Chromium(VI) bioreduction and removal by Enterobacter sp. SL grown with waste molasses as carbon source: Impact of operational conditions

Journal Pre-proofs Chromium(VI) Bioreduction and Removal by Enterobacter sp. SL Grown with Waste Molasses as Carbon Source: Impact of Operational Conditions Yan Sun, Jirong Lan, Yaguang Du, Li Guo, Dongyun Du, Shaohua Chen, Hengpeng Ye, Tian C. Zhang PII: DOI: Reference:

S0960-8524(19)31204-0 https://doi.org/10.1016/j.biortech.2019.121974 BITE 121974

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

21 May 2019 5 August 2019 7 August 2019

Please cite this article as: Sun, Y., Lan, J., Du, Y., Guo, L., Du, D., Chen, S., Ye, H., Zhang, T.C., Chromium(VI) Bioreduction and Removal by Enterobacter sp. SL Grown with Waste Molasses as Carbon Source: Impact of Operational Conditions, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.121974

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.

© 2019 Published by Elsevier Ltd.

Chromium(VI) Bioreduction and Removal by Enterobacter sp. SL Grown with Waste Molasses as Carbon Source: Impact of Operational Conditions

Yan Suna,b,1, Jirong Lana, b, 1, Yaguang Dua, b,*, Li Guoa,b, Dongyun Dua,b, Shaohua Chena,b, Hengpeng Yea,b, and Tian C. Zhangc

aKey

Laboratory of Catalysis Conversion and Energy Materials Chemistry of Ministry of Education; bEngineering Research Center for Control and Treatment of Heavy Metal Pollution of Hubei province , College of Resources and Environmental Science, South Central University for Nationalities Wuhan 430074, P.R. China); cProfessor, Civil Engineering Department, College of Engineering, University of Nebraska-Lincoln, Omaha, NE 68182, USA; *Corresponding author: [email protected] 1Yan Sun and Jirong Lan contributed equally.

Fig. Graphical abstract

1

Abstract: A technology utilizes bacteria Enterobacter sp. SL grown in an anaerobic reactor with waste molasses as carbon source to bio-reduce hexavalent chromium [Cr(VI)] in wastewater and then remove total chromium has been developed. The performance was elucidated through different initial and operating experiments conditions, and the associated mechanism of Cr(VI) reduction was explained. Results show that Cr(VI) removal is 99.91% at 25 h in the anaerobic reactor initially containing bacteria of 5% (v/v), (NH4)2Fe(SO4)2·6H2O of 0.5 gL-1, waste molasses of 2.5 gL-1, Cr(VI) of 100 mgL-1, pH of 6.0, and with the operational temperature of 45 °C. After 120 h reaction, Cr(total) removal reached 91.10%. The major reduction products [FeS, Cr2O3, Cr(OH)3, S0 granules] together with microbes was removed by sludge separation with Cr(VI) in the supernatant (0.027 mgL-1) being much lower than that (not excess 0.2 mgL-1) of Electroplating Pollutant Emission Standard.

Key words: Cr(VI)-containing wastewater; sulfate-reducing bacteria; microbial reduction of chromium; waste molasses; oxidation-reduction reaction

2

1. Introduction Chromium is widely used in leather tanning, electroplating, printing, and dyeing industry, with a large amount of hexavalent chromium [Cr(VI)] containing wastewater being produced annually (Wu et al., 2017). Development of proper processes to treat the Cr(VI)-containing wastes has been the highest priority in order to control possible pollution caused by Cr(VI), which lead to extreme biological toxicity and high mobility in natural water systems (Utgikar et al., 2010). Currently, the most popular treatment strategy for Cr(VI)-containing wastewater is to reduce toxic Cr(VI) to less toxic trivalent chromium [Cr(III)] by adding reductants such as iron sulfate (Lu et al., 2016), calcium polysulfide (Baldrian, 2003; Jiang et al., 2019), and zero-valent iron (Narin et al., 2006; Sahinkaya et al., 2011). However, comparing with chemical reductants (Jobby et al., 2018), microbial reduction of Cr(VI) has the advantages of no secondary pollution (Chen et al., 2016), low energy consumption, and eco-friendly (Pagnanelli et al., 2012), and thus, has become a research hotspot recently (Li et al., 2016). Cr(VI)-containing wastewater has the characteristics of high acidity (Bharagava & Mishra, 2018a), multi-ions (including a large amount of metal ions and sulfate), and fluctuated temperatures (O"Flaherty et al., 1998). These factors make biological treatment of Cr(VI)-containing wastewater difficult and challenging (Nazime Mercan et al., 2011). Existing studies on biological treatment of Cr(VI)-contaminated wastewater mainly target wastewater with low Cr(VI) concentrations (5 mg L-1 to 30 3

mg L-1) (Chen et al., 2019; Haouari et al., 2006; Ozdes et al., 2014; Zhang et al., 2019). Biological treatment typically takes seven days or more (Kieu et al., 2011). The reduction mechanism of Cr(VI) to Cr(III) was also studied (Hwang & Jho, 2018). It is generally believed that the physiological metabolism of bacteria and/or the production of Cr(VI)-reducing protein are important for reducing Cr(VI) to Cr(III) (Dhal et al., 2013). In addition, some reducing substances produced by bacterial metabolism, such as S2-, organic, and inorganic acids can also reduce Cr(VI) (Debabrata Pradhan, 2017). Studies have found that in anaerobic Cr(VI) reduction systems, bacterial survival, and reproduction were inhibited at low pH or in complex systems containing heavy metals or toxic substances (Jobby et al., 2018), resulting in low Cr(VI) reduction efficiency (Jia et al., 2017). To stimulate better performance of Cr(VI) bio-reduction and biomass reproduction, it is necessary to provide more substrate and nutrients, including carbon, nitrogen, and inorganic salts, which also increases the cost of the process (Druhan et al., 2012). Thus, the existing knowledge gap is to develop a cost effective process with a cheap carbon source for efficient bioreduction of Cr(VI). To fill this knowledge gap, a cost effective process that utilizes pure bacteria Enterobacter sp. SL grown in an anaerobic reactor with waste molasses as carbon source to bio-reduce Cr(VI) to Cr(III) and then remove total chromium [Cr(total) = Cr(VI) + Cr(III)] in aqueous solution was developed. This paper describes how to operate this innovative process and evaluate its performance under different 4

conditions and interference factors, and explains the mechanism of Cr(VI) removal in the process. The technology developed in this study may have great application potential for treatment of electroplating wastewater containing sulfuric acid. 2. Materials and Methods 2.1 Enrichment and Isolation of Strains The experimental strains were obtained from the soil near the sewage treatment station of the smelting plant, Huangshi Daye Nonferrous Metals Corporation, Hubei, China. The specific enrichment separation steps are as follows: large particulate matters in soil samples were removed with a sieve of 20-mesh. In order to prepare a soil suspension, 5 g of soil sample was placed into a 100 mL Erlenmeyer flask (GG17, Linyi Co., Ltd. China), diluted with 50 mL sterilized DI water (0.085 μS cm-1, 25°C; Sterilized at 121 °C for 20 minutes in an autoclave) and shaken at 120 rpm for 20 mins. The soil suspension and the liquid medium (Table 1) were inoculated into a 250-mL anaerobic flask (GL45, Shanghai Shupei Experimental Equipment Co., Ltd. China) at a ratio of 1:10 (V:V). Anaerobic incubation was carried out for 3 to 5 days at a temperature of 35 °C. The smell of rotten eggs from the mouth of the flask indicated the generation of H2S, which was caused by the bio-reduction of sulfate. Moreover, black FeS suspension was observed all over the flask. Those phenomena above indicated the termination of bacteria incubation. Then separate the bacteria by using streak plate technique (Su et al., 2019) and culture the plates at 30 °C. When

5

bacteria appeared, select colonies that were sparsely distributed for picking. The picked colonies were quickly placed in a 250-mL anaerobic flask with 150-mL liquid medium (Table 1). The above procedures were repeated until purified bacteria were obtained. The isolated rod-shape bacteria were initially in the form of light yellow round colonies on bacteria solid medium and turn out to be black color after 2 days. The results of Gram staining test were positive. The isolated bacteria SL were enriched by growing them in a batch reactor (a beaker) filled with 1 L LB medium (Sarkar et al., 2017); every week the batch reactor was diluted twofold with fresh medium (Table 1). The concentration of bacteria at 109 colony forming units (CFU) mL-1 was used in subsequent experiments. To calculate bacterial concentration, the dilution plate method (Baskaran & Nemati, 2006) was used. The preservation and identification of bacteria were done by the China Center for Type Culture Collection (CCTCC), Wuhan, Hubei, China. The identification of 16rSDNA showed that the bacteria may belong to Enterobacter sp. SL. The bacterial deposit number is CCTCC M 2018892 (http://www.cctcc.org/). 2.2 Waste Molasses and Bacterial Stock Solution The waste molasses used in the experimental process was taken from Guangxi Qinzhou Lianfeng Sugar Co., Ltd. The main components are shown in Table 2. Among them, fructose, glucose, and reducing substances all have reducing properties. 6

The waste molasses was sterilized once obtain and then stored in a refrigerator (4 oC) for later use. To make bacterial stock solution to be used in the tests, a certain amount of the above-obtained bacteria solution [0 (for control tests), 1, 5, and 10% (v/v) for Test 1 shown in Table 3] were inoculated into a sterile oxygen-free liquid medium (Table 1), which was immediately used in the following tests. 2.3 Experimental Design for Cr(VI) Reduction and Removal Table 3 shows the experimental design for different tests conducted in this study. These tests were conducted to explore the optimal conditions of Cr(VI) removal and to elucidate the associated mechanism. Fig. 1a shows the experimental setting. The anaerobic flasks were cultured in a biochemical incubator (SHP-250, Shanghai Senxin Experimental Instrument Co., Ltd.) at 35 °C (or other temperatures as in Test 7, Table 3) for reduction tests. Under sterile conditions, 250-mL stock solution described in section 2.2 was shaken and dispensed into a 250-mL anaerobic flask (a culture flask). Then, each flask was adjusted as per conditions listed in Table 3 by adding either K2CrO4 solution, adjusting pH, and temperatures, etc. The test solution was then flushed with N2 for 10 mins and then sealed. In each tests, the Cr(VI) content in the culture flask was sampled at 0, 12, 18, 24, 36, and 42 h. In some tests, sampling was shorten to 25 h or extended to 120 h, depending on the purposes of the tests. After each sampling, the flask was flushed N2 for 10 min and then placed back for incubation. The methodology is expressed in Fig 1b.

7

2.4 Analytic Methods Cr(VI) was detected by diphenylcarbazide spectrophotometry (UV/Vis-1100DB, Shanghai mepi da instrument co., LTD, China) as per GB/T 7467-87 (Ching-Yao et al., 2010). Determination of total chromium refers to GB/T 7466-1987 (Ching-Yao et al., 2010). The pH and oxidation-reduction potential (ORP) of the systems (Test 7 in Table 3 at 45°C) was measured with a pH/ORP meter (PHSJ-4F laboratory pH/ORP meter, Shanghai Yidian Scientific Instrument Co., Ltd., China). The Cr(VI) or Cr(total) removal efficiency was calculated as: ηt =

(C0 - Ct) C0

(1)

where ηt is removal efficiency at time t (%); C0 and Ct are the liquid phase concentrations of Cr(VI) or Cr(total) at initial and time t (mgL−1), respectively. Data reported in this paper were the average of replicates (n = 3). The morphological characteristics and elemental composition of freeze-dried bacteria (in bacterial stock solution after 48 h reaction) and precipitates formed after Cr(VI) reduction (tests 8 in Table 3 after 120 h reaction) were analyzed by a scanning electron microscope-energy dispersive spectrometer (SEM-EDS) (Hitach SU8010, Japan). The working conditions of the scanning electron microscope and energy spectrometer were as follows: accelerating voltage is 15 keV; sample current was 1.0 nA; and working distance was 17 mm. The chemical composition and chemical state of the surface elements were analyzed by K-Alpha X-ray photoelectron spectroscopy 8

(XPS) (Thermo Scientific, USA). All the binding energies were referenced to the C1s peak at 284.80 eV of the surface adventitious carbon. In this study, Fourier Transform Infrared Spectrometer (FT-IR, 27 BRUKER TENSOR, German) analysis was used to effectively reflect the functional groups acting on the bacterial surface (the original bacteria and that obtained from Test 7 in Table 3 at 45 °C were freeze-dried) during the reaction and to reveal reaction mechanism (Bharagava & Mishra, 2018). For functional group analysis, the sample to be tested was pretreated with KBr pellets. The wavenumber range was 4000-400 cm-1, and the resolution was 4 cm-1. 3. Results and Discussion 3.1 Effect of Bacterial Inoculum The reduction rate of Cr(VI) increased with an increase in bacterial inoculation (Fig. 2a). While black precipitates were not appeared in the system before 42 h, they appeared in a large amount at 42 h (see below for discussion of Fig. 3). After 42 h the removal efficiency of the system with 0, 1, 5, and 10% inoculation was 54.20, 63.90, 97.60, and 98.40%, respectively, and the higher bacterial inoculation would result in a higher Cr(VI) reduction. The control test (addition of 0% bacteria) results show that waste molasses has a certain reductivity (~42% of Cr (VI) removal) in the system, presumably due to abiotic reduction since the system was sterilized. There is no doubt that 42% of Cr(VI) could be removed by molasses. However, the removal rate of Cr(VI) increased significantly after the addition of microorganisms within the same

9

time. In a short time scale (12 h), reactors with 0, 1, 5, and 10% inoculation resulted in 18.40, 32.20, 40.17, and 46.10% Cr(VI) removal, respectively. After 40 h reaction, the Cr(VI) removal difference between the reactors with 5 and 10% inoculation was negligible. As a result, 5% was selected as an optimal inoculation in later tests with a reaction time within 42 h. 3.2 Effect of Initial Waste Molasses Concentration As shown in Fig. 2b, Cr(VI) reduction efficiency increases as the initial waste molasses concentration increases. After 24 h, the removal of Cr(VI) in the control tests (0 molasses) did not change significantly (at ~ 40%), indicating that without waste molasses as a carbon source, bacteria are difficult to survive and function. When the concentration of waste molasses was 5.5 gL-1, the reduction of Cr(VI) reached the maximum (98.76%) and stabilized at 18 h, much earlier than the other reactors with a lower molasses. Moreover, when the concentration of waste molasses was greater than or equal to 2.5 gL-1, the removal of Cr(VI) can reach above 99% after 42 h of reaction. These results indicate that more waste molasses would not only provide more carbon source to promote microbial activity for Cr(VI) reduction, but also introduce more reducing substances (e.g., glucose, fructose, and others) to promote the reduction of Cr(VI) to Cr(III). 3.3 Effect of Initial Cr(VI) Concentration Fig. 2c shows that the Cr(VI) removal and CFU per mL gradually decreases as 10

the concentration of Cr(VI) increases. For the reactor with an initial concentration of Cr(VI) being 30 mgL-1, the Cr(VI) removal reached a plateau (88.80%) after 18 h (Fig. 2c). With a higher initial Cr(VI) concentration, a longer reaction time would be needed to reach this plateau. Reactors with a Cr(VI) concentration ≤ 100 mgL-1 could reach 99.00% removal of Cr(VI) within 42 h. Compared with the previous research (Bharagava & Mishra, 2018b; Pagnanelli et al., 2012), our method is more efficient. However, for initial Cr(VI) concentration higher than 100 mgL-1, a much longer reaction time was needed to reach the similar or lower removal efficiency (Fig. 2c). If the concentration of Cr(VI) is too high, it would inhibit the growth and activity of bacteria, and thereby, inhibit the bacterial reduction of Cr(VI) (Fig.2d). Under the same conditions of waste molasses concentration and bacterial inoculum, the content of reducing agents in the system was limited. Therefore, the higher the Cr(VI) concentration, the slower the Cr(VI) reduction efficiency. Nevertheless, it seems that, given the test conditions used in this study, it may be possible for all the reactors with different initial Cr(VI) concentrations eventually to reach 99.00% removal with reaction time longer enough. 3.4 Effect of Initial pH At 12 h, the Cr(VI) removal was 60.60% (pH = 3.0), 67.2% (pH = 4.0), 84.2% (pH = 5.0), 92.6% (pH = 6.0), 13.1% (pH = 7.5), 51.9% (pH = 8.0), 46.9% (pH = 9.0), and 22.90% (pH = 10.0) (Fig. 2e). For initial pH of 5 and 6, the removal of Cr(VI) could reach 99% at 18 h. After 42 h, the removal of Cr(VI) corresponding to different 11

initial pH values from low to high was 79.40, 99.14, 99.91, 99.87, 90.94, 88.73, 95.17, and 99.79%, respectively (Fig. 2e). The near-neutral and acidic conditions of pH are more favorable for the reduction of Cr(VI); this finding is advantageous for industrial applications of the process developed in this study as the industrial Cr(VI) wastewater is mostly acidic. At initial pH of 6.0, 5.0, 4.0, the Cr(VI) reduction was relatively faster, which could be because under these conditions, the bacteria were more suitable for survival with enhanced metabolism. In general, results of these pHrelated tests indicate that Enterobacter sp. SL is capable of removing Cr(VI) in the acidic or alkaline environment when the waste molasses is used as a carbon source. 3.5 Effect of Different Carbon Sources The reduction of Cr(VI) by the Enterobacter sp. SL reached the maximum after 114 h. At this time, the removal of Cr(VI) by glucose, sucrose, ammonium acetate, bagasse, and carbon-free source was 41.89, 36.68, 30.78, 33.52, and 33.46%, respectively (Fig. 2f). Among them, bacteria with glucose as the carbon source has the highest removal of Cr(VI), which may be because glucose could be easily utilized by bacteria and would accelerate the metabolism of bacteria and promote the reduction of Cr(VI). The macromolecular substances such as bagasse and sucrose were not readily used by bacteria, and thus the associated removal was low. 3.6 Effect of Coexisting Anions and Cations As shown in Fig. 2g , the removal of Cr(VI) was 94.37 and 67.16% when the 12

system contained 15 and 30 mgL-1 of NO3--N, and was 87.19 and 72.77% when the system had 15 and 30 mgL-1 CO32-, respectively. Bacteria can do anaerobic respiration with NO3- as electron accept in an anaerobic environment (Baba, 2004). In addition, too much carbonate may inhibit the growth of the bacteria, resulting in a decrease in Cr(VI) reduction efficiency. When other conditions were unchanged, cations may slightly inhibit bioreduction of Cr(VI) (Fig. 2h), and such effects were not enhanced by increasing Cu2+ or Zn2+ concentration but by increasing Cd2+ concentration. It was known that Cu2+ and Zn2+ were trace metal ions required for bacterial growth. Appropriate amounts of Cu2+ and Zn2+ were beneficial to bacterial metabolism and enzyme synthesis. However, Cd2+ has strong biotoxicity to the microorganism and inhibits its biological activity. 3.7 Effect of Temperature Fig. 2i shows that the higher the temperature, the better the reduction of Cr(VI) by molasses and bacteria as temperature affects bacterial metabolism and enzyme activity. 3.8 Time Courses of pH and ORP The pH and ORP changes in the system were monitored every 2 h. The pH and ORP showed a downward trend overall during the reduction process (Fig. 3a). Microbial activity led to a continuous decrease in ORP, which helped the bacteria to 13

convert to sulfate (SO42-) breathing (conversion of SO42- to H2S) (see Eq. 1 below). There were two reasons for the decrease in pH. One was because sulfate-reducing bacteria produce acidic organic matter in the process; the other was because the organic matter in waste molasses was decomposed by bacteria, producing a certain amount of weakly acidic substances (e.g., volatile fatty acids). 3.9 Mechanism In order to further clarify the process, the precipitates (sampled from reactors of Test 8 after 120 h) was filtered and dried for XPS analysis (Fig. 5). From the broadspectrum diffraction pattern, characteristic peaks of S, C, O, Cr, and Fe appear in Fig. 4a. The remaining diffraction peaks are characteristic peaks of some of the elements such as N, Na, P, and K contained in the medium. A characteristic peak appears in the narrow peak diffraction pattern of Cr2p. The binding energy is 574.50 eV for Cr2S3, the binding energy is 576.60 eV for Cr2O3, and the binding energy for 578.40 eV is CrOOH (Fig. 4b). Haracteristic peaks of FeS (710.10 eV), Fe2O3 (710.70 eV), and FeOOH (711.19 eV) appeared in the diffraction peak of Fe2p (Fig. 4c), and characteristic peaks of elemental sulfur (163.80 eV) appeared in the diffraction peak of S2p (Fig. 4d). Characteristic peak of FeS (710.10 eV as shown in Fig. 4c) further validates the above described reduction process. At the same time, the reduction process was completed in a closed environment to avoid the danger of generating H2S. Figure 4e shows that the surface of bacteria has a large number of carboncontaining organic functional groups, which would shift as bacteria reduced Cr(VI) to 14

Cr(III) (Fig. 4f). This means that the surface of the bacteria adsorbed or chemically reacted with Cr(VI). Fig. 3b shows that no black precipitates were produced as long as Cr(VI) still existed in the solution (e.g., reaction time < 42 h, depending on the test conditions in the reactor), which is called the Cr(VI) reduction stage in this study. However, Fig. 3b also shows that as soon as the removal of Cr(VI) reached ~100%, the black precipitates were formed. These phenomena indicate that the bacteria reduce sulfate to produce S2- (Eq. 1), which then reacts with chromate (CrO42-) in the solution to form Cr(III) and element sulfur (Eq. 4). At the same time, the bacteria also reduce part of the Cr(VI) by its own physiological metabolism (Eq. 5). When Cr(VI) was completely reduced, the remaining S2- reacts with Fe2+ and Cr(III) in the solution to form black FeS (Eq. 6) and Cr2S3 (a very small part) precipitates (Eq. 7), which explains why Cr(total) in the solution would continuously reduce with time after 42 h (Fig. 3b) and why the solution would change to black color (as black FeS formed). These conjectures were confirmed by Fig. 4b as it shows that the chemical forms of Cr after black precipitates formed are Cr2O3 and CrOOH. In addition, the organic functional groups on the surface of bacteria changed, indicating that various forms of Cr(III) formed complexation or adsorbed on the bacterial surface, which were proved by the characterization of SEM, FTIR, and XPS. Figure 5 summarizes the proposed mechanism for Cr removal in the molasses + Enterobacter sp. SL system. 8x

C H O + 2H + + SO24 - → H2S + y - 2z + 4x x 𝑦 z 15

8xy

8x

H2O +  y - 2z + 4xCO2 2y - 4z + 8x

Eq. (1)

4x + y + 4z + 1 8

H2S → H + + HS -

Eq. (2)

HS - →H + + S2 -

Eq. (3)

2CrO24 - +16H + +3S2 - →3S + 2Cr3 + + 8H2O

Eq. (4)

CxHyOz + CrO

24

+ H + → Cr3 + +

4xy + 4yz + y + y2 + 8 16

H2O +

x + xy + 4xz + 4x2 8

CO2

Eq. (5) S2 - + Fe2 + →FeS

Eq. (6)

3S2 - +2Cr3 + →Cr2S3

Eq. (7)

3.10 Implications The study could be potentially applied to Cr [Cr(VI) & Cr(III)] containing wastewater, which have similar characteristics of the experimental conditions. Weak acid chromium smelting wastewater and electroplating wastewater have high amount of Cr(VI) and complex heavy metals(Luo et al., 2019), which are difficult to be treated by ordinary microorganisms. However, these Cr(VI)-containing wastewater may be treated with the technology developed in this study in a much more efficient way than the biological Cr(VI) reduction processes reported in the literature. Batch tests indicate that the formed black precipitates and the bacteria can be easily separated with gravity sedimentation as the sludge volume index (SVI) would be in the range of 75 to 110 mLg-1, and Cr(VI) in the supernatant was as low as 0.027 mgL-1. The standard specified in the first-level standard for electroplating pollutant discharge standards 16

(GB21900-2008) is < 0.1 mgL-1. Thus, the chromium discharge after treatment and sludge separation in this study would meet the first-class standard of electroplating pollutant emission standards. However, limited by the score of this study, the kinetics related to Eqs. 4, 5, and 6 were not evaluated. Thus, whether the system would allow Eq. 4 occur first, followed by Eqs. 6 and 7, or allow them occur at the same time were not clear; the exact roles of microbes in these reactions was unclear as well, which warrant future studies. 4. Conclusion Under anaerobic conditions with the reaction time of 25 h, initial pH of 6.0, molasses of 2.5 gL-1, Cr(VI) of 100 mgL-1, and the reaction temperature of 45 °C, the respective removal of Cr(VI) and the total chromium can reach 99.91 and 91.10% from wastewater. Thus, the chromium-wastewater after treatment and sludge separation met the first-class standard of electroplating pollutant discharge standards. In addition, Cr(VI) could be efficiently reduced by bacteria Enterobacter sp. SL in an acidic and alkaline environment (pH 3.0 to 10.0), making the technology have great industrial application potential.

E-supplementary data for this work can be found in e-version of this paper online. Acknowledgement 17

This work was financially supported by the National Sci-Tech Support Plan, (2015BAB01B03), which is greatly appreciated; Major Innovation Projects of Hubei Province of China (2019ACA156); And the National Natural Science Foundation of China(51708561). Also, we are highly thankful to Dr. Rao Y. Surampalli for his great advises and supports on this paper.

References [1] Baba, A.N.Y. 2004. Mechanism of hexavalent chromium adsorption by persimmon tannin gel. Water Res, 38(12), 2864. [2] Baldrian, P. 2003. Interactions of heavy metals with white-rot fungi. Enzyme Microb Tech, 32(1), 78-91. [3] Baskaran, V., Nemati, M. 2006. Anaerobic reduction of sulfate in immobilized cell bioreactors, using a microbial culture originated from an oil reservoir. Biochem Eng J, 31(2), 148-159. [4] Bharagava, R.N., Mishra, S. 2018a. Hexavalent chromium reduction potential of Cellulosimicrobium sp. isolated from common effluent treatment plant of tannery industries. Ecotox Environ Safe, 147, 102-109. [5] Bharagava, R.N., Mishra, S. 2018b. Hexavalent chromium reduction potential of Cellulosimicrobium sp. isolated from common effluent treatment plant of tannery industries. Ecotox Environ Safe, 147, 102-109. [6] Chen, G., Bai, Y., Zeng, R.J., Qin, L. 2019. Effects of different metabolic pathways and environmental parameters on Cr isotope fractionation during Cr(VI) reduction by extremely thermophilic bacteria. Geochim Cosmochim Acta, 256, 135-146. [7] Chen, G., Feng, J., Wang, W., Yin, Y., Liu, H. 2016. Photocatalytic removal of hexavalent chromium by newly designed and highly reductive TiO 2 nanocrystals. Water Res, 108, 383-390. [8] Ching-Yao, H., Shang-Lien, L., Ya-Hsuan, L., Ya-Wen, H., Kaimin, S., Chin-Jung, L. 2010. Hexavalent chromium removal from near natural water by copper-iron bimetallic particles. Water Res, 44(10), 3101-3108. [9] Debabrata Pradhan, L.B.S., Matthew Sawyer, Pattanathu K.S.M. Rahman. 2017. Recent bioreduction of hexavalent chromium in wastewater treatment:A review. J Ind Eng Chem, 55(25), 1-20. [10] Dhal, B., Thatoi, H.N., Das, N.N., Pandey, B.D. 2013. Chemical and microbial remediation of hexavalent chromium from contaminated soil and mining/metallurgical solid waste: A review. J Hazard Mater, 250-251(30), 272-291. [11] Druhan, J.L., Steefel, C.I., Sergi, M., Williams, K.H., Conrad, M.E., Depaolo, D.J. 2012. Timing the onset of sulfate reduction over multiple subsurface acetate amendments by measurement and modeling of sulfur isotope fractionation. Environ Sci Technol, 46(16), 18

8895-8902. [12] Haouari, O., Fardeau, M.L., Tholozan, J.L., Hamdi, M., Ollivier, B. 2006. Isolation of sulfate-reducing bacteria from Tunisian marine sediments and description of Desulfovibrio bizertensis sp. nov. Int J Syst Evol Micr, 56(12), 2909-2913. [13] Hwang, S.K., Jho, E.H. 2018. Heavy metal and sulfate removal from sulfate-rich synthetic mine drainages using sulfate reducing bacteria. Sci Total Environ, 635(1), 1308-1346. [14] Jia, Y., Khanal, S.K., Zhang, H., Chen, G.H., Lu, H. 2017. Sulfamethoxazole degradation in anaerobic sulfate-reducing bacteria sludge system. Water Res, 119(1), 12-20. [15] Jiang, B., Gong, Y., Gao, J., Sun, T., Liu, Y., Oturan, N., Oturan, M.A. 2019. The reduction of Cr(VI) to Cr(III) mediated by environmentally relevant carboxylic acids: State-of-theart and perspectives. J Hazard Mater, 365, 205-226. [16] Jobby, R., Jha, P., Yadav, A.K., Desai, N. 2018. Biosorption and biotransformation of hexavalent chromium [Cr(VI)]: A comprehensive review. Chemosphere, 207(9), 255-266. [17] Kieu, H.T.Q., Müller, E., Horn, H. 2011. Heavy metal removal in anaerobic semi-continuous stirred tank reactors by a consortium of sulfate-reducing bacteria. Water Res, 45(13), 3863-3870. [18] Li, X., Dai, L., Zhang, C., Zeng, G., Liu, Y., Zhou, C., Xu, W., Wu, Y., Tang, X., Liu, W. 2016. Enhanced biological stabilization of heavy metals in sediment using immobilized sulfate reducing bacteria beads with inner cohesive nutrient. J Hazard Mater, 324(15), 340-347. [19] Lu, X., Wang, N., Wang, H., Deng, Y., Zhang, Y. 2016. Molecular Characterization of the Total Bacteria and Dissimilatory Arsenate-Reducing Bacteria in Core Sediments of the Jianghan Plain, Central China. Geomicrobiol J, 34(5), 467-479. [20] Luo, Q., Chen, Z., Li, Y., Wang, Y., Cai, L., Wang, L., Liu, S., Wang, Z., Peng, Y., Wang, Y. 2019. Highly Efficient and Recyclable Shewanella xiamenensis-Grafted Graphene Oxide/Poly(vinyl alcohol) Biofilm Catalysts for Increased Cr(VI) Reduction. ACS Sustain Chem & Eng, (7) 12611-12620 [21] Narin, I., Surme, Y., Soylak, M., Dogan, M. 2006. Speciation of Cr(III) and Cr(VI) in environmental samples by solid phase extraction on Ambersorb 563 resin. J Hazard Mater, 136(3), 579-584. [22] Nazime Mercan, D., Cetin, K., Sibel, G., Dodge, C.J., Banu Coskun, Y., Mehmet Ali, M. 2011. Chromium(VI) bioremoval by Pseudomonas bacteria: role of microbial exudates for natural attenuation and biotreatment of Cr(VI) contamination. Environ Sci Technol, 45(6), 2278-2285. [23] O"Flaherty, V., Mahony, T., O"Kennedy, R., Colleran, E. 1998. Effect of pH on growth kinetics and sulphide toxicity thresholds of a range of methanogenic, syntrophic and sulphate-reducing bacteria. Process Biochem., 33(5), 555-569 [24] Ozdes, D., Gundogdu, A., Kemer, B., Duran, C., Kucuk, M., Soylak, M. 2014. Assessment of kinetics, thermodynamics and equilibrium parameters of Cr(VI) biosorption ontoPinus brutiaTen. Can J Chem Eng, 92(1), 139-147. [25] Pagnanelli, F., Viggi, C.C., Cibati, A., Uccelletti, D., Toro, L., Palleschi, C. 2012. Biotreatment of Cr(VI) contaminated waters by sulphate reducing bacteria fed with ethanol. J Hazard Mater, 199(2), 186-192. 19

[26] Sahinkaya, E., Gunes, F.M., Ucar, D., Kaksonen, A.H. 2011. Sulfidogenic fluidized bed treatment of real acid mine drainage water. Bioresour Technol, 102(2), 683-689. [27] Su, C. Q., Li, L.Q., Yang, Z.H., Chai, L.Y., Liao, Q., Shi, Y., Li, J.W. 2019. Cr(VI) reduction in chromium-contaminated soil by indigenous microorganisms under aerobic condition. T Nonferr Metal Soc, 29(6), 1304-1311. [28] Thatoi, H., Das, S., Mishra, J., Rath, B.P., Das, N. 2014. Bacterial chromate reductase, a potential enzyme for bioremediation of hexavalent chromium: A review. J Environ Manage, 146, 383-399. [29] Utgikar, V.P., Harmon, S.M., Navendu, C., Tabak, H.H., Rakesh, G., Haines, J.R. 2010. Inhibition of sulfate-reducing bacteria by metal sulfide formation in bioremediation of acid mine drainage. Environ Toxicol,, 17(1), 40-48. [30] Wu, M., Ye, X., Chen, K., Li, W., Yuan, J., Jiang, X. 2017. Bacterial community shift and hydrocarbon transformation during bioremediation of short-term petroleum-contaminated soil. Environ Pollut, 223, 657-664. [31] Zhang, Y., Li, M., Li, J., Yang, Y., Liu, X. 2019. Surface modified leaves with high efficiency for the removal of aqueous Cr (VI). Appl Surf Sci, 484, 189-196.

20

Figure Caption Fig. 1. (a)Experimental setup, The anaerobic flask loaded with sterilized 250-mL test solution was cultured in a biochemical incubator at 35 °C (or other temperatures) round-about shaking at 120 rpm. Before each test, the test solution was flushed with N2 for 10 min; (b) The methodology of this study. Fig. 2. Effects of different conditions on Cr(VI) removal: (a) Enterobacter sp. SL inoculum (Test 1 in Table 3); (b) initial waste molasses concentration (Test 2 in Table 3); (c) initial Cr(VI) concentration on time courses of Cr(VI) removal (Test 3 in Table 3); (d) bacterial concentration (Test 3 in Table 3); (e) initial pH (Test 4 in Table 3); (f) different carbon sources (Test 5 in Table 3); (g) coexisting anions (Test 6 in Table 3); (h)coexisting cations (Test 6 in Table 3); (i) temperatures (Test 7 in Table 3). Fig. 3. (a) Changes in pH and ORP over time during reduction, (Test 8 in Table 3); (b) Time course of Cr(VI) and Cr(total) removal and formation of black precipitates in the system changes. (Test 8 in Table 3); ( The reactor pictures were taken at t = 0, 12, 24 to 120 h, corresponding to the dash lines.)

Fig. 4. (a) XPS survey spectra: surface element content of black precipitate; (b) high resolution XPS spectra of Cr 2p; (c) Fe2p; and (d) S2p; (e) high resolution 21

XPS spectra of C1s bacteria; and (e) bacteria + Cr(VI). Samples for analyzed were taken from reactors of Test 8 after 120 h.

Fig. 5. Proposed Cr removal mechanism by Enterobacter sp. SL.

Table 1 Medium composition and content for bacterial culture (Thatoi et al., 2014). Component

Concentration (g·L-1)

(NH4)2Fe(SO4)2·6H2O

0.50

MgSO4·7H2O

2.00

NH4Cl

1.00

K2HPO4

0.50

Na2SO4

0.50

NaCl

2.00

Yeast extract

1.00

Waste molasses

2.50

* All chemicals used were analytically grade. The pH of the medium was adjusted to 7.5 with 0.10 M H2SO4 and 0.10 M NaOH solutions. The medium was then boiled and sterilized at 121 °C under 100 kPa pressure, and then deoxygenated with N2 before using. This medium is culture solution. 22

Table 2 Molasses composition analyzed by Guangxi Qinzhou Lianfeng Sugar Co., Ltd. (%, by weight).

Sucrose

33.00

Fructose

15.00

Glucose

9.00

Reducing substance 8.00

23

Colloid

Polysaccharide

Others

6.42

8.58

20.00

Table 3. Experimental design and test conditions for chromium(VI) bioreduction and removal by Enterobacter sp. SL. Test # 1

Test conditions

Test objectives

the Enterobacter sp. SL inoculum size = 0 (control), 1%, 5%, and 10% (v/v),

Effects of bacterial inoculum

respectively; initial pH = 7.5; Cr(VI) = 100 mg L-1; temperature = 35 °C + 250-mL

size on Cr(VI) removal

liquid medium (Table 1) 2

3

4

The initial concentration of waste molasses = 0 (control), 1.0, 1.5, 2.5, 3.5, 4.5 and 5.5

Effects of initial molasses

g L-1, respectively; initial pH = 7.5; the Enterobacter sp. SL inoculum size = 5%;

concentration on Cr(VI)

Cr(VI) = 100 mg L-1; temperature = 35 °C + 250-mL liquid medium (Table 1)

removal

The initial Cr(VI) concentration = 30, 50, 100, 150, 200 mg L-1, respectively; initial

Effects of initial Cr(VI)

pH = 7.5; the Enterobacter sp. SL inoculum size = 5%; temperature = 35 °C + 250-

concentration on Cr(VI)

mL liquid medium (Table 1)

removal

The initial pH = 3.0, 4.0, 5.0, 6.0, 7.5, 8.0, 9.0 and 10.0, respectively; the

Effects of initial pH on

Enterobacter sp. SL inoculum size = 10%; Cr(VI) = 100 mg L-1 temperature = 35 °C

Cr(VI) removal

+ 250-mL liquid medium (Table 1) 5

6

The initial substrate concentration = 2.5 g L-1 of glucose, sucrose, ammonium acetate,

Verifying the advantages of

bagasse, or carbon-free sources, respectively; the Enterobacter sp. SL inoculum size =

waste molasses as a bacterial

10%; Cr(VI) = 100 mg L-1; temp = 35 °C + 250-mL liquid medium (Table 1)

carbon source

The initial concentration = 15 and 30 mg L-1, respectively for NO3—N, CO32- , Cu2+,

Effects of other anions and

Zn2+, and Cd2+; initial pH = 7.5; the Enterobacter sp. SL inoculum size = 5%; Cr(VI)

cations on Cr(VI) removal

= 100 mg L-1; temperature = 35 °C + 250-mL liquid medium (Table 1) 7

The temperature = 25, 35, and 45 oC; The initial concentration of waste molasses =

Effects of temperature on

2.5 g L ; initial pH = 7.5; the Enterobacter sp. SL inoculum size = 5%; Cr(VI) = 100

Cr(VI) removal

-1

mg L-1; temperature = 35 °C + 250-mL liquid medium (Table 1) 8

Inoculum size = 5 %; initial Cr(VI) = 30 mg L-1; initial pH of 7.5; and temperature =

Changes in pH and ORP over

45°C+ 250-mL liquid medium (Table 1); The initial concentration of waste molasses

time during reduction; Time

= 2.5 g L

courses of color change and

-1

Cr(VI) and Cr(total) in the system Note: 1) Tests 5, Tests 6, Tests 7 and Tests 8 had replicates (n = 3). Others are single reactor only.

24

Fig. 1.

25

Fig. 2.

26

Fig. 3.

27

Fig. 4.

28

Fig. 5.

Highlights



A high-efficiency process for bioreducing/removal of Cr(VI) was developed



The Enterobacter sp. SL can use waste molasses as carbon source for growth



The process works well in a wide pH range and with the coexistence of polyions



The major reduction products and microbes can be removed by sludge separation



The treated Cr-containing wastewater is of high quality

29

30