Growth-dependent Cr(VI) reduction by Alteromonas sp. under haloalkaline conditions: toxicity, removal mechanism and effect of heavy metals

doi:10.21203/rs.3.rs-3590297/v1

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Growth-dependent Cr(VI) reduction by Alteromonas sp. under haloalkaline conditions: toxicity, removal mechanism and effect of heavy metals

https://doi.org/10.21203/rs.3.rs-3590297/v1

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Bacterial reduction of hexavalent chromium (Cr(VI)) to trivalent chromium (Cr(III)) is a sustainable bioremediation approach. However, Cr(VI) by bacteria is severely impeded by Cr(VI) toxicity and complex environmental conditions like salt, alkaline pH and heavy metals. Hence, there is a need for Cr(VI) reducing bacteria to thrive as well as to metabolize under complex conditions. This study investigated Cr(VI) reduction, toxicity and removal mechanisms under complex conditions using an Alteromonas sp. isolated from the aerobic granular sludge cultivated from seawater-borne microorganisms. Rapid and complete removal of 100 mg/L Cr(VI) was achieved within 24 h under haloalkaline conditions (salinity: 3.5 to 7.5; pH 8 to 11). This strain exhibited high tolerance to heavy metals under haloalkaline conditions and reduced 100 mg/l Cr(VI) within 24 h in the presence of 100 mg/L As(V), 100 mg/L Pb(II), 50 mg/L Cu(II) or 5 mg/L Cd(II). The toxicity of Cr(VI) on the bacterial cells was evident by the increased reactive oxygen species levels and inhibition of esterase activity. Regardless of Cr(VI) toxicity, the cells grew and efficiently reduced Cr(VI) to Cr(III). The bacterial Cr(VI) reduction was strongly dependent on the growth, necessitating actively growing cells and growth medium. While, resting cells and spent medium barely contributed to Cr(VI) reduction. The biochemical assays revealed efficient Cr(VI) reduction using a cytosolic protein fraction from Alteromonas sp. and an exogenous reducing agent (e.g., NADPH). This study demonstrates an efficient Cr(VI) reduction system for potential Cr(VI) bioremediation applications under complex conditions including extreme haloalkaline conditions and toxic heavy metals.

Bioreduction

Chromate reduction

Salinity

Alkaline pH

Heavy metals

Chromate reductase

Chromium (Cr) is used in various industries including tanneries, textile, chrome-plating, steel, mining, and chemical industries. As a result, Cr is encountered as a common emerging pollutant in various environmental settings (Prasad et al., 2021; Reddy and Nancharaiah, 2018). Environmental discharge of Cr laden wastewater without proper treatment poses an ecological threat, and health hazard (Mortada et al., 2023). The toxicity, and environmental impact of Cr is dependent on its oxidation state. Cr majorly exists in two oxidation states such as hexavalent Cr (Chromate (CrO₄²⁻), Cr(VI)) and trivalent Cr (Cr(III)) in environmental settings. Cr(VI) is highly soluble, mobile, toxic to biota and a known carcinogen. While, Cr(III) is 100 times less toxic and 1000 times less mutagenic as compared to Cr(VI) (Gu et al., 2023). Additionally, Cr(III) serves as a trace element for balanced carbohydrate, lipid and amino acid metabolism (Malaviya and Singh 2016). The genotoxic, mutagenic, and teratogenic effects of Cr(VI) are well known. The toxicity of Cr(VI) has been linked to respiratory disorders, lung carcinoma, accumulation in the placenta, and foetal damage (He et al., 2020). Due to the severity of health hazard, Cr(VI) has been classified as human carcinogen and priority pollutant by various international agencies (Goswami et al., 2023). To avoid environmental pollution, Cr(VI) release to environment is regulated with a discharge limit 0.05 mg/L. This necessitates effective treatment of Cr(VI)-containing wastewaters prior to environmental discharge. Various physicochemical and biological methods have been proposed for the treatment of Cr laden wastewaters (Singh et al., 2022). Mechanistic interactions such as biosorption, bioaccumulation, bioreduction and chromate efflux are involved in microbial Cr(IV) detoxification (Gutiérrez-Corona et al., 2016). Among these, microbial reduction of toxic Cr(VI) to less toxic Cr(III) is an effective biotechnological solution for the remediation (Nancharaiah et al., 2015). The reduction of Cr(VI) has been reported among phylogenetically diverse microorganisms including Bacillus sp., Shewanella sp., Klebsiella sp., Pseudomonas sp., Rhodobacter sp., and Cellulomonas sp. (Malaviya and Singh 2016; Kabir et al., 2018; Tamindžija et al., 2019; He et al., 2020; Chen and Tian 2021; Su et al., 2023). Under aerobic conditions, bacterial Cr(VI) reduction is catalysed by soluble enzymes using electron donors such as NADH, NADPH or other endogenous compounds (Malaviya and Singh 2016; Thatoi et al., 2014). Although Cr(VI) reduction is widely reported among phylogenetically distinct microorganisms, biological treatment is challenging due to the toxic effects of Cr(VI) on metabolism and viability of microorganisms (Nancharaiah et al., 2015).

Cr containing wastewaters are often characterised with high salt ions, alkaline pH and toxic heavy metals (Lefebvre et al., 2005; Watts et al., 2015; Chowdhury et al., 2015). For example, tanneries use high concentration of salt in hide preservation and pickling result in generation of saline effluents (Lefebvre et al., 2005). Tannery soak liquor can contain up to 7.23% of NaCl (Lefebvre et al., 2006). Cr containing saline wastewater is also generated in chrome plating industries (Mubeena and Muthuraman 2015). Chromite ore processing residue (COPR) is a solid waste generated during Cr(VI) extraction from the chromite ore. The COPR must be treated properly to avoid Cr(VI) contamination of water and soil. Due to the alkaline nature, processing of COPR produces Cr(VI) containing leachate with a pH from 11 to 13 (Watts et al., 2015). Leather processing steps such as soaking and beamhouse (unhairing-deliming) generates alkaline effluents with pH 8.1 to 12.3 (Cooman et al., 2002). The tannery effluents collected from the industrial sites are alkaline with pH 8.5 (± 0.5) (Sarankumar et al., 2020). Besides Cr, presence of metals such as Fe, Mn, Na, Ca, Pb, Cd, As, Co, Cu, Zn, and Ni are commonly reported in tannery, textile and chrome plating wastewaters (Kaya et al., 2016; Chowdhury et al., 2015; Deepali 2009, Li et al., 2021). High salinity and high solution pH are detrimental to growth of microbes as these conditions interfere with osmatic balance, enzyme catalysis, metabolic activity and cell survival. Additionally, toxic heavy metals such as As, Cr, Pb, Cd and Cu can affect microbial metabolism and cell survival through diverse mechanisms (Igiri et al., 2018). The reduction of Cr(VI) by bacteria can be strongly influenced by the prevailing salt ions, alkaline pH and heavy metal co-contaminants. Haloalkaliphilic bacteria can convert toxic Cr(VI) to nontoxic Cr(III) under alkaline pH and in the presence of excess salt ions. Although phylogenetically distinct bacteria exhibit Cr(VI) reduction, there are limited reports of Cr(VI) reduction in the presence of salt, alkaline pH and heavy metals (Table 1). The salts ions, alkaline pH and heavy metals can strongly inhibit the bacterial growth and Cr(VI) reduction potential.

Table 1

Summary of studies related to bacterial Cr(VI) reduction under haloalkaline conditions and heavy metals.
Bacteria	Salt/salinity	pH	Co-heavy metals	Initial Cr(VI) concertation, (% removal)	Reference
Pseudochrobactrum saccharolyticum	Up to 2% NaCl	7-10.7	--	55–360 mg/L, 100% at 55 mg/L in 72 h.	Long et al., 2013
Sporosarcina saromensis W5	--	9–12	--	50–400 mg/L, 100% at 200 mg/L in 12 days	Huang et al., 2021
Vigribacillus sp.	Up to 12% NaCl	8	--	50–250 mg/L, 99.2% at 100 mg/L, 6% NaCl in 70 h	Mishra et al., 2012
Bacillus subtilis	--	8	--	50–200 mg/L, 100% at 50 mg/L in 72 h	Mangaiyarkarasi et al., 2011
Cellulosimicrobium funkei AR8	Up to 3% NaCl	5–9	--	100–250 mg/L, At 200 mg/L Cr(VI) and pH 7, 100% with 0% NaCl, 70% with 2% NaCl	Karthik et al., 2017
Chelatococcus daeguensis TAD1	--	7	5 mg/L each of Cu(II), Ni(II), and Zn(II)	15 mg/L, 96.5% with Cu(II), 83% with Ni(II) and 68% with Zn(II)	Li et al., 2016
Bacillus sp. FM1	--	8	0.1 mM each of Cd(II), Zn(II), Cu(II), Co(II), Ni(II)	100 mg/L, 35% with Cd(II), 50% with Zn(II), 55% with Cu(II), 100% with Co(II) and Ni(II).	Masdood and Malik 2011
Alteromonas sp.	Up to 7.5% Salinity	8–11	Each of 100 mg/L Pb(II), 100 mg/L As(V); 50 mg/L Cu(II), 5 mg/L Cd(II)	100 mg/L, 100% at pH 7.6–11 and 5.5% salinity in 24 h. Complete Cr(IV) removal at the mentioned concentrations of heavy metals	Current study

It is hypothesized that marine bacteria thriving under haloalkaline conditions are suitable for achieving efficient Cr(VI) reduction under complex environmental conditions. Hence, a halophilic and selenite-reducing bacterium (Reddy et al., 2023) isolated from the aerobic granular sludge cultivated from seawater-borne microorganisms (Sarvajith et al., 2020) was chosen for determining Cr(VI) reduction, toxicity and reduction mechanisms under complex conditions. This study for the first time investigated the Cr(VI) reduction potential of Alteromonas sp. in the presence of salt ions, alkaline pH and heavy metals (As, Cd, Cu, and Pb). The growth, Cr(VI) tolerance, and Cr(VI) reduction potential were characterized at varied salinities (3.5 to 11.5), pH (5 to 12) and different heavy metals (5 to 100 mg/L). Experiments were performed with resting cells, growing cells, cell free supernatant and cellular protein fractions for elucidating Cr(VI) reduction mechanisms. The metabolic activity and intracellular reactive oxygen species (ROS) were determined for evaluating Cr(VI) induced toxicity on Cr(VI) reducing bacteria.

2.1. Bacterial source, chemicals, culture media, and conditions

Halophilic aerobic granular sludge (hAGS) cultivated from the seawater microbiome in a laboratory scale sequencing batch reactor treating saline wastewater was used as the inoculum source for isolating metal(loid) reducing bacteria (Sarvajith and Nancharaiah 2020a,b). An Alteromonas sp. obtained from hAGS exhibited efficient reduction of selenite and tellurite to Se(0) and Te(0), respectively, in the presence of salt ions (Reddy et al., 2023). The bacterial strain was maintained on Zobell marine agar (ZMA) by regular subculturing. For liquid cultures, strain was inoculated in sterile Zobell marine broth (ZMB) and incubated at 30°C and 120 RPM in a temperature controlled orbital shaker. Alteromonas sp. cells grown for overnight were used as the inoculum for Cr(VI) reduction experiments. ZMB was used for the Cr(VI) reduction experiments. A 10000 mg/L Cr(VI) stock solution was prepared by dissolving K₂Cr₂O₇ in sterile demineralized water.

2.2. Growth of Alteromonas sp. under different conditions

2.2.1. Growth in the presence of different Cr(VI) concentrations

Growth of Alteromonas sp. was determined in the presence of 20 to 200 mg/L Cr(VI) in microtiter well plates. Alteromonas sp. cells were grown in ZMB for overnight, harvested by centrifugation, washed, resuspended in sterile ZMB and adjusted to an optical density (OD) of ~ 0.05 at 600 nm (corresponds to 3–5×10⁷ cells/mL). Chromate stock solution was added to ZMB by a 2-fold dilution method to prepare 25 to 200 mg/L Cr(VI). The ZMB with cells and Cr(VI) was disbursed as 200 µL aliquots per well with five replicate wells for each concentration. The plates were incubated at 30°C in a microplate reader (Tecan, Infinite® 200 PRO) and measured the growth by continuously recording the absorbance at 600 nm over a period of 48 h.

2.2.2. Growth at different salinities

ZMB having a salinity of 3.5% was supplemented with NaCl to obtain a final salinity in the range of 3.5 to 11.5% and sterilized by autoclaving. ZMB with different salinities were inoculated with Alteromonas sp. as mentioned earlier. Suspensions were aliquoted into 96 well plates in 6 replicates per salinity and growth kinetics were obtained in multimode reader as explained previously.

2.2.3. Growth at different pH

The pH of the ZMB was determined to be 7.6. The pH of the ZMB was adjusted from 5 to 12 using 1N HCl or 1N NaOH in Erlenmeyer flasks to 50 mL final volume. Sterilized ZMB media with different pH was inoculated with bacterial cells from an overnight grown culture and adjusted to a final OD600 of ~ 0.05. Two replicate flasks per each pH condition was used and these suspensions were incubated as earlier. Kinetics of Alteromonas sp. growth under these conditions were determined by aliquoting the samples at regular time intervals and by measuring the absorbance at 600nm.

2.2.4. Growth in the presence of different heavy metals

Tolerance and growth of Alteromonas sp. in presence of toxic heavy metals (Pb, As, Cu, Cd) was performed in 96 well plate over a period of 48 h. Different concentrations (3.125 6.25, 12.5, 25, 50, 100 mg/L) of heavy metals (Pb, As, Cu) were prepared in sterile ZMB through 2-fold dilution method. Due to high toxicity, Cd concentration was prepared between 0.325 to 20 mg/L. Similar to earlier experiments, heavy metal spiked ZMB was adjusted with actively growing Alteromonas sp. cells at a final OD of ~ 0.05. These suspensions were aliquoted into 96 well plates at 200 µL per well in 6 replicates per concentration and growth kinetics were determined as earlier.

2.3. Cr(VI) removal by Alteromonas sp.

2.3.1. Cr(VI) removal at different initial concentrations

Cr(VI) removal by Alteromonas sp. was carried out in 100 mL Erlenmeyer flasks with 50 mL sterile ZMB. Chromate stock solution was directly added to obtain a final Cr(VI) concentration of 12.5, 25, 50 and 100 mg/L. Two replicates were setup for each concentration. The flasks were inoculated with Alteromonas sp. at a final density of 3–5×10⁷ cells/mL. ZMB containing 100 mg/L Cr(VI) without inoculation was setup to determine effect of medium components on Cr(VI) removal and served as an abiotic control. ZMB containing 100 mg/L Cr(VI) was inoculated with heat killed cells to determine the role of growth on Cr(VI) removal. All the flasks were incubated at 30°C in a temperature controlled orbital shaker at 120 RPM. Liquid samples were collected at regular time intervals, centrifuged and stored at 4°C. These samples were analysed for residual Cr(VI) and total Cr.

2.3.2. Cr(VI) removal at different salinities

ZMB was prepared with different salinities from 3.5 to 11.5% was transferred as 50 mL aliquots to 100 mL Erlenmeyer flasks and autoclaved. Known volume of filter-sterilized Cr(VI) stock solution was directly added to the flask to obtain a final concentration of 100 mg/L. The flasks were inoculated with Alteromonas sp. at a final density of 3–5×10⁷ cells/mL as described above. Two replicate flasks were used per each salinity. Control flasks with heat killed cells and without cells were setup as described above. The flasks were incubated at 30℃, 120 RPM and sampled at regular time intervals until 96 h for monitoring Cr(VI) removal. The samples were centrifuged and stored at 4°C until analysis.

2.3.3. Cr(VI) removal at different pH

The pH of ZMB was adjusted to 5 to 12, distributed as 50 mL aliquots in 100 mL Erlenmeyer flasks and sterilized by autoclaving. Filter sterilized chromate stock solution was added to the sterile media to obtain 100 mg/L Cr(VI) in the flasks. Abiotic controls were kept at each pH condition to assess the Cr(VI) removal through precipitation, if any. Experiment was carried with two replicates for each pH condition and all the flasks were incubated at 30℃ and 120 RPM. Samples were collected at regular time intervals for over a period of 72 h, centrifuged and stored in the refrigerator until analysis.

2.3.4. Cr(VI) removal in presence of heavy metals

Erlenmeyer flasks with sterile ZMB (50 mL per each 100 mL flask) were prepared with 12.5 to 100 mg/L Cr(VI) as mentioned above. Based on the growth kinetics in the presence of heavy metals, 100 mg/L each of As, Pb, Cu or 5 mg/L Cd was added to sterile ZMB containing 12.5 to 100 mg/L Cr(VI). These flasks containing Cr(VI) and one of the heavy metal were inoculated with Alteromonas sp. cells and incubated at 30°C and 120 RPM for determining Cr(VI) removal. Appropriate controls (abiotic and heat killed) were included. A minimum of two flasks were setup for each heavy metal and controls. Liquid samples were collected at periodic time intervals, centrifuged and stored at 4°C prior to analysis.

2.4. Cr(VI) removal mechanisms

2.4.1. Cr(VI) reduction by higher inoculum cell densities, resting cells, and spent media

Overnight grown cells were harvested, washed and re-suspended in fresh sterile ZMB for preparing regular and higher cell densities for inoculation. Sterile ZMB with 100 mg/L Cr(VI) was prepared in the flasks as described above. These flasks were inoculated with ~ 10¹² cells/mL (OD at 600 nm: ~1.4) for determining influence of higher initial cell number on chromate removal. For comparison, separate set of flasks were inoculated with 10⁷ cells/mL (OD at 600 nm: ~0.05) commonly used in the Cr(VI) removal experiments. Corresponding abiotic and heat killed cell controls were also kept with 100 mg/L Cr(VI).

For resting cells experiment, sterile seawater was used in place of ZMB. Overnight grown cells in ZMB were washed, suspended in sterile seawater and adjusted the OD at 600 nm to obtain a final cell density of 10¹² cells/mL. To this, a known volume of chromate stock was added to obtain 100 mg/L Cr(VI). Sterile seawater with 100 mg/L Cr(VI) without inoculation of Alteromonas sp. cells was used as control. The flasks were incubated at 30°C and 120 RPM. Liquid samples were collected at periodic time intervals for monitoring Cr(VI) removal.

Alteromonas sp. cells were grown in ZMB with and without 100 mg/L Cr(VI) at 30°C and 120 RPM for 24 h. The grown cultures were centrifuged at 10,000 rpm for 10 min to collect spent medium. The supernatant was filtered through 0.2 µm sterile syringe filter to remove the residual cells, if any. Chromate stock was added to the prepared cell free supernatants (spent media) to obtain 100 mg/L Cr(VI). The spent media with 100 mg/L Cr(VI) were incubated normal culture conditions (30°C, 120 RPM) for determining Cr(VI) removal kinetics.

2.4.2. Cr(VI) reduction mechanism by Alteromonas sp.

Alteromonas sp. cells grown for 24 h were harvested by centrifugation at 8000 RPM at 4°C for 5 min and processed for protein extraction on ice bath (Abo-Alkasem et al., 2022). Cells were washed, suspended in pre-cooled Tris-HCl buffer (50 mM, pH 7.2) and subjected to ultrasonication at 25% amplitude for 10 min (20 sec pulse on and 10 sec pulse off). From this crude extract, a sample was collected and stored in freezer at -20°C. The crude extract was centrifuged at 6000 rpm for 5 min to separate the cell debris. The supernatant was collected and centrifuged again at 14,000 rpm at 4°C for 1 h. The pellet was suspended in a known volume of buffer and served as the cell membrane protein fraction. While, the supernatant collected during the centrifugation step served as cytosolic protein fraction. The protein concentration in each of these two protein extracts was estimated by Bradford method against bovine serum albumin standard. Normalized protein concentration (190 µg per reaction) from each fraction was incubated with 10 mg/L Cr(VI) and 2 mM NADPH at 30 ⁰C. Appropriate controls lacking protein, containing heat killed protein and without NADPH were included. Samples were drawn at regular time intervals and analyzed for residual Cr(VI).

2.5. Cr(VI) toxicity

2.5.1. Oxidative stress and metabolic state of cells cultured with Cr(VI)

Alteromonas sp. cells cultured in ZMB with and without 100 mg/L Cr(VI) for 24 h were harvested by centrifugation, washed and re-suspended in potassium phosphate (60 mM, pH 7.6) buffer. The OD (600 nm) of these cell suspensions were adjusted to 0.3 and incubated with 20 µg/mL 2',7'–dichlorodihydrofluorescein diacetate (DCH2FDA) for quantifying intracellular reactive oxygen species (ROS). The cell suspensions with DCH2FDA were incubated at 30 ⁰C and 120 rpm for 30 min. The reduced DCH2FDA gets oxidized in the presence of ROS to yield green fluorescence emanating 2',7'–dichlorofluorescein, indicative of oxidative stress. Intracellular fluorescein fluorescence was measured using 480 nm and ≥ 530 nm, excitation and emission settings.

2.5.2. Esterase activity

Cellular esterases present in metabolically active cells cleave FDA to produce green fluorescein fluorescence, which can be quantified. The Alteromonas sp. cells cultured in ZMB with and without 100 mg/L Cr(VI) were harvested by centrifugation, washed and suspended in potassium phosphate buffer. These harvested cells were incubated with 10 µg/mL FDA and incubated at 30 ⁰C and120 RPM for 1 h. Fluorescein formed due to cleavage of FDA by esterases was quantified by fluorimeter.

2.6. Analytical methods

Adjustment of turbidity of cultures for obtaining desired cell densities was carried out with UV-Vis spectrophotometer (Lovibond XD-7500). Growth kinetics of Alteromonas sp. was monitoring by measuring turbidity at 600 nm using Infinite® 200 PRO (Tecan, Switzerland) multimode plate reader. Fluorescence emanating from fluorescein was measured using 480 nm and 530 nm, respectively, for excitation and emission with Infinite® 200 PRO multimode reader. The cell free liquid samples collected under different experimental conditions were analyzed with 1,5-diphenylcarbazide (DPC) method for determining Cr(VI) (Nancharaiah et al. 2010). In this method, Cr(VI) reacts with DPC dye and forms reddish purple 1, 5 diphenyl carbazone (DPCA) in acidic environment. The reddish purple colour is measured at 540 nm for quantifying Cr(VI). Total Cr was determined using Ultima 2 inductively coupled plasma-atomic emission spectrometer ((ICP-AES), Horiba Jovin Yvon, France).

2.7. Statistical analysis

Experimental data obtained from multiple replicates of Cr(VI) removal was processed and presented as mean ± standard deviation (SD). Student’s t-test was performed to determine the statistical significance between control and Cr(VI) treatment samples. The values were considered to be significant at P-values < 0.05.

3.1. Growth in presence of Cr(VI) and its reduction

Figure 1A shows the growth kinetics of Alteromonas sp. in the presence of different initial Cr(VI) concentrations. Growth of Alteromonas sp. was observed in the presence of 25 to 100 mg/L Cr(VI). A final OD of 0.83 was achieved within 48 h in the control as against ~ 0.65 at 100 mg/L Cr(VI). The overall growth of the bacterium was lower by 21% in the presence of 25 to 100 mg/L. Nevertheless, the growth rate of the bacterium was similar in the presence of 25, 50 or 100 mg/L Cr(VI) with that of control without Cr(VI). Thus, a marginal effect on growth in terms of a reduction in growth yield was evident in the presence of up to 100 mg/L Cr(VI). In contrast, a complete retardation in growth was observed in the presence of 150 or 200 mg/L Cr(VI). Based on these observations on growth, concentrations of up to 100 mg/L were chosen for Cr(VI) removal experiments. Figure 1B shows Cr(VI) removal by Alteromonas sp. at all the tested Cr(VI) concentrations. The removal kinetics were dependent on the initial Cr(VI) concentration. Near complete removal of 12.5, 25, 50 and 100 mg/L Cr(VI) was achieved in 6, 8, 10 and 24 h, respectively (Fig. 2B). Cr(VI) removal was not noticed in the abiotic (ZMB without inoculum) and heat killed (ZMB with heat killed inoculum) controls. Cr(VI) was removed, but the total Cr concentrations remained constant without much change indicating no net removal from the medium (Fig. 1C).

3.2. Salinity tolerance and Cr(VI) removal at varied salt concentrations

Growth of Alteromonas sp. at different salinities ranging from 3.5 to 11.5% was shown in Fig. 2A. The growth was observed at all the salinities ranging from 3.5 to 9.5%. As compared to the growth at 3.5% salinity, the growth was slightly lower at salinities of 5.5, 7.5 and 9.5%. However, a complete inhibition in growth was observed at 11.5% salinity. Figure 2B shows time course removal of 100 mg/L Cr(VI) by Alteromonas sp. at different salinities. The Cr(VI) removal potential was strongly dependent on the salinity. At 3.5 and 5.5% salinity, removal of 100 mg/L Cr(VI) was fast and complete within 24 h. At increased salinity of 7.5%, near complete removal was achieved in 72 h. However, only a 40% of 100 mg/L Cr(VI) was removed at 9.5% salinity. A complete inhibition in Cr(VI) removal was observed at 11.5% salinity (Fig. 2B).

3.3. Growth and Cr(VI) removal at alkaline pH

Figure 3A shows the growth of Alteromonas sp. at different medium pH. Normal growth was observed at pH from 7 to 11 (Fig. 3A). At pH 5 and 6, extended lag phase was observed. Growth was observed after 24 and 12 h of inoculation at pH 5 and 6, respectively. At pH 12, a complete inhibition in growth was seen. Higher absorbance (0.4) values noticed at pH 12 (at 0 h) were due to pH adjustment and not because of cells or growth (Fig. 3A and Fig. S1). Further increase in absorbance was not observed throughout the incubation period indicating no growth of Alteromonas sp. These results suggested pH 6 to 11 are suitable for the growth of Alteromonas sp.

Time course removal of 100 mg/L Cr(VI) by Alteromonas sp. at different media pH from 5 to 12 was shown in Fig. 3B. Cr(VI) removal was mainly observed under alkaline pH from 8 to 11. Although growth of the strain was normal at pH 7 in the absence of Cr(VI), growth was severely impacted in presence of Cr(VI) at pH 7 (Fig. S1). This has led to no Cr(VI) removal at pH 7. Near complete removal of 100 mg/L Cr(VI) was achieved within 24 h at pH 8 to 11 (Fig. 3B). At pH 12, removal of Cr(VI) was not observed. Cr(VI) removal from the medium without the inoculum was found to be ~ 20% at pH 5 and 6. But, Cr(VI) removal was negligible from the medium without inoculum at pH 7 to 12 (Fig. S2).

3.4. Tolerance and Cr(VI) removal in presence of heavy metals

Time course of Alteromonas sp. growth at different concentrations of Pb, As, Cu and Cd was shown in Fig. 4. Good growth of the strain was observed even in the presence of 100 mg/L of Pb, As or Cu. In case of Cd, growth was strongly inhibited in the presence of > 5 mg/L. Based on this data, 100 mg/L of Pb, As or Cu and 5 mg/L of Cd were chosen for determining Cr(VI) removal ability of the strain in the presence of heavy metals.

At a fixed concentration of heavy metals, removal of 12.5 to 100 mg/L Cr(VI) by Alteromonas sp. was shown in Fig. 4. Near complete removal of 12.5, 25, 50 and 100 mg/L Cr(VI) was achieved in 15, 15, 24 and 39 h, respectively, in the presence of 100 mg/L of Pb (Fig. 5A). Cr(VI) removal was moderately inhibited in the presence of Pb. For example, Cr(VI) removal was about 82% in 24 h in the presence of 100 mg/L Pb as compared to near complete removal without Pb. Removal of Cr(VI) by Alteromonas sp. was similar with or without As (Fig. 5B). The lag phase was absent in the Cr(VI) removal profiles indicating higher tolerance of Alteromonas sp. to As. In contrast, Cr(VI) removal was strongly inhibited in the presence of Cu and Cd (Fig. 5C and D). Although near complete removal of 12.5 to 50 mg/L Cr(VI) was achieved in the first 24 h, complete removal of 100 mg/L Cr(VI) was not observed in presence of Cu. Even with 48 h incubation, only 40% Cr(VI) removal was observed with 100 mg/L Cu (Fig. 5C). Among all the tested heavy metals, Cd showed strongest inhibition on Cr(VI) removal by Alteromonas sp. (Fig. 5D). Near complete removal of 12.5, 25 and 50 mg/L Cr(VI) was observed in 38, 48 and 96 h, in the presence of 5 mg/L Cd. Extended lag phase was evident in the removal of 12.5 to 50 mg/L Cr(VI). A complete inhibition in the removal 100 mg/L was noticed in the presence of 5 mg/L Cd.

3.5. Cr(VI) removal in presence of high cell numbers, resting cells and cell free supernatant

The effect of inoculum cell density on Cr(VI) removal was shown in Fig. 6. Complete removal of 100 mg/L Cr(VI) was observed in 15 and 24 h, respectively, with 10¹² and 10⁷ cells/mL inoculum densities (Fig. S3A). After 11 h of inoculation in ZMB, the Cr(VI) removals were 15% and 80%, respectively, with 10⁷ and 10¹² cells/mL (Fig. S3B). The removal rate of Cr(VI) from ZMB was dependent on the initial cell density used for inoculation. However, Cr(VI) removal was found to be minimal at 20% even after 6 d incubation when the experiment was performed with 10¹² cells/mL in natural seawater (Fig. 6 and Fig. S4). This was corroborated with negligible growth of Alteromonas sp. in the seawater. Removal of Cr(VI) by ZMB or cell free spent medium was found to be negligible.

3.6. Oxidative stress and metabolic activity in Cr(VI) challenged cells

Figure 7 shows intracellular ROS and esterase activity in Alteromonas sp. cells grown with and without 100 mg/L Cr(VI). Production of ROS was detected by DCH2FDA in the Alteromonas sp. cells grown in ZMB with and without Cr(VI). A significant increase in the ROS levels was evident when the cells grown in the presence of 100 mg/L Cr(VI) (Fig. 7A). ROS generation was about 2-fold higher in the cells grown in Cr(VI)-containing medium. Esterase activity indicates metabolic activity and viability of the cells. A 50% decrease in esterase activity was observed in the cells grown with 100 mg/L Cr(VI) containing medium as compared to control cells grown without Cr(VI) (Fig. 7B). The ROS generation and esterase activity measurements by DCH2FDA and FDA staining assays, respectively, indicates that Cr(VI) exerts toxicity and affects cellular metabolism.

3.7. Mechanism of Cr(VI) reduction

Cr (VI) removal by crude, cytosolic and membrane protein fractions under different conditions was summarized in Table 2. Efficient Cr(VI) removal was achieved using crude and cytosolic protein fractions by supplying NAPDH as electron donor (Fig. S5). In the absence of exogenous NADPH, the Cr(VI) removal by crude or cytosolic fraction was low at 15% and 35%, respectively, in 15 to 24 h. In contrast, about 99% of Cr(VI) was removed with crude or cytosolic fraction by supplying NADPH. Autoclaving of crude and cytosolic fractions inhibited their Cr(VI) removal activity. Only a marginal Cr(VI) removal was observed with membrane protein fraction. Cr(VI) removal was not observed in the control experiments carried out with the plain buffer and reaction mixture without protein extract. These results confirmed the requirement of crude or cytosolic protein fraction along with NADPH for exhibiting Cr(VI) removal activity.

Table 2

Reduction of 10 mg/L Cr(VI) by different protein fractions of *Alteromonas* sp. under different experimental conditions.
Hours	Blank	Supernatant without NADPH	Protein factions with NADPH			Autoclaved protein fractions with NADPH
Hours	Blank	Supernatant without NADPH	Crude	Cytosolic	Membrane	Crude	Cytosolic	Membrane
0	10.2*	11.2	10.1	10.3	10.6	12.7	12	12
15	10.1	8.5	1.2	0.95	7	10.9	11	6.5
24	10.4	6.5	1.3	0.77	6.6	11.5	10.9	8.1
*values represent Cr(VI) concentrations.

4.1. Cr(VI) removal by Alteromonas sp. under haloalkaline conditions

Alteromonas sp. was originally isolated from the aerobic granular sludge cultivated from seawater microbiome at pH 8 and 3.5% salinity in a sequencing batch reactor (Sarvajith and Nancharaiah 2020). The strain grew well in the presence of salt ions at 3.5 to 9.5% salinity and pH from 6 to 11. It had optimum growth at 3.5 to 9.5% salinity and 7 to 11 pH, hence deemed to be considered as halophilic alkaliphile (Mesbah and Wiegel 2012). Alteromonas sp. is a commonly reported marine bacterium with versatile metabolic potential and adaptability to the changing environmental conditions (Cusick et al., 2020). The members of Alteromonas genus are identified with higher tolerance to heavy metals including Co(II), Cu(II) and Zn(II) (Cusick et al., 2020). Recently, Alteromonas sp. was characterized with high tolerance and reduction of metal(loid) oxyanions such as selenite and tellurite under saline conditions (Reddy et al., 2023). In spite of its high metal tolerance, there are no previous studies on the use of Alteromonas sp. for chromate removal.

This study demonstrated that Alteromonas sp. can grow efficiently up to 100 mg/L Cr(VI) in the presence of salt and alkaline pH. Growth of Alteromonas sp. was completely retarded at 150 mg/L Cr(VI) and above, indicating potential toxicity at higher concentrations. Besides growth, complete removal of 12.5 to 100 mg/L Cr(VI) was achieved within 24 h. The Cr(VI) removal rates were determined to be 2.1, 3.1, 5 and 4.1 mg/L h^− 1, respectively, from 12.5, 25, 50 and 100 mg/L Cr(VI). The variation in removal rates was related to initial Cr(VI) concentration and the length of initial lag phase. Nevertheless, fast removal kinetics and near complete removals were attractive for determining removal kinetics in the presence of heavy metals and understanding Cr(VI) reduction mechanisms.

It is widely reported that Cr(VI) removal activity is strongly influenced by salts ions and pH. For example, Cr(VI) removing ability of Cellulosimicrobium funkei was decreased by 50% in the presence of 3% salt ions (Karthik et al., 2017). Similarly, many of the Cr(VI) removing bacteria that fail to grow in alkaline pH are not suitable for bioremediation of alkaline waters and soils (Mishra et al., 2012; Karthik et al., 2017). For ex, efficient Cr(VI) reducing Bacillus sp. FM1 was sensitive to high pH, wherein rapid decrease in Cr(VI) reduction was observed at pH above 9. The optimum growth of Alteromonas sp. in the presence of high salt (3.5 to 7.5% salinity) and alkaline pH (7.6 to 11) facilitated efficient Cr(VI) removal. The ability of the strain to grow in the presence of salt ions was decreased by Cr(VI). This was clearly evident by growth inhibition in the medium with 9.5% salt in the presence of 100 mg/L Cr(VI). Due to halophilic nature, complete removal of 100 mg/L Cr(VI) was achieved with an extended incubation time of 96 h at 7.5% salinity. Therefore, Alteromonas sp. used in this study can perform efficient Cr(VI) removal apart from its high salinity tolerance. The growth of Alteromonas sp. was inhibited at pH 5 to 7 and 12 when the medium contained 100 mg/L Cr(VI). Profuse growth and efficient Cr(VI) removal at pH 8 to 11 indicates the suitability of Alteromonas sp. for biological treatment of saline and alkaline wastes.

4.2. Cr(VI) removal in the presence of heavy metals

Metal ions such as Cu(II), As(V), Pb(II), and Cd(II) are most commonly reported in Cr containing effluents (Chowdhury et al., 2015; Kaya et al., 2016). The metal tolerance of Alteromonas sp. strains have been demonstrated using several metal ions including Cu(II), Co(II), Mn(II), Zn(II), and Hg(II) (Cusick et al., 2020; Math et al., 2012). Independent growth experiments in the presence of each of 100 mg/L Cu(II), As(V) or Pb(II) showed normal growth (Fig. 4), indicating high tolerance to heavy metals. However, growth was strongly inhibited in the presence of Cd(II) at > 5 mg/L. Similar effects of Cd(II) on growth inhibition was previously reported (Abo-Alkaasem et al., 2022). Complete removal of 12.5 to 100 mg/L Cr(VI) was not affected by the presence of 100 mg/L As(V) or Pb(II) (Fig. 5A, 5B). In the presence of Cu(II) and Cd(II), removal of higher concentrations of Cr(VI) was strongly affected. The decrease in Cr(VI) removal efficiencies in the presence of Cu and Cd may be due to either direct toxicity or inhibition of enzymes/molecules involved in bacterial growth and/or Cr(VI) removal. It was earlier reported that Cr(VI) removal by Bacillus sphaericus, Exiguobacterium mexicanum, and Salipaludibacillus agaradhaerens is significantly decreased in the presence of Pb(II) or Cd(II) (Pal and Paul 2004; Abo-Alkasem et al., 2021; Das et al., 2021). Presence of divalent metal cations such as Ni(II), Zn(II), Cd(II), and Pb(II) lowered the Cr(IV) reduction rates by Acinetobacter sp. Cr-B2 (Hora and Shetty 2014). This decrease in Cr(VI) removals was corroborated with the growth inhibition, indicating that Cu(II) and Cd(II) exerts a direct toxicity on cells. The effect of the heavy metals on growth was more prominent at higher Cr(VI) concentrations suggesting that combined toxicity was responsible for extended lag phase or complete inhibition of growth. Cr(VI) removal was enhanced in the presence of Cu(II) in E. mexicanum as Cu(II) may presumably act as a co-factor for chromate reductase. In contrast, presence of 6 mg/L Cu(II) significantly (~ 50%) affected Cr(VI) reduction by Bacillus sp. FM1 (Masood and Malik 2011). Even in the current study, decrease in Cr(VI) removal in the presence of Cu(II) was observed and was attributed to growth inhibition due to toxicity.

Alteromonas sp. cells grown in presence of Cr(VI) experienced severe oxidative stress (Fig. 6B). In bacterial systems, ROS generation is mainly linked to the Cr(VI) detoxification mechanism under aerobic conditions. The generated ROS could damage cellular components such as membranes, nucleic acids, proteins and enzymes to reduce the metabolic activity as seen from the lower metabolic activity in the cells grown with Cr(VI).

4.3. Cr(VI) removal mechanism

Removal of Cr(VI) was negligible in the abiotic, heat killed and spent medium (cell free supernatant) controls. Efficient Cr(VI) removal was achieved by inoculating growth medium with Alteromonas sp. cells. The Cr(VI) removal was minimal at < 25% from seawater inoculated at similar cell densities. Thus, growth medium (ZMB) and actively growing cells are essential for achieving Cr(VI) removal. The time course profiles of growth, Cr(VI) and total Cr, revealed (i) conversion of soluble Cr(VI) to Cr(III) and (ii) strong association between the growth and Cr(VI) reduction. The rate of Cr(VI) reduction was significantly enhanced by increasing the inoculum cell density to 10¹² cells/mL from 10⁷ cells/mL in the growth medium (Fig. 6). About 80% reduction of Cr(VI) was achieved with 10¹² cells/mL within 11 h as against < 20% reduction by 10⁷ cells/mL (Fig. 6). The faster kinetics of Cr(VI) by higher cell densities may be related to ready availability of biochemical/enzymatic machinery and negligible lag phase in the growth curve in nutrient rich medium. Faster and efficient reduction of Cr(VI) or metal(loid)s has been reported using pure cultures and mixed culture biofilms (Reddy and Nancharaiah, 2018; Reddy et al., 2023). The lag phase was evident in the Cr(VI) removal profiles recorded with and without heavy metals. At higher inoculum densities, elimination of lag phase result in faster Cr(VI) removal kinetics. The lower Cr(VI) removals achieved with higher cell densities in seawater was related to unavailability of nutrients required for growth. Therefore, growth medium and actively growing cells are prerequisites for efficient reduction of Cr(VI) by Alteromonas sp. cells.

The protein fractions obtained from lysed Alteromonas sp. cells gave insights into the Cr(VI) tolerance and removal mechanisms. Cr(VI) reduction under aerobic conditions involve the use of enzymatic reductases (Chen and Tian 2021). These can be membrane bound or soluble cytosolic in nature, which utilises reducing equivalents (NADPH or NADH) for Cr(VI) reduction (Cervantes et al., 2001). Aerobic Cr(VI) reduction involves the formation of transient intermediate Cr(V). Reoxidation of Cr(V) generates ROS, as observed from the Alteromonas sp. cells grown with Cr(VI). Reduction of Cr(V) by reductases yields stable Cr(III). The Cr(III) can be either accumulated inside the cells (Karthik et al 2017) or transported out of the cells (Ramírez-Díaz et al. 2007). The protein fractions obtained from Alteromonas sp. cells confirmed involvement of intracellular soluble reductases in Cr(VI) reduction (Table 2). Both crude and cytosolic protein extracts exhibited Cr(VI) reducing activity using NADPH as electron donor. In contrast, Cr(VI) reducing activity was marginal with the membrane protein fraction. A significant decrease in Cr(VI) reducing activity was observed in the absence of NADPH. Based on these results, involvement of NADPH dependent chromate reductases of cytoplasm are responsible for efficient and growth dependent Cr(VI) reduction by Alteromonas sp.

4.4. Practical implications

Microbial reduction of toxic Cr(VI) to non-toxic Cr(III) is an effective method for bioremediation of Cr(VI)-contaminated waters and soils. The salt ions, alkaline pH and heavy metals can strongly influence the growth, metabolism and Cr(VI) reduction potential of microorganisms considered for bioremediation. In this context, identification of microorganisms and characterization of reduction mechanisms is desirable for developing robust treatment technologies. The marine bacterial strain, Alteromonas sp. reported in the current study is shown to tolerate and grow under haloalkaline conditions and in presence of several toxic heavy metals. Additionally, this bacterium is previously shown to tolerate other metals and metalloids which are potentially toxic in nature. Aerobic mode of growth, efficient Cr(VI) removal at high salinity, alkaline pH and in presence of toxic heavy metals can make it an candidate organism for bioremediation of complex wastes.

This study, for the first time, demonstrates efficient Cr(VI) reduction by an Alteromonas sp. under extreme haloalkaline conditions and in the presence of heavy metals. The Alteromonas sp. strain was halophilic, alkaliphilic and exhibited robust growth at 3.5 to 9.5% salinity and pH 5 to 11. The strain had an obligate requirement for alkaline pH for performing Cr(VI) reduction. At 3.5% salinity and pH 7.6, complete removal of 100 mg/L Cr(VI) was achieved in 24 h. The growth and Cr(VI) removal were not inhibited in the presence of up to 100 mg/L each of Pb(II), As(V), Cu(II) or 5 mg/L Cd(II) supplied individually. The toxicity of Cr(VI) on Alteromonas sp. cells was evident in terms of ROS generation and inhibition of esterase activity. In-spite of toxicity, the cells exhibited profuse growth and Cr(VI) reduction. Cr(VI) was removed from the medium by converting to non-toxic Cr(III). Actively growing cells and growth medium were essential for achieving Cr(VI) removal. The cytosolic protein fraction was identified to be responsible for converting Cr(VI) to Cr(III) using NADPH as electron donor. Tolerance to multiple stressors and efficient reduction toxic Cr(VI) to no-toxic Cr(III) in the presence of harsh environmental conditions (salinity, alkaline pH and heavy metals) suggest potential use of Alteromonas sp. in bioremediation and biological treatment of Cr(VI)-containing wastes.

Acknowledgements

Authors acknowledge and thank Abdul Nishad of Bhabha Atomic Research Centre (BARC) for his support in total Cr estimations using ICP-AES.

Funding

This work was supported by Bhabha Atomic Research Centre, Department of Atomic

Energy, Government of India through CG VISION PROJECT 2020-2023.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by G. Kiran Kumar Reddy, K. Kavibharathi and Anuroop Singh. The first draft of the manuscript was written by G. Kiran Kumar Reddy and Y.V. Nancharaiah commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability statement

All data generated or analysed during this study are included in this published article and its supplementary information file.

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Growth-dependent Cr(VI) reduction by Alteromonas sp. under haloalkaline conditions: toxicity, removal mechanism and effect of heavy metals

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Bacterial source, chemicals, culture media, and conditions

2.2. Growth of Alteromonas sp. under different conditions

2.2.1. Growth in the presence of different Cr(VI) concentrations

2.2.2. Growth at different salinities

2.2.3. Growth at different pH

2.2.4. Growth in the presence of different heavy metals

2.3. Cr(VI) removal by Alteromonas sp.

2.3.1. Cr(VI) removal at different initial concentrations

2.3.2. Cr(VI) removal at different salinities

2.3.3. Cr(VI) removal at different pH

2.3.4. Cr(VI) removal in presence of heavy metals

2.4. Cr(VI) removal mechanisms

2.4.1. Cr(VI) reduction by higher inoculum cell densities, resting cells, and spent media

2.4.2. Cr(VI) reduction mechanism by Alteromonas sp.

2.5. Cr(VI) toxicity

2.5.1. Oxidative stress and metabolic state of cells cultured with Cr(VI)

2.5.2. Esterase activity

2.6. Analytical methods

2.7. Statistical analysis

3. Results

3.1. Growth in presence of Cr(VI) and its reduction

3.2. Salinity tolerance and Cr(VI) removal at varied salt concentrations

3.3. Growth and Cr(VI) removal at alkaline pH

3.4. Tolerance and Cr(VI) removal in presence of heavy metals

3.5. Cr(VI) removal in presence of high cell numbers, resting cells and cell free supernatant

3.6. Oxidative stress and metabolic activity in Cr(VI) challenged cells

3.7. Mechanism of Cr(VI) reduction

4. Discussion

4.1. Cr(VI) removal by Alteromonas sp. under haloalkaline conditions

4.2. Cr(VI) removal in the presence of heavy metals

4.3. Cr(VI) removal mechanism

4.4. Practical implications

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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