Sugar Co., Taiwan), and ES served as the carbon sources for Cr6+ bioreduction. KC bottles contained 500 mg/L NaN3 and 250 mg/L HgCl2 for bacterial growth control. At each time point, triplicate microcosms were sacrificed for analyses. In substrate addition microcosms (Groups C, D, and E), 0.5 mL of substrate 7

solution (containing either NB, CM, or ES) was added in each microcosm bottle, and the initial total organic carbon (TOC) concentrations were in the range from 385 to 432 mg/L in Groups C to E microcosms. In LC and KC microcosms (Groups A and B), the initial TOC concentrations were around 18 mg/L. This could be due to leakage of wastewater into the subsurface. In each microcosm bottle, the initial Cr6+ concentration was about 39 mg/L. In KC microcosm, groundwater and soils used were further autoclaved. N2 gas was filled in the headspace of the microcosms.

2.2 Water and soil sample analyses Soil samples from the microcosms were used for bacterial diversity analyses. Water samples were analyzed for Cr6+, Cr3+, TOC, and geochemical indicators including pH, sulfate, sulfide, ferrous iron, nitrate, and methane. The ICP-AES (inductively coupled plasma atomic emission spectrometer) (The Optima 7000 DV, PerkinElmer, USA) was applied for the composition of the components and the total Cr analyses. The ESEM/EDS (Environmental Scanning Electron Microscope/Energy Dispersive Spectrometer) (FEI Quanta 200, FEI, USA) and XRD (X-ray diffractometer) (Siemens D5000, KS Analytical Systems, USA) were applied to perform surface analysis of metal particles to understand the surface composition and metal structure of the surface deposited metal. The Cr6+ and Cr3+ analyses were performed following the procedures described in Huang et al. (2014 and 2017). The Cr6+ concentration was determined colorimetrically at 540 nm using the diphenylcarbazide method with a UV–vis spectrophotometer (Model DR 6000, Hach Co., USA) (Huang et al., 2014 and 2017). The concentration of total Cr was analyzed by ICP-AES. The concentration of Cr3+ was obtained by subtracting the Cr6+ concentration from the total Cr concentration.

2.3 Illumina MiSeq sample preparation and data analyses

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The total genomic DNA in microcosm soils was extracted following the procedures described in Langmead and Salzberg (2012) and Bolger et al. (2014). In this study, the sample preparation, library construction, and DNA sequencing were performed by Welgene Biotech Co., Ltd. (Taiwan). The Illumina MiSeq data analyses were performed following the processes in (Magoč and Salzberg, 2011; Schloss et al., 2009). Taxonomy assignment of Operational Taxonomic Units (OTUs) sequences was conducted via mothur with the SILVA database v123 (Yilmaz et al., 2013). Alpha diversity indices (Richness, Good’s coverage, ACE, Chao1, Shannon diversity, and Simpson evenness) were calculated using mothur. Rarefaction curves and species accumulation curves were calculated using the VEGAN R package, as was the Beta diversity indices (PCoA) (Dixon, 2003). OTU analysis and annotation was performed by Welgene Biotech Co., Ltd. (Taiwan).

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3. Results and discussion 3.1 Microcosm experiment The changes of Cr6+ concentrations in microcosms during the 80-day experiment are presented in Fig. 1. Figures 2a and 2b illustrate the variations in Cr3+ and total Cr (Cr6+ + Cr3+) concentrations in different microcosms during the 80-day experiment. Compared to Groups A and B (control), Groups C, D, and E microcosms with the supplements of NS, CM, and ES, respectively, had higher Cr6+ removal efficiencies. Results indicate that all three carbon sources could serve as the carbon sources for the enhancement of Cr6+ reduction via biological processes. Tables 4a and 4b present the averages of analytical results for the five groups of microcosms after 10 and 70 days of the experiments, respectively. Substrate biodegradation caused the complete depletion of DO and nitrate in Groups C to E microcosms. This reveals that the anaerobic bioreduction could be the possible process caused Cr6+ removal.

3.1.1 Variations in Cr concentrations Table 5 shows the Cr removal efficiencies and variations in Cr3+ in five groups of microcosms. In live control microcosms (Group A), slight Cr6+ removal was observed. The intrinsic bioreduction process could be the cause of Cr6+ removal. Moreover, in killed control microcosms (Group B), a slight drop of Cr6+ was detected with some Cr3+ production, i.e., abiotic reduction might occur in this group of microcosms. Because Fe2+ was detected in site groundwater, it could serve as e- donor for Cr6+ reduction under abiotic conditions (Němeček et al., 2015). The TOC in natural groundwater was approximately 18 mg/L, thus, it could be consumed as the carbon source by intrinsic bacteria. Therefore, intrinsic bioreduction mechanism could be the cause of slight Cr6+ removal in microcosms (Group A). However, intrinsic Cr6+ bioreduction could not be applied for engineered remediation as the in situ

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treatment process is slow and limited due to the low intrinsic organic carbons in the natural system. Increased Cr3+ concentrations in Groups C to E microcosms indicate that Cr6+ was reduced to Cr3+ under anaerobic conditions. However, the decrease in total Cr (Cr6+ + Cr3+) concentrations was also observed during the incubation period. This implies that part of the produced Cr3+ was precipitated onto the soil particles after the bioreduction, which accounted for the reduction of total Cr in dissolved phase. Significant drops of Cr6+ concentrations in microcosms (Groups D and E) were detected after 60 and 80 days of operation, respectively, with the observed increase in Cr3+ concentrations during the operational processes. Up to 78% of Cr6+ was removed after 80 days of incubation in Group C microcosms using NB as the substrate. However, Cr6+ removal efficiencies were approximately 2% and 8% in Groups A and B microcosms after 60 and 80 days of operation, respectively. Results indicate that CM was more applicable and more biodegradable by indigenous bacterial consortia including Cr6+ reduction bacteria. This would result in a more effective Cr6+ bioreduction. Compared to Group D with CM supplement, slightly lower Cr6+ removal was detected in microcosms with ES supplement. This was because that the ES mainly contained vegetable oil, which was less biodegradable compared to CM. However, ES is more applicable for field applications because ES can be used for a long-term source of substrate release (Kuo et al., 2014; Sheu et al., 2016). Thus, the operational and maintenance cost can be reduced for the control of Cr6+ plume. Because NB was mainly used for bacterial selection for specific functions, it might not be effectively used for the electron donor supplement. Thus, relatively lower Cr6+ reduction efficiency was obtained. The adsorption mechanism might be the cause of a slight Cr6+ removal in Group B microcosms (killed control) because Cr3+ was not detected in the water phase. The observed decrease in Cr6+ concentrations in LC microcosms was attributed to the occurrence of both adsorption and

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intrinsic bioreduction mechanism (using natural TOC as the substrate) because slight Cr3+ was observed.

3.1.2 Analyses water quality indicators Variations in TOC concentrations in different microcosms are shown in Fig. 3. The initial TOC concentrations in different microcosms were 17.9 (Group A), 18.1 (Group B), 436 (Group C), 415 (Group D), and 389 (Group E) mg/L. Results show that TOC concentrations in five Groups (A to E) dropped to 14.2, 18, 298, 219, and 286 mg/L after 10 days, respectively. TOC concentrations in five Groups (A to E) further dropped to 7.1, 17.9, 69, 58, and 134 mg/L after 70 days of operation, respectively. Results indicate that an obvious drop of TOC concentrations was detected in Groups C to E with NB, CM, and ES addition, respectively. This indicates that all three substrates were biodegradable and could serve as the primary substrates for bacterial consortia. Higher TOC decay rate was observed in Group D with CM addition. Results suggest that CM was more biodegradable than NB and ES, which resulted in the higher TOC decay rate during the incubation period. Because ES contained less biodegradable carbon sources (vegetable oil), relatively higher TOC concentrations were detected in Group E with ES supplement after 80 days of operation. This could be beneficial to a long-term control of Cr6+ release. Results from Table 4 show that a drop of DO, ORP, and a continuous pH decline were observed in microcosms with substrate addition (Groups C to E). The pH values dropped to 6.3, 6.2, and 6.5 and 5.9, 5.7, and 6.1, respectively, after 10 and 70 days of incubation in Groups C, D, and E microcosms, respectively. The results indicate that the biodegradation of NB, CM, and ES resulted in a pH drop (acidification) in the microcosm solution due to the production of fatty acids. This also confirms that the supplied substrates accelerated the anaerobic biodegradation mechanisms and caused the drops of TOC, DO, and pH. Results

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from Table 4 also show that a drop of sulfate and nitrate concentrations was detected, and ferrous iron (Fe2+), sulfide, and methane concentrations were also increased in microcosms with substrate addition (Groups C to E). This demonstrates that the intrinsic bacterial consortia could apply nitrate, sulfate, ferric iron, and CO2 as electron donors and proceeded denitrifying, iron-reducing, sulfate-reducing, and methanogenic processes, respectively. These mechanisms played important roles in the substrate biodegradation after DO consumption. Because CM contained a larger amount of easily biodegradable components, higher substrate biodegradation activities were observed with higher methane production. Thus, complete Cr6+ removal via the anaerobic bioreduction could be obtained. The decreased TOC concentrations in microcosms with substrate addition (Groups C, D, and E) corresponded with the increased concentrations of CO2, Fe2+, sulfide, and CH4 and decreased concentrations of DO, nitrate, and sulfate (electron donors) (Tables 4a and 4b). Results suggest that methanogenesis became the dominant biodegradation process, and thus, anaerobic Cr6+ bioreduction could be enhanced.

3.1.3 SEM and EDS analyses Figures 4a and 4b present the SEM image and EDS analyses for the soil particles collected from the Group D microcosms after 70 days of incubation. SEM analyses reveal that a bacterial population was increased and the soil bacteria became rough along with surface depression in Cr6+-containing environment. Because a higher Cr6+ bioreduction efficiency was observed in Group C microcosms, more Cr precipitation and bacteria could accumulate in microcosms in this group. Thus, two SEM images with the same magnification at different locations were selected for presentation. Results show that bacterial morphons contained rod and spherical-shaped bacteria, which might be the dominant Cr6+ bioreduction

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bacteria (Husain et al., 2019; Ziadi et al., 2020). In Fig. 4a, the rod and spherical-shaped bacteria are circled and shown in (b) and (c) images, respectively. The weight and area percentages of Cr in Group D soil samples collected after 10 days incubation were 25% and 29%, respectively. The percentages of weight and area increased to 49% and 54%, respectively, after 70 days of incubation. This indicates that the soil surface and metal structure were mainly chromium, which was coated on the surface of soil particle. The EDS results show that Ni, Cu, and Cr were three main components in the precipitates, and the increased Cr on the soil surface was due to the formation of Cr3+ precipitates after Cr6+ reduction (Kumari et al., 2016; Sengupta et al., 2017). The chromium precipitates might be associated with the presence of Cr3+ species or complexation of Cr3+ species with cell surface molecules.

3.2 The responses of bacterial community Metagenome was used to assess variations in bacterial diversity in microcosms with different substrate amendments during Cr6+ bioreduction (Kao et al., 2016; Wang et al., 2007). Supplementary Table S1 shows the OTU richness, coverage and diversity richness index of the 16S rRNA gene obtained from metagenomic sequencing. Supplementary Fig. S1 shows the rarefaction curves of the bacterial 16S rRNA gene metagenomics data sets from soils at 97% similarity. DNA sequencing from soil samples generated a total of 52,341 to 66,518 high quality reads that clustered into 138 to 738 OTUs in four groups (A, C, D, and E) of microcosms (Supplementary Table S1). Rarefaction Curve (Supplementary Fig. S1) and Good’s coverage values (>99%) (Supplementary Table S1) reveal that the bacterial communities had fair sampling activities. Alpha diversity is the measure of diversity within a sample using different estimators such as Shannon’s richness and Simpson’s evenness, which demonstrate

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that soil sample from Group D had the least species richness and evenness compared to the other samples (Supplementary Table S1). Taxonomic profiling data provided information about the dominant bacterial communities, which could bioreduce or tolerate the target contaminant (Cr6+). Figure 5 presents the bacterial diversity in Group A, C, D and E microcosms after 80 days of operation. There was a major difference between NB, CM, and ES microcosms on day 80 due to various supplied substrates. The top 40 most abundant taxa (combined across samples) were selected, and then heatmaps were constructed. Figure 6 presents the heatmaps of soil samples collected from four Groups of microcosms (A, C, D, E) for bacterial species at genus level. The bacterial communities in microcosms receiving NB displayed less bacterial community, whereas microcosms amended with ES had higher bacterial community diversity among these treatments (Fig. 5). Compared to Group E microcosms, Group D with CM addition had less microbial diversity. However, an increase in the percentages of Cr6+ bioreduction bacteria was observed. This would be beneficial to the Cr6+ reduction efficiency. Results imply that ES caused an encyclopedic effect on bacterial communities. This could be due to the fact that the ES contained different components including soybean oil, two surfactants (Simple GreenTM and lecithin), and lactate. These components had different biodegradable characteristics, and thus, the complexity of the ES had positive effects on bacterial diversity. In Group C, NB supplement caused the increased genera of Bacillus, Acinetobacter, and Delftia. Delftia sp. JD2 had been reported as a chromium-reducing bacterium (Wang et al., 2015). Researchers also reported that Bacillus cereus had the capability for Cr6+ bioreduction (Kumari et al., 2016). Acinetobacter lwoffii was found in chromate-contained wastewater and sludge, and it had the potential for Cr6+ bioreduction (Thatheyus and Ramya, 2016). In Group D, supplement of CM caused an increase in some bacterial species related to

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Cr6+ bioreduction including Delftia, Cupriavidus, Pseudomonas and Ensifer (Han et al., 2019; Oves et al., 2017; Suja et al., 2018). After 80 days of incubation, the percentages of Sporolactobacillus, Clostridium sp., and Ensifer increased from 0%, 0% and 0% on day 0 to 86%, 8%, and 0.35%, respectively, indicating that that these three bacterial genera played important roles in Cr6+ bioreduction after CM addition. Sporolactobacillus could utilize carbohydrates to produce D-lactic acid (De la Torre et al., 2019). It has been recognized that D-lactic acid could be used as a substrate for the growth of Ensifer, which was capable of Cr6+ bioreduction (Poehlein et al., 2017). Clostridium sp. could also utilize D-lactic acid to produce hydrogen, which could be used by CRB to enhance Cr6+ bioreduction (Kongjan et al., 2019). Thus, the Cr6+ bioreduction efficiency could be enhanced in Group D microcosms. In Group E, the addition of ES caused the increase in Cupriavidus, Caulobacter, Bacteroides, Acidovorax, Serratia, Pseudomonas, Ensifer, Stenotrophomonas, Sphingomonas, Cellulomonas, Enterobacter, Methylotenera, and Eubacterium genera. Except for Bacteroides and Methylotenera which had less Cr6+ bioreduction capabilities, the other bacterial genera were able to tolerate Cr6+ and reduce Cr6+ under biological processes (Lu et al., 2018). After incubation, the percentages of Cupriavidus, Serratia, Stenotrophomonas, Cellulomonas, Enterobacter, and Pseudomonas increased from 0%, 0%, 0%, 0%, 0%, and 0% on day 0 to 18%, 3.64%, 2.81%, 1.71%, 1.04% and 10% on day 80, respectively, indicating that more Cr6+ could be reduced after ES addition (Baldiris et al., 2018; Cason et al., 2017; He et al., 2018; He et al., 2019; Huang et al., 2019; Sun et al., 2019; Yung et al., 2014). The increased diversity and richness of Cr6+ bioreduction bacteria would result in Cr6+ removal in microcosms with ES addition. Figures 7 presents the variations in correlation analyses using PCoA Plot. Results show that a more significant divergence between the bacterial communities in Group E and Groups C/D was observed. The addition of substrates resulted in significant divergence between the

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bacterial communities in Group A and Groups C to E. However, the difference between the bacterial communities in Group C and Group D was not obvious. This might be due to the fact that both NB and CM were water-soluble substrates, and ES (in Group E) was oil-based substrate, which formed emulsified oil globules in the solution. The variations in the substrate characteristics resulted in the changes of the bacterial communities after incubation. Overall, the metagenomics analyses could provide us significant information for remedial strategies selection to enhance the Cr6+ bioreduction process.

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4. Conclusions In this study, the effectiveness of Cr6+ reduction by the chromium-reducing bacteria was evaluated to remediate Cr6+-contaminated groundwater under different treatment conditions. Microcosms

were

constructed

using

indigenous

microbial

consortia

from

a

Cr6+-contaminated aquifer as the inocula and NB, CM, and ES as the primary substrates. Conclusions of this study include the following: (1) In microcosms with substrates addition, complete consumption of DO and nitrate as well as the production of sulfide, ferrous iron, and methane were observed. Moreover, no significant Cr6+ removal was observed in killed control microcosms. This indicates that the bioreduction process could be the major mechanism causing the Cr6+ removal under anaerobic conditions. (2) Increased Cr3+ concentrations in microcosms with substrate addition. This reveals that Cr6+ was reduced to Cr3+ under anaerobic conditions. However, decreased total Cr (Cr6+ + Cr3+) concentrations were also observed during the incubation period. This indicates that part of the produced Cr3+ was precipitated onto the soil particles after the bioreduction process, which resulted in the reduced total Cr concentrations in the water phase. (3) Complete Cr6+ reduction via the biological mechanism was observed within 80 days under anaerobic conditions with the increased Cr3+ concentrations during the operational processes. Cr6+ removal efficiencies were 83% and 59% in microcosms using ES and NB as the substrates, respectively. CM was more applicable and more biodegradable by indigenous bacterial consortia including Cr6+ reduction bacteria. This would result in a more effective Cr6+ bioreduction. ES mainly contained vegetable oil, which was less biodegradable compared to CM. Thus, slightly lower Cr6+ removal efficiency was observed in microcosms with ES addition. NB was mainly used for bacterial selection for specific functions, and thus, relatively lower Cr6+ reduction efficiency was obtained.

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(4) Increased bacterial communities associated with Cr6+ bioreduction was observed in microcosms treated with CM and ES addition. However, decreased bacterial communities was observed in NB microcosms. Results indicate that CM was more applicable by indigenous Cr6+ reduction bacteria, which resulted in effective Cr6+ bioreduction possibly due to the growth of Cr6+-reduction related bacteria including Sporolactobacillus, Clostridium, and Ensifer. NB was appropriate for specific bacterial selection, and thus, it might not be appropriate for electron donor application. (5) The metagenomics analyses provide us significant information for dominant bacterial species and bacterial community dynamics of Cr6+ bioreduction. Results would be helpful in designing an in situ, green, and sustainable Cr6+ bioreduction process to cleanup the Cr6+-contaminated groundwater.

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Table Captions Table 1. Characteristics of the groundwater sample collected from the studied site. Table 2. Characteristics of the soil sample collected from the studied site. Table 3. Components of each group of anaerobic microcosm. Table 4. (a) Averages of analytical results for the five groups of microcosms after 10 days of the experiment (on day 10). (b) Averages of analytical results for the five groups of microcosms after 65 days of the experiment (on day 70). Table 5. Cr6+ removal efficiencies and variations in Cr3+ in five groups of microcosms.

27

Table 1. Characteristics of the groundwater sample collected from the studied site. Parameters pH

Value 6.8±0.2

DO (mg/L)

0.1±0.1

TOC (mg/L)

18.3±2 -

Nitrate (mg NO 3 /L)

0.3±0.2

3-

0.9±0.1

Sulfate (mg SO 4 /L)

4.3±1.6

Ferrous iron (mg Fe2+/L)

3.7±1.6

Phosphate (mg PO 4 /L) 2-

28

Table 2. Characteristics of the soil sample collected from the studied site. Parameters pH Total organic content (%) Cation exchange capacity (meq per 100 g) Iron (Fe) (mg/kg) Soil texture

29

Value 6.7±0.2 0.6±0.1 3.9±1.2 22.5±3.6 sandy loam

Table 3. Components of each group of anaerobic microcosm. Microcosm Anaerobic A Live control (LC) Anaerobic B Killed control (KC) Anaerobic C NB addition (NB) Anaerobic D CM addition (CM) Anaerobic E ES addition (ES)

Inocula Groundwater + soils Groundwater + soils Groundwater + soils Groundwater + soils

Components 15 g soils + 50.5 mL Cr6+-contained groundwater 15 g soils + 50.5 mL Cr6+-contained groundwater + 250 mg/L HgCl2 + 500 mg/L NaN3 15 g soils + 50 mL Cr6+-contained groundwater + 0.5 mL NB solution 15 g soils + 50 mL groundwater + 0.5 mL CM solution

Groundwater + soils

15 g soils + 50 mL Cr6+-contained groundwater + 0.5 mL ES solution

30

Table 4a. Averages of analytical results for the five groups of microcosms after 10 days of the experiment (day 10). DO mg/L

ORP sulfate sulfide mv mg/L mg/L

Group A Group B Group C Group D

6.8 6.8 6.3 6.2

0.1 0.1 <0.1 <0.1

-14 -23 -102 -146

2.8 4.4 3 1

0.6 <0.1 3 5

2.4 1.7 7.4 9.8

2.3 1.5 7.2 9.7

14.2 18.0 272 219

0.1 <0.1 1.3 2.5

Group E

6.5

<0.1

-101

5

2

4.4

4.1

305

1.9

Microcosm

Total Fe mg/L

Fe2+ TOC CH4 mg/L mg/L mg/L

pH

(Analytical results on day 0 before the experiment: pH = 7.2, DO = 1.8 mg/L, ORP = 87 mv, sulfate = 19.2 mg/L, sulfide < 0.1 mg/L, total Fe = 0.1 mg/L, Fe2+ < 0.1 mg/L, CH4 < 0.1 mg/L; TOC in Groups A, B, C, D, and E microcosms were 18.1, 17.8, 436, 415, and 389 mg/L, respectively.)

Table 4b. Averages of analytical results for the five groups of microcosms after 70 days of the experiment (on day 70). DO mg/L

Group A Group B

6.7 6.8

0.1 0.1

-32 -28

2.2 4.1

0.9 <0.1

2.6 1.8

2.4 1.7

7.1 17.9

0.2 <0.1

Group C Group D Group E

5.9 5.7 6.1

<0.1 <0.1 <0.1

-126 -193 -165

1.4 1.2 3.1

4.2 6.5 3.9

11.9 13.4 11.7

11.7 13.4 11.5

69 58 134

3.7 6.1 4.9

Microcosm

ORP sulfate sulfide mv mg/L mg/L

31

Total Fe mg/L

Fe2+ TOC CH4 mg/L mg/L mg/L

pH

Table 5. Cr6+ removal efficiencies and variations in Cr3+ in five groups of microcosms. Cr3+ (0

Cr6+ (0

Total Cr

Cr3+ (80

Cr6+ (80

Total Cr Reduction

day) (mg/L)

day) (mg/L)

(0-day) (mg/L)

day) (mg/L)

day) (mg/L)

(80 day) (mg/L)

Cr6+ rate (%)

A

1.3

40

40

4

34.4

38.4

14

B C D E

0.2 0.9 0 6.1

40 40 40 40

40 40 40 40

0.2 3.6 0.8 3.1

38.4 8.4 0 0

38.6 12 0.8 3.1

4 79 99.9 99.9

32

Figure Captions Figure 1. Variations in Cr6+ concentrations in five groups of microcosms during the 80-day incubation period. Figure 2. Variations in (a) Cr3+ and (b) total Cr concentrations in five groups of microcosms during the 80-day incubation period. Figure 3. Variations in TOC concentrations in five groups of microcosms during the 80-day incubation period. Figure 4a. SEM image for the soil samples collected from the Group D microcosms after 70 days of the microcosm experiment (a) 200X, (b) 20,000X, and (c) 20,000X. (The rod and spherical-shaped bacteria are circled and shown in (b) and (c) images, respectively.) Figure 4b. Results of EDS analyses for the soil particles collected from the Group D microcosms after (a) 10 and (b) 70 days of the microcosm experiment. Figure 5. Bacterial diversity in Group A, C, D and E microcosms after 80 days of incubation. Figure 6. Heatmaps of soil samples collected from Groups A, C, D, E microcosms for bacterial species at genus level. Figure 7. Variations in correlation analyses using PCoA Plot (Group A D

, Group E

).

33

, Group C

, Group

Cr6+ Concentration (mg/L)

40

30

20

A (LC) B (KC) C (NB) D (CM) E (ES)

10

0 0

20

40

60

80

Time (Day)

Figure 1. Variations in Cr6+ concentrations in five groups of microcosms during the 80-day incubation period.

34

(a)

10 A (LC) B (KC) C (NB) D (CM) E (ES)

Cr3+ Concentration (mg/L)

8

6

4

2

0 0

20

40

60

80

Time (Day)

(b)

Total Cr Concentration (mg/L)

40

30

20

A (LC) B (KC) C (NB) D (CM) E (ES)

10

0 0

20

40

60

80

Time (Day)

Figure 2. Variations in (a) Cr3+ and (b) total Cr concentrations in five groups of microcosms during the 80-day incubation period.

35

TOC concentration (mg/L)

500 Group A Group D

400

Group C Group E

300 200 100 0 0

10

20

30

40

50

60

70

80

Day

Figure 3. Variations in TOC concentrations in microcosms during the 80-day incubation period.

36

Figure 4a. SEM image for the soil samples collected from the Group D microcosms after 70 days of the microcosm experiment (a) 200X, (b) 20,000X, and (c) 20,000X. (The rod and spherical-shaped bacteria are circled and shown in (b) and (c) images, respectively.)

Figure 4b. Results of EDS analyses for the soil particles collected from the Group D microcosms after (a) 10 and (b) 70 days of the microcosm experiment.

37

Relative abandance (%)

100 80 60 40 20 0

a

d

c

e

Group

Cupriavidus Caulobacter Bacteroides Others Acidovorax Prevotella_9 Serratia Pseudomonas Ensifer Stenotrophomonas Lachnospiraceae_NK4A136_group Escherichia-Shigella Sphingomonas Cellulomonas Enterobacter Clostridium_sensu_stricto_1 Ruminococcus_1 Sporolactobacillus Acinetobacter Bacillus Ruminiclostridium_9 Staphylococcus Ruminococcaceae_UCG-010 Delftia Clostridium_sensu_stricto_12 Clostridium_sensu_stricto_11 Methylotenera

Figure 5. Microbial diversity in Group A, C, D and E microcosms after 80 days of incubation.

38

Figure 6. Heatmaps of soil samples collected from Groups A, C, D, E microcosms for bacterial species at genus level.

39

Figure 7. Variations in correlation analyses using PCoA Plot (Group A D

, Group E

).

40

, Group C

, Group

Highlights Title: Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: microcosm and microbial diversity studies




Bioreduction was the main mechanism causing Cr6+ removal under anaerobic conditions.




Produced Cr3+ was precipitated onto the soil particles after the bioreduction process.




Substrate addition caused the increased bacterial communities for Cr6+ bioreduction.




Sporolactobacillus, Clostridium, and Ensifer were species caused Cr6+ bioreduction.

1

Novelty Statement

Title: Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: microcosm and microbial diversity studies

In this study, the effectiveness of Cr6+ bioreduction by the chromium-reducing bacteria was evaluated. Complete Cr6+ reduction via the biological mechanism was observed using cane molasses and emulsified substrate as the carbon sources under anaerobic processes with the increased trivalent chromium production. Part of the produced Cr3+ was precipitated onto the soil particles after the bioreduction process. Substrates addition caused the increased bacterial communities for Cr6+ bioreduction. Sporolactobacillus, Clostridium, and Ensifer were the dominant species caused for Cr6+ bioreduction under anaerobic conditions. Results are helpful in designing an in situ Cr6+ bioreduction process to cleanup the Cr6+-contaminated groundwater.

1

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: