Bacterial assisted treatment of anaerobically digested distillery wastewater

Abstract

The present study focused on the treatment of anaerobically digested distillery wastewater (ADSW) by a bacterial method as the high COD (30–35 000 ppm), BOD (8–10 000 ppm), total solids (60–65 000 ppm) and presence of organic compounds even after anaerobic treatment of wastewater indicated that the conventional treatment methods are far from reaching the safe limits for disposal. ADSW consists organic and inorganic compounds that need to be degraded. This study was carried out with a culture rich in Pseudomonas sp., which was more effective and would make the further treatment process easier. The consortia of bacterial culture was taken from DEE soil and ADSW contaminated soil, and the bacteria were grown in Kings B media to enrich the Pseudomonas sp. The bacterial system caused a reduction of the organic pollution load (COD) of ADSW up to 26.05% for ADSW contaminated soil bacteria and 18.15% for DEE soil bacteria. Conventional immobilization of bacteria in sodium alginate also performed to determine the reduction level of pollution in ADSW and the result was compared with free bacterial treatment. This bacterial system can further be combined with other aerobic treatment for enhanced COD reduction.

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1. Introduction

Central Pollution Control Board (CPCB) has listed distilleries among the top 17 most polluting industries of India (http://www.cpcb.nic.in). According to the Environment (Protection) rules 1986, CPCB has prescribed safe disposal limits for distilleries (Table 1). Distillery wastewater (spent wash) treatment is being carried out generally by three routes in this industry: (a) concentration followed by incineration; (b) direct oxidation by air at a high temperature followed by aerobic treatment, and (c) anaerobic digestion with biogas recovery followed by aerobic polishing (http://www.environmental-expert.com). Out of the three routes, anaerobic digestion is mostly used in South Indian Distilleries because of economic and environmental factors. However, the characteristics of treated wastewater through this route require further treatment to reach a safe disposal limit (Table 2). Physical and/or chemical methods are costlier and are less employed. In recent years, wastewater treatment using biological systems have attracted the attention of researchers and have helped in the development of an efficient low cost wastewater treatment system.¹ Biological processes have been utilized for the removal of organic compounds from effluent. Microorganisms, such as fungi,^2,3 yeast,^4,5 algae⁶ and bacteria, have been used for the degradation and decolorization of distillery effluent. Various bacterial groups, such as Lactobacillus plantarum,⁷ Bacillus licheniformis, Bacillus sp., Alcaligenes sp.,⁸ Klebsiella oxytoca, Serratia marcescens, Citrobacter sp.,⁹ Pseudomonas aeruginosa PAO1, Stenotrophomonas maltophilia, Proteus mirabilis,¹⁰ and Pseudomonas putida, Aeromonas sp., are reported as potential organisms to treat distillery wastewater.¹¹ This approach can remove most of the biologically removable organics, CODs, and color.^12–15 Identification and optimization of biotechnological treatment methods is a current necessity.¹⁶

Table 1 Safety disposal limits prescribed by CPCB for distilleries

S. no.	Parameter	Standard
1.	pH	5.5–9
2.	Total suspended solids	100 ppm
3.	BOD, 27 °C, 3 days inland water (river, lakes, streams)	30 ppm
4.	BOD, 27 °C, 3 days disposal on land for irrigation	100 ppm
5.	COD	250 ppm

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Table 2 Characteristics of anaerobically digested distillery wastewater (ADSW)

S. no.	Parameter	Value
1.	pH	7.5–8.0
2.	Total suspended solids	1800–3000 ppm
3.	BOD	8000–10 000 ppm
4.	COD	25 000–32 000 ppm

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Many reports deal only with treatment of distillery wastewater with dilution or artificial creation of wastewater for obtaining better treatment efficiency or for successful experiments. However, real wastewater characteristics largely differ from the experimental ones. In our study, real anaerobically digested distillery wastewater (ADSW) directly taken for the experiments with very minimal deviation from their original characteristics were utilized. The results showed in this study are very close to real time treatment of distillery wastewater, which help as an input for scaling up of an operation. We experimented and compared ADSW treatment with a culture rich in Pseudomonas sp. isolated from ADSW contaminated soil and from DEE soil. Conventional immobilization was also performed for finding closeness/deviation of results with free bacterial treatment.

2. Materials & methods

2.1. Soil sample collection

Soil from two different locations was collected for understanding the potential change of bacteria to degrade pollutants in the effluent as the source changes. The sources were soil near the Department of Energy & Environment and soil contaminated with ADSW around Trichy Distilleries & Chemicals Ltd, Tiruchirappalli.

2.2. Methodology for isolation of bacteria

2.2.1. Serial dilution method. Bacteria were isolated from the soil by a serial dilution method. The technique used to carry out a single dilution was repeated sequentially using more and more dilute solutions as the “stock” solution. At each step, 1 mL of the previous dilution was added to 9 mL of distilled water. Each step resulted in a further 10-fold change in the concentration from the previous concentration. Serial dilution is an inexpensive and most common method used for isolating microorganisms. The water used for the dilution was autoclaved so as to sterilize the water completely. 1 mL of solution from each test-tube were aseptically transferred using sterile pipettes into nutrient agar plates and all the processes were performed in a laminar air flow to maintain a sterile environment. The agar plates were made airtight by covering its sides with a parafilm and it was incubated for 24 hours at room temperature so as to obtain colonies. Even slants of the same were made for future use, which was stored at −20 °C. The colonies from these plates were picked by a sterile loop and were streaked onto the plates containing Kings B agar media so as to promote the growth of Pseudomonas species predominantly. The plates were incubated for 24 hours at 32 °C in a sterile environment.

2.2.2. Agar plate preparation. Agar bacterial plates were prepared by a pour plate method. Herein, we prepared 2 sets of agar plates: one having nutrient agar and the other a selective media for Pseudomonas species, i.e. Kings B media. The composition of both the media is given in Tables 3 and 4.

Table 3 Composition of nutrient agar

Ingredients	g L^−1
Peptone	5.000
Sodium chloride	5.000
Beef extract	1.500
Yeast extract	1.500
Agar	15.000
Final pH (at 25 °C)	7.4 ± 0.2

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Table 4 Composition of Kings B agar media

Ingredients	g L^−1
Proteose peptone	20.000
Dipotassium hydrogen phosphate	1.500
Magnesium sulphate heptahydrate	1.500
Agar	20.000
Final pH (at 25 °C)	7.2 ± 0.2

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The directions followed while preparing the plates for nutrient agar were as follows: suspended 43.41 grams in 950 mL distilled water; heated to boiling with frequent agitation to dissolve the medium completely; sterilized by autoclaving at 15 lbs pressure (121 °C) for 15 minutes. Cooled to 45–50 °C. Mixed well before dispensing. The plates into which the media was poured were completely sterile and the entire transferring activity was carried out in an aseptic environment in a laminar air flow to avoid cross contamination (Himedia technical data).

The procedure for preparing the plate for Kings B agar media was as follows: suspended 42.23 grams of dehydrated medium into 1000 mL distilled water containing 15 mL of glycerol; boiled to dissolve the medium completely; mixed well; sterilized by autoclaving at 15 lbs pressure (121 °C) for 15 minutes and finally it was aseptically poured into sterile Petri plates (Himedia technical data).

2.2.3. Bacterial cultivation. The colonies from the agar plate having Kings B agar media was picked and aseptically transferred to sterile Kings B broth. The medium was incubated in an incubator at 32 °C in order to grow the bacteria. The bacterial growth analysis was done using a spectrophotometer (Merck Spectroquant Pharo 300) at 600 nm.

2.3. ADSW treatment

The bacterial culture when at the exponential phase was inoculated into the effluent and the treatment studies were done for 72 h. COD analysis was carried out for the initial and final treatment time. COD was measured at 605 nm by a dichromate method using a thermal digester (TR 420) and spectrophotometer (Merck Spectroquant Pharo 300) instruments.¹⁷

2.4. Bacterial immobilization

Bacteria were immobilized using sodium alginate. 4 mL of bacterial culture in Kings B media was dispersed into a sterile autoclaved sodium alginate solution and thoroughly mixed using a magnetic stirrer at 600 rpm. After homogenizing the solution, this sodium alginate solution was added in a dropwise manner into a 0.1 M CaCl₂ solution. The beads were kept suspended in CaCl₂ solution for 3 h after which the CaCl₂ solution was decanted and the beads were washed with distilled water. These beads were further inoculated into the effluent for treatment of the effluent (ADSW).¹⁸

3. Results & discussion

3.1. Bacterial growth study

Bacteria were isolated from soil contaminated with ADSW. After serial dilution, the bacteria was allowed to grow on a common medium (nutrient agar) for 24 hours at room temperature as per standard microbiological methods.^19,20 Grant & Holt²¹ picked the colonies from the common media agar plates and streaked them onto selective media agar plates (Kings B) and incubated them for 24 h at 32 °C. In a similar way, the colonies were transferred to Kings B plate in our experiments. The colonies from the plate were picked and further inoculated into Kings B broth. 500 mL of Kings B broth was prepared and autoclaved to make it completely sterile and the colonies were picked and inoculated into the media aseptically in a laminar air flow to carry out the bacterial growth analysis as carried out by Johnsen & Nielsen.²² The analysis was done by measuring the OD (600 nm), bacteria wet weight and dry weight and substrate concentration at regular one hour intervals for 24 h until the stationary phase was reached. Fig. 1(a)–(c) shows the bacterial growth. Fig. 1(a) indicates the increase in the absorbance value because of an increase in cell density and reduction in the absorbance of the substrate due to the substrate utilization by bacteria. Increase in bacterial wet, dry weight and reduction in substrate concentration are shown in Fig. 1(b) & (c), which are indications of bacterial growth.

Fig. 1 (a) Graph showing the absorbance of bacterial growth and substrate with regard to time (b) showing the changes in the substrate concentration and bacterial wet weight with regard to time (c) showing the availability of substrate and changes in the dry weight of bacteria with regard to time.

3.2. Bacterial treatment of ADSW with ADSW contaminated soil born bacteria

3.2.1. Bacterial inoculum optimization. As the bacteria showed the potential to degrade the pollutants in the effluent in the preliminary studies, there is a need to optimize the amount of bacterial inoculum to improve the economy of the process and obtain maximum degradation.

Gay et al., (1996)²³ found the significance of inoculum concentration and pre-incubation temperature on Listeria monocytogenes. Through their approach, we too utilized 2 sets of 6 conical flasks each containing same amount of ADSW (1000 mL) and the conical flasks were inoculated with 100, 120, 140, 160, 180 and 200 mL of bacterial culture. All the inoculated cultures were maintained at room temperature and 180 rpm in an incubator cum shaker. Measurements such as biomass (wet), chemical oxygen demand (COD) and total dissolved solids (TDS) were taken every 3 hours. Measurements showed that the conical flask inoculated with 180 mL of bacterial culture had maximum degradation after 72 h (3 days) (Fig. 2–4, the average value is plotted in the figure). Furthermore, ADSW reduction slightly decreased and so no further concentration of bacterial inoculum was added. Fig. 2 shows the increase in wet biomass value (g) with respect to time, which indicates the bacterial growth on ADSW was in the similar way as experimented by Eroglu et al., 2010 (ref. 24) for measuring biomass accumulation as a function of growth time of R. sphaeroides under continuous illumination. In our study, inoculum concentrations of 180 mL and 200 mL resulted in the maximum biomass value (grams); however, for the 200 mL concentration, the stationary phase started at 69 hours, whereas for 180 mL concentration, it was started only in 72 hours. COD and TDS reduction were also at a maximum, i.e. 21.29% & 26.00% for the 72 hours of the treatment study with 180 mL concentration of bacterial inoculum (Fig. 3 and 4).

Fig. 2 Bacterial growth with varying inoculum concentration during ADSW treatment.

Fig. 3 COD Reduction in ADSW treatment with varying bacterial inoculum concentration.

Fig. 4 Total dissolved solids (TDS) reduction in ADSW treatment with varying bacterial inoculum concentration.

3.2.2. ADSW treatment study. The soil from the Trichy Distilleries & Chemicals Ltd, which was contaminated with the ADSW, was collected and the bacteria were isolated by the same method of serial dilution followed by growing it in a common media and then in Kings B media. The bacterial colonies obtained from the plates were further picked and allowed to grow in Kings B broth. 180 mL of optimized bacterial culture of OD 0.69 was inoculated into the effluent (1 litre ADSW) and the treatment study was carried out for 72 hours with the results shown in Fig. 5(a)–(d). Similarly one more trail was performed and the average value is plotted in the Fig. 5. The increase in biomass (wet and dry) with respect to treatment time are shown in Fig. 5(a), indicating the growth of bacteria rich in Pseudomonas sp. on ADSW.²⁴ Fig. 5(b)–(d) show the reduction of COD, total carbon (TC) and TDS values before and after 3 days of treatment. The treatment efficiencies were COD, TC and TDS up to 26.05%, 21.50% and 26.18%, respectively.

Fig. 5 (a) Bacterial biomass growth (wet and dry weight) with regard to time during the treatment of ADSW (b) COD reduction due to reduce in organic load (reduction percentage – 26.05%) (c) reduction in the total carbon (reduction percentage – 21.50%) (d) reduction in total dissolved solids concentration during bacterial treatment (reduction percentage – 26.18%) – source: ADSW contaminated soil.

3.3. Bacterial treatment of ADSW with DEE soil born bacteria

The soil from the Department of Energy & Environment was collected and serially diluted to isolate the bacterial colonies. The sample was poured into agar plates and further transferred to Kings B agar media to obtain culture rich in Pseudomonas sp. in the same way performed for ADSW contaminated soil bacteria. To carry out the treatment study, the colonies were transferred to Kings B broth. 180 mL of optimized culture was used as inoculum for the treatment of 1 litre of ADSW. The results are reported in Fig. 6(a)–(d). An increase in biomass (wet and dry) with respect to treatment time are shown in Fig. 6(a), indicating the growth of bacteria rich in Pseudomonas sp. isolated from DEE soil on ADSW.²⁴ Fig. 6(b)–(d) show the reduction of COD, total carbon (TC) and TDS values before and after three days of treatment respectively. The treatment efficiency for COD, TC and TDS were 18.15%, 13.94% and 14.50%, respectively.

Fig. 6 (a) Bacterial biomass growth (wet and dry weight) with regard to time during the treatment of ADSW (b) COD reduction due to decrease in organic load (reduction percentage – 18.15%) (c) reduction in the total carbon (reduction percentage – 13.94%) (d) reduction in total dissolved solids concentration during bacterial treatment (reduction percentage – 14.50%) – source: DEE soil.

3.4. ADSW treatment by bacterial immobilization

A conventional method of immobilization¹⁸ was also performed with sodium alginate to determine the deviations of parameters in the bacterial treatment of ADSW. The results are shown in Fig. 7(a) and (b). It was observed that the reduction of COD and TDS occurred to a lower extent than that of free bacterial treatment isolated from ADSW contaminated soil (19.87% and 23.15%). However, immobilization possesses the advantages of reusability of culture and easier separation processes. It could be further improved by having optimum inoculum concentration in the immobilized cell and treatment repetition.

Fig. 7 Treatment of ADSW with bacterial culture rich in Pseudomonas sp. in immobilized form (a) COD reduction with respect to days (reduction percentage – 19.87%) (b) total dissolved solids reduction with respect to days (reduction percentage – 23.15%).

4. Conclusion

Bacterial culture rich in Pseudomonas sp. was effective in the treatment of ADSW with a COD 25–32 000 ppm pollution load. The effect of inoculum concentration showed that a minimum of 180 mL mother culture is required for the treatment of 1 L of ADSW. It also showed the ability of the organism to grow on ADSW for 3 days as the lag phase is long. The bacteria those are isolated from ADSW contaminated soil show higher treatment efficiency of the parameters such as COD, TDS, total carbon, wet and dry weight measurement of biomass as compared to the DEE soil bacteria. Furthermore, if the experiment was repeated continuously, the pollution reduction of ADSW shown in the article could be achievable within a day as the bacteria wholly adapted for the treatment environment and hence the economy associated with the treatment will greatly improve. Thus, the organic compounds of ADSW are appreciably treated by a bacterial culture rich in Pseudomonas sp.

Abbreviations

COD	Chemical oxygen demand
DEE	Department of Energy & Environment
TDS	Total dissolved solids

Acknowledgements

Authors gratefully acknowledge the Algal Biotechnology Laboratory, Dept. of Energy & Environment, NITT for the facilities provided to carry out the project.

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