Biogenic sulfides for sequestration of Cr (VI), COD and sulfate from synthetic wastewater

Peer review under responsibility of National Water Research Center.

Abstract

The aim of this work is to explore the possibility of using mixed culture of mesophilic sulfate-reducing bacteria (SRB) for retrieval of toxic and carcinogenic Cr (VI) from synthetic solution. In order to treat Cr (VI) containing wastewater effectively, SRB culture was adapted to 50 mg/L Cr (VI) and maintained through repeated sub-culturing to enhance the growth and activity of SRB. Batch biosorption experiments were carried out in glass serum vials by cultured SRB, accomplishing the removal of 82.1% Cr (VI), 76.9% sulfate, 85.7% COD under the following optimized conditions: pH 7, hydraulic retention time (HRT) 7 days, temperature 37 °C and initial Cr (VI) concentration of 50 mg/L. Further sorption experiments were conducted on synthetic wastewater under optimal operational conditions and resulted in 89.2% Cr (VI), 81.9% COD and 95.3% sulfate reduction from simulated wastewater. The results of this work contributed to a better understanding of metal uptake by biogenic sulfides and would be beneficial in the development of potential biosorbents that possess high capacities for Cr (VI) uptake from aqueous environments.

Keywords:

• Cr (VI)
• Sulfate-reducing bacteria (SRB)
• Hydrogen sulfide (H₂S)
• Chemical oxygen demand (COD)
• Hydraulic retention time (HRT)
• Wastewater

1 Introduction

In natural environment, chromium can exist in several oxidation states ranging from Cr (II) to (VI) among which trivalent Cr (III) and hexavalent Cr (VI) are the most dominant chromium species in the environment (Dogan et al., 2011; Vaiopoulou and Gikas, 2012; Cirik et al., 2013). The Cr (VI) compounds are used in metallurgical and chrome plating industries for chrome alloy and chromium metal production. It is used as an oxidizing agent and in the production of other chromium compounds in chemical industry. About 80–90% of leather tanning process utilizes chromium chemicals; out of which 40% of chromium used is discharged in the effluent as Cr (VI) and Cr (III) (Saha and Orvig, 2010). Cr (III) salts are used less widely, yet are being employed in photography, textile dyeing, ceramics and glass industry. Cr (III) is required in trace amount for living organisms as it decreases body fat, cholesterol and triglyceride levels, activating enzyme reactions, and increasing the muscle mass (Dhal et al., 2013). Cr (VI) is not only highly toxic to all forms of living organisms but also mutagenic and carcinogenic in humans and animals (Losi et al., 1994). The U.S. Environmental Protection Agency (EPA) has classified Cr (VI) as a Group ‘A’ human carcinogen. So due to its high toxicity, stringent regulations are applied on the discharge of Cr (VI) to surface water. The EPA has set a limit of 100 μg Cr(III) and 50 μg Cr (VI)/L for drinking water and a limit of ≤0.05 mg/L (Baral and Engelken, 2002) for its discharge into surface water, while total chromium, including Cr (III), Cr (VI) and its other forms, should be lower than 2 mg/L (Zayed and Terry, 2003). So treatment of wastewater is one of the growing concerns in environmental cleaning. Conventional methods for treatment of metal contaminated wastewater are restricted, because of technical or economical constrains (Kiran and Kaushik, 2012; Dhal et al., 2013). Therefore, recent studies have concentrated on the development of low cost processes. The use of microorganisms including bacteria, fungi, algae and yeast has gained much more attention in recent years since they carry a wide range of binding sites for heavy metal ions (Mona et al., 2011; Kiran and Thanasekaran, 2012; Singh et al., 2013). Cr (VI) reduction by biogenic sulfides is one of the major approaches used for detoxification of Cr (VI) containing wastewater. Cr (VI) can be reduced in biological treatment processes to Cr (III), which is almost insoluble and less-toxic chromium form (Agrawal et al., 2006). The best way to reduce the mobility of heavy metals is to transform them into insoluble compounds such as sulfides, which are more stable forms. It has been reported that certain heavy metals such as Cr (VI) may be reduced under sulfate reducing conditions (Singh et al., 2011; Sahinkaya et al., 2013; Cirik et al., 2013). Sulfate-reducing bacteria (SRB) under anaerobic conditions oxidize simple organic compounds (such as acetic acid and lactic acid) by utilizing sulfate as an electron acceptor and generate hydrogen sulfide. Hydrogen sulfide reacts with heavy metal ions to form insoluble metal sulfides that can be easily separated from solution (Steed et al., 2000; Luptakova and Kusnierova, 2002; Lloyd, 2003). The potential advantages of metal precipitation by biogenic sulfides include the production of lower sludge volumes and products with lower solubility as compared to hydroxide precipitation (Gadd, 2004). In addition, valuable metals can be recovered from metal sulfide sludges (Boonstra et al., 1999). Several studies have reported process optimization, organic carbon (electron donor) consumption coupled with sulfate reduction, hydrogen production and the efficiency of metal precipitation in the biological treatment with biogenic sulfide (Bertolino et al., 2012; Zhou et al., 2013; Martins and Pereira, 2013; Bai et al., 2013). In the present work, the effect of varying pH, temperature, metal dose and incubation time were optimized so as to attain high removal efficiency of Cr (VI) by SRB. Thus this effort provides optimum conditions for efficient removal of Cr (VI) using consortium of sulfate-reducing bacteria. These optimized parameters were further applied to investigate efficiency of biogenic sulfides produced from sulfate-reducing bacterial consortia in sequestration of chromium (VI), COD and sulfate to treat simulated wastewater in a small scale bioreactor.

2 Materials and methods

2.1 Growth media and conditions

A modified Postgate growth medium was used in all experiments. The composition of the Postgate growth medium used was as (g/L): KH₂PO₄ 0.5; Na₂SO₄ 1.0; NH₄Cl 2.0; CaCl₂ 0.06; FeSO₄ 0.005; sodium citrate 0.3; yeast extract 0.1; sodium lactate 15 mL; Resazurin 0.1%. All incubations were done at 37 °C in the dark. All chemicals were of analytical grade, and solutions were prepared with sterile deionized water.

2.2 Source of inoculums

The mixed culture of SRB used in the present study was obtained from the sludge sample collected from anaerobic digester sludge of sewage treatment plant. One gram of the screened sludge was inoculated in small scale bioreactor (1000 mL) containing 800 mL Postgate (1984) isolation selective medium. The composition of the Postgate isolation media was as (g/L): Na₂SO₄ 1.0; KH₂PO₄ 0.5; NH₄Cl 1.0; CaCl₂ 0.7; FeSO₄ 0.1; yeast extract 1.0; agar 15.0; mercaptoacetic acid 0.1; ascorbic acid 0.1; ethanol (absolute) 4.5 mL; and Resazurin 0.1%. Reactor was kept in anaerobic conditions for the growth of anaerobic bacteria for a period of 7 days at 37 °C till the color of media changed to blackish gray. After 7 days of anaerobic incubation, the 200 mL inoculum of isolates was further transferred into 800 mL sterilized Postgate growth medium in another 1000 mL reactor. The Postgate medium is partially selective for SRB, with sodium lactate as electron donor and carbon source. The H₂S generation and blackening of the medium were observed as positive indication for the presence of sulfate-reducing bacteria.

2.3 Batch experiments

Experiments were conducted in batch system under anaerobic conditions. Glass serum vials (120 mL) were used for all the experiments. 80 mL of modified Postgate medium with pH 7 was added in the vials for batch tests. After inoculation, the bottles were sealed with butyl rubber stoppers and aluminum crimp seals, and incubated at temperature 37 °C under static condition. Oxygen was replaced from the gaseous phase with nitrogen. Bacteria used in batch experiments were cultivated in closed serum vials using standard procedures for SRB (Postgate, 1984). Subsequently the vials were sealed and 5 mL inoculum of bacteria cultivated in Postgate medium was added by a sterile syringe. The change in color from yellowish to colorless indicated the growth of the consortium (Singh et al., 2011). All tests were performed in duplicates; average values were reported. Later optimized parameters were applied for treatment of synthetic wastewater. The reactors were kept in BOD incubator at optimized conditions for 7 days under static conditions. Sampling from the reactors was performed at 0 h and day 7 of incubation.

2.4 Analysis for various parameters

Liquid samples were centrifuged using a Hettich Rotofix 32 centrifuge at 3000 rpm for 10 min, prior to determination of COD, Cr (VI) and sulfate in the supernatant. Spectralab COD Digester (2015 M) and COD Titrator (CT-15) were used to determine soluble chemical oxygen demand in the supernatant. Total residual chromium was quantified by Atomic Absorption Spectrophotometer (Shimadzu AA-6300, Japan). The sulfate in the diluted sample was measured using standard methods of APHA-1995. pH was also measured according to the standard methods recommended by (APHA, 1995). The removal (%) of heavy metal was determined using the following equation:(1) $\text{Removal}\%=\frac{C_i-C_f}{C_i}\times 100$(1) where C_i is the initial heavy metal concentration; C_f is the final heavy metal concentration.

3 Results and discussion

Effectiveness of the process was evaluated through diagnosis of sulfate, metals ions and COD bioreduction in simulated wastewater. Formation of black precipitate is indicative of sulfide as a reduction of sulfate (SO₄²⁻) to sulfide (S₂⁻) (Herbert and Gilbert, 1984). In batch biosorption study, experiments were conducted for the optimization of different parameters such as pH, temperature, Cr (VI) concentration, and HRT. The results are explained in the following sections.

3.1 Effect of pH

The pH had a key effect on the biogenic sulfide ions in solution and effected the reduction of Cr (VI). The experiments were conducted at constant initial Cr (VI) concentration of 50 mg/L at temperature 37 °C, by varying pH from 5 to 9. As evident from the results, at pH 7, maximum COD removal was 55% on day 7. As shown in Fig. 1, there was a progressive increase in biosorption of Cr (VI) with increase in pH up to 7.0 and later decreasing trend of Cr (VI) removal was investigated with further increase in pH. The maximum removal percentage of Cr (VI) and sulfate was found to be 84.78% and 49.9% respectively at pH 7; then a reduction in efficiency of sulfate removal was observed above pH 7.0. These results are coincided with that found by Pagnanelli et al. (2012). They found that pH 7.6 was the optimum value for removal of Cr (VI) contaminated waters by sulfate-reducing bacteria. Bratkova et al. (2013) reported similar findings as maximum rate of sulfate reduction was achieved at pH 7.25. It is reported that optimum pH for growth of SRB is between pH 5.5 and 9 (Barton, 1995). Mostly thermophilic bacteria grow near neutral pH from 6 to 8 but pH value exceeding the bounds is liable to cause deactivation of SRB activity (Widdel, 1988; Zhao et al., 2011).

Fig. 1 Effect of pH on reduction of various parameters at constant metal dose 50 mg/L, temperature 37 °C and time 7 days.

3.2 Effect of temperature

Optimum temperature was obtained for the isolated consortia by conducting experiments at temperature ranging from 30 to 40 °C for 7 days of incubation at constant initial metal ion concentration Cr (VI) of 50 mg/L. As shown in Fig. 2, maximum reduction in COD and sulfate was observed to be 45.8% and 76.5% respectively. The utmost removal of Cr (VI) was 90.3% at an optimum temperature of 37 °C and pH 7.9 but with further increase in temperature, the removal efficiency was decreased. Thus it can be concluded from the results that at 37 °C, maximum growth of SRB and maximum reduction in parameters such as COD, sulfate and metal ion concentration will be obtained. Zhou et al. (2013) outlined similar findings as 37 °C was found to be the optimum temperature for sulfate reduction and Cu (II) removal from wastewater.

Fig. 2 Effect of incubation temperature on reduction of various parameters at optimized pH 7, constant metal dose 50 mg/L and incubation time 7 days.

3.3 Effect of heavy metal dosing

Initially, increasing dose of chromium (VI) up to 50 mg/L increased the microbial growth indicated that chromium acts as essential nutrient for growth. Beyond 50 mg/L of Cr (VI), microbial growth was inhibited due to its toxicity. The activity and growth of bacteria were strongly affected by metals as it interferes with nucleic acids and enzyme active sites (Sani et al., 2001). Many enzymes and co-enzymes depend on a minimal amount of certain traces of metals for their activation and activity. When present in large amounts, they cause an inhibitory or toxic effect to micro-organisms (Li and Fang, 2007). Thus 50 mg/L concentration was considered as the optimum metal loading dose for further experiments. The maximum metal and COD removal efficiency for Cr (VI) at 50 mg/L were determined to be 82.1% and 85.7% but it decreased with further increase of metal ion dosing as shown in Fig. 3. The maximum sulfate removal efficiency was observed to be 76.9% due to more H₂S production but it decreased with further metal ion dosing beyond 50 mg/L. This may be due to the increase in the number of metal ion competing for available binding sites in the biosorbent and also due to lack of binding sites for the complexation at higher concentration level.

Fig. 3 Effect of initial metal dose on reduction of various parameters at optimized pH 7 and temperature 37 °C and incubation time 7 days.

3.4 Effect of hydraulic retention time (HRT)

Hydraulic retention time (HRT) is the key factor that affects proficiency of bioreactors and has been reported extensively (Rockhold et al., 2002; Kaksonen et al., 2004; Singh et al., 2011). A shorter HRT may not allow adequate time for SRB activity to neutralize acidity and precipitate metals. A longer HRT may imply depletion of either the available organic matter source or the sulfate source for SRB (Dvorak et al., 1992). The reactors were set up for 7 days at optimum conditions temperature 37 °C with sodium lactate as carbon source. As shown in Fig. 4, it could be concluded that pH of the reactor fluctuates between 7.0 and 7.49, which was optimum for the activity of the SRB. The continuous COD reduction was observed and 86.7% was achieved after 168 h of incubation period. Cr (VI) and sulfate removal were noticed to be 82.7% and 91.5%, respectively on day 7 of incubation period.

Fig. 4 Effect of hydraulic retention time on various parameters pH (secondary X axis), removal of COD, sulfate and Cr (VI) by SRB consortium at pH 7, metal dose 50 mg/L and temperature 37 °C.

3.5 Treatment of simulated water under optimum conditions

The treatment of simulated wastewater was performed under the optimized condition of Cr (VI) 50 ppm, temperature 37 °C and pH 7 for 7 days. The two reactors each of 1000 mL capacity were used in this study for Cr (VI) removal from simulated wastewater. Out of these two reactors one was set as a control and was run parallel to check the removal efficiency without microbial growth (SRB). 100 mL SRB innoculum was added into the 900 mL mixture in 1:1 ratio (v/v) of Postgate growth media and wastewater in the reactors. The wastewater treatment study was carried out in duplicate and the mean value was reported. One reactor was inoculated with 100 mL SRB inoculum, while the second was kept as control having Postgate media only. The anaerobic conditions were created by maintaining all reactors air-tight. The reactors were coupled with aspirated bottles filled with labeled water level. Decreased water level in aspirated bottle indicated production of gas which was due to growth of SRB. The level of the water was continuously monitored while water level in control reactor remained the same. The reactors were kept in BOD incubator at optimized conditions i.e. temperature 37 °C with Cr (VI) 50 mg/L for seven days under static conditions. Sampling from reactors was performed at the time of incubation (0 hr) and after seven days. The increase in pH of effluent from 6.35 to 7.82 indicated growth of SRB due to formation of hydroxyl ions. At this pH, SRB were capable of removing 95.3% sulfate, 81.9% COD and 89.2% Cr (VI) from the simulated wastewater in an anaerobic bioreactor operated under alkaline condition with lactate as organic carbon source. The microorganisms present in the wastewater convert this sulfate into biogenic sulfides which helps in heavy metal precipitation.

4 Conclusion

The study concludes that the maximum removal of chromium was found to be 89.2% in simulated wastewater with initial 50 mg/L of Cr (VI) loading and sodium lactate as carbon source for 7 days of HRT. Treatment of synthetic wastewater under optimized conditions resulted in high removal efficiency for Cr (VI), sulfate and COD because SRB utilized organic substrate from wastewater as a carbon source. The obtained results confirmed that consortium of SRB was found suitable for development of an efficient and economic biosorbent for removal of sulfate and heavy metals from wastewater.

Conflict of interest

Authors wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Notes

Peer review under responsibility of National Water Research Center.