Physicochemical, Biochemical and Statistical Analysis of Beverages Industry Effluent

Abhinav K Srivastava¹, Ashish Kumar Gupta², Tithi Mehrotra¹, Ronit Choudhury¹, Rachana Singh^1*

¹Water Quality Monitoring and Bioremediation Lab, Amity Institute of Biotechnology, Amity University Uttar Pradesh-201313

²Patanjali Research Institute, Patanjali Ayurveda Limited, Haridwar, Uttarakhand-249405

*Corresponding Author E-mail: rsingh2@amity.edu

ABSTRACT:

The beverages industry faces growing scrutiny of responsible use and protection of the natural environment through conservation and sustainable practices. These industries cause high probability of polluting the environment by discharging untreated wastewater in to the soil and water which lead to agricultural loss and human health hazards. The aim of this study was to characterize various physiochemical parameters such as pH, temperature, TS, BOD, COD and DO etc. from effluent samples collected from four different beverage industries located in Delhi-NCR along with the biochemical characterization of isolated bacterial strains from effluent. Analysis of the collected samples states that the pH values were 4.57, 4.91, 5.17 and 5.11; Total Dissolved Solids were 1175 mg/l, 1210mg/l, 1270 mg/l and 1250 mg/l; total hardness was measured as 226, 223, 244 and 220 mg/l and alkalinity were 130, 138, 145 and 141 mg/l, respectively. Chloride test was found negative for effluent samples revealing the absence of chlorine molecule. Dissolved Oxygen were 3.3, 2.8, 3.1 and 3.2 mg/l, Biological Oxygen Demand were 211, 256, 237 and 272 mg/l where as Chemical Oxygen Demand was found 1067, 1098, 1078 and 1092 mg/l, respectively. Results suggested that pH values, DO, total hardness, alkalinity; BOD and COD were exceeded to the permissible standard limits. This study provides a detailed insight that beverage industries do produce waste linked problems and there is a high probability of polluting the environment by these industries as a result of discharge of untreated wastewater into the water body or soil that may lead to death of crops or reduction in crops yield, contamination of drinking water supplies and/or accumulation and dissemination of toxic chemicals that may further endanger ecosystems and threaten public health. It is hoped that waste water management practices being used in beverages and other food industries will benefit to sustain the natural environment.

KEYWORDS: Physicochemical analysis, biochemical analysis, beverage industry effluent, waste water .

INTRODUCTION:

Water pollution is, introduction of chemical, physical, biological materials into fresh water bodies or ocean water which degrades the quality of the water and affects the organism living in it. A report states that the discharge of untreated sewage is single most important cause for pollution of surface and ground water in India¹. The Beverage and food processing industries in India is a sunrise sector that has gained prominence in recent years. The Indian beverages and food processing industry is one of the largest in the world in terms of production, consumption, export and in growth prospects². Increase industrialization with literacy and affluence has given a considerable push to the beverage and food processing industry growth. Mechanical life style and crave for comfort is pushing people towards ready to eat services. It is very good for the progress of the industry but it also leads to the generation and consumption of water in tremendous volumes.

Wastewater generated from these industries depicts wide variation in strength and characteristics. Variation due to the amount of water usage, type of raw material used, type of product and different additives like salt, sugar, gelatin, colors, oil, dyes and added preservatives also leads to the pollution encumbrance in the wastewater but this wastewater is non toxic in nature because it comprises less hazardous compounds. Almost 50% of the water utilized in beverages and food processing industry is for washing and rinsing purposes³. Water being the primary ingredient is widely used as a cleaning agent in food processing industry. This wastewater has been reported to be treated by aerobic and anaerobic biological techniques⁴. Due to biological system malfunctions and fluctuating load and characteristics of wastewater fluctuates widely, it is hence preferable to provide physicochemical treatment first to this wastewater prior subjecting it to the biological treatment. Characteristics of industrial wastewater vary from industry to industry and within industries also. There are variations in the quality depending upon the processes, for example quality of wastewater coming out from a cooling tower will be quite different then the wastewater coming out from any chemical process. On the other hand there are limited variations in the quality of sewage depending upon season, sewerage system and lifestyle of people⁵. Wastewater contains offensive and potentially dangerous substances which cause pollution and contamination of receiving water bodies⁶.

Wastewater is the primary area of concern at the beverage industry. With the exclusion of some toxic cleaning products, wastewater from beverages and food-processing facilities is generally organic and can be treated by conventional biological technologies⁷. Primary issues associated with food and beverage industrial wastewater are biochemical oxygen demand (BOD); chemical oxygen demand (COD); total suspended solids (TSS); excessive nutrient loading viz. nitrogen and phosphorus compounds; pH of the water; total alkalinity and pathogenic organisms. Solid wastes from the food and beverage industries include both organic and packaging waste. Organic wastes from raw materials such as food grain, flavoring and coloring agents result out from processing operations. Inorganic waste typically includes excessive packaging items like plastic, glass, and metal⁸. The effluents from the food and beverage industry are contaminated with toxic metals, these can affect adversely on human health as either acute or chronic diseases. Livestock and agricultural production around the industry and its disposal site can also be hampered. Many industrial organic substances found in water can cause death or reproductive failure in fish, shellfish and wildlife⁹. In addition, they can accumulate in animal and fish tissue, be absorbed in sediments, or find their way into drinking water supplies, posing long-term health risks to human¹⁰. The present work is undertaken with a view to study the nature of effluents from different beverage industries and hence to make a comparison of the quality of effluent.

MATERIALS AND METHODS:

Samples:

Effluent samples were collected from four different beverage industries, located in Delhi-NCR (28°40′N 77°13′E). The samples were collected in a clean sterile container and stored at 4ºC until the physicochemical and biochemical analysis was carried out according to the methods of APHA 2005¹¹.

Physicochemical analysis:

The samples were analyzed physically for parameters such as pH, DO (dissolved Oxygen), TDS (Total dissolved Solids), TS (Total Solids), TSS (Total Suspended Solids), BOD (Biological Oxygen Demand), COD (Chemical Oxygen Demand), Hardness and Alkalinity. The pH of samples was determined by pH meter and temperature in Degree Celsius on scientific thermometer. TS, TDS and TSS were estimated by gravimetric method. Chloride, DO determined by titration method. Chemical oxygen Demand (COD) analyzed and Biological Oxygen Demand (BOD) analyses from incubation at 20ºC for 5 days.

Statistical analysis:

Results obtained from the effluent samples characterization were further analyzed statistically. Statistical analysis was done by using ‘SPSS 16’ package program.

Isolation and characterization of bacteria:

Isolation of bacteria was done by tenfold serial dilution of effluent. After dilution, spreading over nutrient agar plates and overnight incubation at 37°C was done. The single colonies of bacteria were identified from the growth on spread plate and further streaked on nutrient agar plates to obtain the pure culture of bacteria¹². For gram staining, Smear was prepared and heat fixed. The smear was treated with crystal violet for 1min and was then removed by Gram’s iodine to react for 1min. The smear was then washed with water and treated with 95% ethanol for 15 sec. Smear was washed with water and again treated with safranin for 30 seconds. Finally smear was washed, dried and examined under microscope¹³. The bacterial isolates were maintained on nutrient agar slants at 4°C and glycerol stocks of bacterial culture were also prepared.

Biochemical characterization:

Biochemical activities of the isolated bacteria were analyzed by different biochemical tests such as amylase production, cellulase production, and degradation of pectin, hydrolysis of gelatin, casein hydrolysis, urease test, hydrogen sulfide production, carbohydrate catabolism, IMVic tests, citrate utilization, catalaste test and oxidase test¹⁴.

RESULTS AND DISCUSSION:

The objective of this study was to investigate the pollution parameters of wastewater from beverages industry. Total four samples of effluent were collected from four different industries in the same season and stored at 4ºC for analysis. Temperature was recorded on the site and effluent samples were analyzed for physicochemical parameters like DO, TDS, TSS, TS, BOD, COD, hardness and alkalinity.

Physicochemical characteristics of effluent samples are presented in Table 1. Mean values of physical characteristics such as Temperature, pH, TDS, TSS and TS are 34.5ºC, 4.94, 1226.25mg/L, 180 mg/L, 1406.25mg/L, respectively and mean values of chemical characteristics such as DO, BOD and COD are 3.1 mg/L, 244 mg/L and 1083.75 mg/L, respectively. The average, minimum and maximum values were obtained from the analysis of the wastewater effluent samples. The values obtained after analysis are found higher than the standard permissible limits given by the Central Pollution Control Board (India). The standard permissible limits for parameters for pH, TDS, Total Hardness, DO, BOD and COD are 6.5 – 8.5, 2100 mg/L, 61 mg/L, 50 mg/L and 250 mg/L, respectively.

In Table 2 standard permissible values of parameters according to the Central Pollution Control Board, India is included along with the calculated values of mean, range, standard error and standard deviation. The range shown in table as for pH it is 0.6 with minimum value 4.57 and maximum value 5.17, for temperature it is 2 with minimum value 34 degree centigrade and maximum value 36 degree centigrade, for total hardness it is 6 with minimum value 220 and maximum value 226, for alkalinity it is 15 with minimum value 130 and maximum value 145, for TDS it is 95 with minimum value 1175 and maximum value 1270, for TSS it is 60 with minimum value 140 and maximum value 200, for DO it is 0.5 with minimum value 2.8 and maximum value 3.3, for BOD it is 61 with minimum value 211 and maximum value 272 and for COD it is 31 with minimum value 1067 and maximum value 1098. Standard error calculated defines the calculative error of mean in range of minimum value to maximum value like for pH, Standard Error 0.135 in range 4.57 to 5.17 and so on for other parameters included in this study. Standard deviation stated in table shows the deviation on mean value. Generally, the standard deviation should be less than one third of the mean. Here, the standard deviation values for parameters pH, temp, TH, alkalinity, TDS, TSS, DO, BOD and COD are 0.270, 1, 2.5, 6.350, 42.303, 27.080, 0.216, 26.242 and 13.961, respectively.

In this study, total four bacterial isolates were obtained on the basis of visual difference of colonies on spread agar plates. All these four isolates (ISB1, ISB2, ISB3 and ISB4) were further observed for their morphological characteristics. ISB1 was observed thin, off-white, smooth regular and translucent on nutrient agar plate. It was found gram positive and its shape was cocci. ISB2 was thin, golden, smooth, regular and opaque on the nutrient agar plate. It was found gram negative and shape was bacilli. ISB3 was observed as thin, white, waxy and clear on the plate while it was gram positive with cocci shape. ISB4 on nutrient agar plate was thin, white, granular and clear. Its shape was cocci and it was found to be gram positive.

Table 1: Physicochemical characteristics of effluent sample collected from four different beverage industries

Samples	pH	Temp (ºC)	TH (mg/L)	Alkalinity (mg/L)	TDS (mg/L)	TSS (mg/L)	TS (mg/L)	DO (mg/L)	BOD (mg/L)	COD (mg/L)
Site 1	4.57	34	226	130	1175	140	1315	3.3	211	1067
Site 2	4.91	34	223	138	1210	190	1400	2.8	256	1098
Site 3	5.17	36	224	145	1270	200	1470	3.1	237	1078
Site 4	5.11	34	220	141	1250	190	1440	3.2	272	1092

Table 2: Standard parameter and calculated values as per the CPCB, India

Parameters and Values	pH	Temp (ºC)	TH (mg/L)	Alkalinity (mg/L)	TDS (mg/L)	TSS (mg/L)	DO (mg/L)	BOD (mg/L)	COD (mg/L)
Standard	6.5-8.5	34-36	61	100	2100	100	5	50	250
Mean	4.94	34.5	223.25	138.5	1226.25	180	3.1	244	1083.75
Range	0.6	2	6	15	95	60	0.5	61	31
Std Error	0.135	0.5	1.25	3.175	21.151	13.54	0.108	13.121	6.98
Std Deviation	0.27	1	2.5	6.35	42.303	27.08	0.216	26.242	13.961

Table 3: Characteristics of bacteria isolated from beverage industry effluent

S. No.	Strain	Colony on Plate	Shape	Gram Test
1	ISB 1	Thin, Off white, Smooth, Regular, Translucent	Cocci	Positive
2	ISB 2	Thin, Golden, Smooth, Regular, Opaque	Bacilli	Negative
3	ISB 3	Thin, White, Waxy, Clear	Cocci	Positive
4	ISB 4	Thin, white, Granular, Clear	Cocci	Positive

All the four bacterial isolates were characterized by biochemical tests (Table 4 and Figure 1). In the amylase production test; ISB1 and ISB4 were found positive while ISB2 and IS3 were found negative. In carbohydrate catabolism all four isolates (ISB1, ISB2, ISB3 and ISB4) were found positive. In casein hydrolysis test, only ISB4 was found positive while ISB1, ISB2 and ISB3 were found negative. All the four isolates were found positive for Catalase test. In citrate utilization, ISB1, ISB3 and ISB4 were found positive while ISB2 was observed negative. Gelatin hydrolysis was found positive for ISB2, ISB3 and ISD4 while it was found negative for ISB1. In hydrogen sulphide production test, ISB2 was positive while ISB1, ISB3 and ISB4 were found negative. Indole test was positive for only ISB4 while negative results were found for ISB1, ISB2 and ISB3. ISB1 and ISB2 were positive for MR test and ISB3 and ISB4 were negative. All the four isolates were positive for oxidase test. In urease and VP test, all of them (ISB1, ISB2, ISB3 and ISB4) were found negative.

Table 4: Biochemical characteristics of the bacterial isolates obtained from beverage effluent

S.No

Strain

Amy

Car

Cas

Cat

Cit

Gel

H₂S

Ind

Oxi

Ure

ISB 1

ISB 2

ISB 3

ISB 4

(Amy-Amylase prduction, Car-Carbohydrate catabolism, Cas-Casein hydrolysis, Cat-Catalse test, Cit-Citrate utilization, Gel-Gelatin hydrolysis, H₂S-Hydrogen sulphide production, Ind-Indole, MR-Methyl Red test, Oxi-Oxidase test, Ure-Urease test, VP-Voges-Proskauer test)

Figure 1: Result of biochemical characterization (A) Amylase production test (B) Carbohydrate catabolism test (C) Casein hydrolysis test (D) Catalase test (E) Citrate utilization test (F) Gelatin hydrolysis test (G) H₂S production test (H) Indole test (I) MR test (J) Oxidase test (K) Urease test (L) VP test

The isolated bacterial strains ISB1, ISB2, ISB3 and ISB4 were further studied for their growth parameters at different conditions. Under the significant variation of pH and temperature, the growths of these four strains were studied after 24 hours of incubation. All the four strains were acting same and showing similar results at different range of pH and temperature. Optimum pH range for growth was found 6.5 to 7.5 for all the four strain (Figure 2). Optimum temperature range for growth of all the four strain was found between 35ºC to 40ºC (Figure 3).

Figure 2: Bacterial growth at different pH range (OD at 600nm, incubation time 24hours)

Figure 3: Bacterial growth at different temperature range (OD at 600nm, incubation time 24hours)

The relationship between the physicochemical parameters like pH, Temp, TH, Alkalinity, TDS, TSS, TS, DO, BOD and COD in between the samples from four different industries were calculated by the Pearson Correlation Index using SPSS16 package program. The Pearson Correlation Index was also calculated to identify the relationship of obtained data in between the four different industries. Industry wise recorded significant relations are given in Table 5 and all recorded significant relations according to water quality parameters are given in Table 6. In Table 5, site wise Pearson Correlation Index result shows that all the data obtained from four different beverages industries are highly correlated at the level of significance 0.01 i.e. p<0.01. The effluent characteristics depend on the quality of water used in the industry and the product which is being manufactured.

The Industry wise correlations coefficient results are as Site 2 with Site 1-Highly Correlated (0.999**), Site 3 with Site 1-Highly Correlated (0.998**), Site 4 with Site 1-Highly Correlated (0.999**), Site 3 with Site 2-Highly Correlated (0.999**), Site 4 with Site 2-Highly Correlated (1.0**) and Site 4 with Site 3-Highly Correlated (1.0**).

Table 5: Pearson correlation index coefficient - Industry wise

	Site 1	Site 2	Site 3	Site 4
Site 1	1
Site 2	0.999**	1
Site 3	0.998**	0.999**	1
Site 4	0.999**	1.0 **	1.0 **	1

** Correlation is significant at the 0.01 level (p<0.01)

In Table 6, water quality parameter wise Pearson Correlation Index result shows that all the certain parameters obtained from industries are highly correlated at the level of significance 0.01 and 0.05 i.e. p<0.01 and p<0.05. The parameter wise correlations coefficient results are as Alkalinity with pH-Highly Correlated (0.985*), TDS with pH-Highly Correlated (0.977*), TS with pH-Highly Correlated (0.996**), BOD with TH-Highly Correlated (-0.965*), TDS with Alkalinity-Highly Correlated (0.977*), TS with Alkalinity-Highly Correlated (0.997**), TS with TDS-Highly Correlated (0.981*) and TS with TSS-Highly Correlated (0.952*). This result indicates that the pH shows the high correlation with alkalinity, TDS and TS; BOD is highly correlated with Total Hardness; Alkalinity is in correlation with TDS; and TS is highly correlated with TDS and TSS.

Table 6: Pearson correlation index coefficient - Parameter wise

	pH	Temp	TH	Alkalinity	TDS	TSS	TS	DO	BOD	COD
pH	1
Temp	0.567	1
TH	-0.685	0.2	1
Alkalinity	0.985*	0.682	-0.556	1
TDS	0.977*	0.689	-0.571	0.977*	1
TSS	0.946	0.492	-0.64	0.95	0.873	1
TS	0.996**	0.632	-0.617	0.997**	0.981*	0.952*	1
DO	-0.274	0	0.185	-0.316	-0.109	-0.57	-0.298	1
BOD	0.704	-0.178	-0.965*	0.598	0.557	0.741	0.649	-0.435	1
COD	0.516	-0.275	-0.771	0.445	0.322	0.688	0.48	-0.752	0.905	1

* Correlation is significant at the 0.05 level (p<0.05);

** Correlation is significant at the 0.01 level (p<0.01).

CONCLUSION:

As soft drink industries are using large amount of fresh water especially in bottling plants where used bottles are washed with caustic solutions to make them ready for reuse .Therefore it is suggested that wastewater of soft drink industry can be treated and reused to save burden on fresh natural water resources and energy. The objective of this study was to investigate the main pollution parameters of wastewater in beverage industry. The water quality parameters viz; pH, Temperature, BOD, COD, DO, TSS and alkalinity values were found above the permissible limits as recommended values given by CPCB. Statistical analysis was also performed on the results of water quality parameters of samples of four beverage industries. Site wise Pearson Correlation Index result shows that all the data obtained from four different beverages industries are highly correlated. This reveals the effluent characteristics of an industry are dependent on the water quality used in manufacturing process and type of product being manufactured. On the basis of the result found, we can also conclude that if the environmental and geographical conditions are same, one kind of industry may produce similar kind of effluent. The results indicated that pollution parameter levels in effluents of the selected beverage industries were found high. To avoid the environmental pollution and to protect public health, wastewater treatment systems are recommended for beverage industry to treat the effluent properly before its discharge.

ACKNOWLEDGEMENT:

The authors are thankful to the beverage industries located in Delhi-NCR for providing waste water samples as and when required. The authors are also thankful to Amity Institute of Biotechnology, Amity University Uttar Pradesh for providing infrastructure and scientific support to conduct this study.

REFERENCES:

1. Agrawal A, Pandey RS, Sharma B. Water pollution with special reference to pesticide contamination in India. Journal of Water Resource and Protection. 2(5); 2010: 432.

2. Wilkinson J. The food processing industry, globalization and developing countries. The transformation of agri-food systems: globalization, supply chains and smallholder farmers. 2008: 87-108.

3. Haroon, H. A. J. I. R. A., Waseem, A., and Mahmood, Q. Treatment and reuse of wastewater from beverage industry. J Chem Soc Pak. 35; 2013: 5-10.

4. Van der Zee FP, Villaverde S. Combined anaerobic–aerobic treatment of azo dyes—a short review of bioreactor studies. Water Research. 39(8); 2005: 1425-1440.

5. Shivsharan VS, Wani M, Khetmalas MB. Characterization of dairy effluents by physicochemical parameters. British Biotechnology Journal. 3(4); 2013: 575.

6. Osho A, Mabekoje OO, Bello OO. Preliminary evaluation of wastewater effluents from two food companies in Nigeria. African Journal of Microbiology Research. 4(13); 2010: 1395-1399.

7. Kleerebezem R, Macarie H. Treating industrial wastewater: anaerobic digestion comes of age: anaerobic treatment systems offer important advantages over conventionally applied aerobic processes for removing organic pollutants from water-based streams. Chemical Engineering. 110(4); 2003: 56-65.

8. Maxime D, Marcotte M, Arcand Y. Development of eco-efficiency indicators for the Canadian food and beverage industry. Journal of Cleaner Production. 14(6); 2006: 636-648.

9. Atkinson BW, Bux F, Kasan HC. Considerations for application of biosorption technology to remediate metal-contaminated industrial effluents. Water S. A. 24(2); 1998: 129-135.

10. Barron MG. Bioconcentration. Will water-borne organic chemicals accumulate in aquatic animals? Environmental Science and Technology. 24(11); 1990: 1612-1618.

11. Federation WE. American Public Health Association. Standard methods for the examination of water and wastewater. American Public Health Association (APHA). Washington, DC, USA. 2005.

12. Friedemann M. Enterobacter sakazakii in food and beverages (other than infant formula and milk powder). International Journal of Food Microbiology. 116(1); 2007: 1-10.

13. Gregersen T. Rapid method for distinction of Gram-negative from Gram-positive bacteria. European Journal of Applied Microbiology and Biotechnology. 5(2); 1978: 123-127.

14. Aneja KR. Experiments in Microbiology, Plant Pathology and Biotechnology. New Age International. 2003.

Received on 05.05.2016 Modified on 20.05.2016

Research J. Pharm. and Tech. 2016; 9(7):887-892

DOI: 10.5958/0974-360X.2016.00169.4