Biological wastewater treatment is economically feasible and ecofriendly. This study was aimed at isolating bacteria from brewery wastes and evaluating their bioremediation potential as individual isolate and/or their consortium in reducing the pollutants of brewery effluents. A total of 40 bacterial isolates were recovered and of these the three best isolates were selected. The selected bacteria were identified to genus level by using morphological and biochemical characteristics. Accordingly, the isolates were identified as
Industries are major source of pollution in all environments. Brewery plants have been known to cause pollution by discharging effluent into receiving stream, ground water, and soil in Ethiopia [
Untreated brewery effluents typically contain suspended solids in the range 10–60 mg/l, BOD in the range 1,000–1,500 mg/l, COD in the range 1800–3000 mg/l, and nitrogen in range 30–100 mg/l [
Currently, in Ethiopia, there are ten breweries. It is reported that the majority of brewery industries in Ethiopia discharge their wastewaters into nearby water bodies and open land with little or no prior treatment [
One of the wastewater treatment systems which is economically feasible and environmentally sound is a biological treatment using microorganisms [
Brewery and other industries are blooming in Ethiopia; as a result, pollution of the environment is increasing. Thus, isolation and identification of effective microorganisms are a novel approach to minimize the current environmental problems. The objective of this study was to assess the potential of bacterial isolates in the treatment of brewery effluents and also to confirm removal of pollutants by conducting germination test on seeds of beet root
The study was conducted at Bedele Brewery Microbiology and Wastewater Treatment Laboratory. The industry was founded in 1993, which is located at 483 km south west of Addis Ababa in Buno Bedele zone, Oromia Regional State. Bedele town is located at a longitude and latitude of 8°27′N 36°21′E and with an elevation between 2,012–2,162 meters (6,601–7,093 ft) above sea level.
Brewery waste samples were collected from three sources. These were brewery effluent, sludge of waste, and contaminated soil with brewery effluents. One liter of effluent, two kg of sludge, and soil samples were collected aseptically using sterile glass bottles. Samples of soil, sludge, and effluent were labeled and analyzed separately. The samples were collected three times (3x) from three sources by the interval of 15 days to get different bacterial isolates. The samples were stored at 4°C until further analysis.
The samples were serially diluted in physiological saline (0.9% NaCl w/v solution) and 0.1 ml of aliquots from appropriate dilution was spread plated on nutrient agar (NA) (Hi-Media, Mumbai, India). All the plates were incubated aerobically at 30°C for 48–72 hrs. The plates were observed for bacterial growth. Morphologically different colonies were selected from plates with countable colonies and transferred to nutrient broth (NB) (Hi-media, Mumbai, India) and checked for purity by repeated streaking on NA plates. The pure isolates were designated based on their sources of isolation, from soil (SO), effluent (EF), and sludge (SL) followed by number codes. The pure cultures were preserved on NA slant at 4°C and 50% glycerol at −10°C in duplicate for further study. The day to day experimentation was carried out with cultures maintained on plates and slants. The isolates were periodically checked for purity.
Inoculum was prepared following the method outlined by Krishnaswamy et al. [
After the OD of the suspension was adjusted to OD0.5; 0.1 ml was spread plated on nutrient agar to estimate number of viable cells per ml of suspension. The number of bacterial cells used for inoculation is presented in Table
Treatment design and number of bacteria cells (CFU) estimated per ml of suspension.
Treatment | Isolate code | Designation | Mean colony forming unit per ml of suspension × 106 CFU/ml |
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T1 | EF-01(A) | A | 2.75 ± 10 |
T2 | SL-10(C) | C | 2.85 ± 17 |
T3 | SL-25(D) | D | 2.77 ± 14 |
T4 | Comb 1 | C + D | 2.76 ± 24 |
T5 | Comb 2 | A + C + D | 2.76 ± 26 |
T6 | Control | Not inoculated | — |
T: treatment and Comb: combination.
Experiment on pollutant removal of bacterial isolates was conducted at laboratory scale batch culture in bottle under shaking at 150 rpm. The brewery wastes were sterilized by autoclaving separately. The bacterial isolates were inoculated aseptically to that of sterilized wastes in different bottles in three duplicates. The bottles were incubated at room temperature on a shaker maintained at 150 rpm. Bottles with only brewery wastes (without addition of microbes) were used as a control. In this study, three different potential bacterial isolates were selected. Selection of potential bacterial isolates was based on pollutant removal efficiency after 12th day of incubation. These three isolates EF-01(A), SL-10(C), and SO-25(D) showed the best ability to degrade the brewery wastewater and were selected to constitute their combinations (Table
The three isolates that showed the best performance in removal efficiency of the brewery wastewater pollution profile were revived from their stock cultures and subcultured. A loopful of each individual isolate of 24 hrs old on NA was aseptically inoculated into sterile nutrient broth medium (10 ml) in test tubes. The inoculated test tubes were incubated for 48 hrs at 30°C. The bacterial broth cultures were adjusted at OD0.5. Thereafter, well-mixed cell suspension was added to sterilized brewery wastewater. The inoculated bottles containing wastewater were incubated.
In this study, pH, EC, BOD, COD, TN, TP, TSS, TS, and TDS were selected to measure pollutants removal activity of bacterial isolates. The parameters were measured before and after inoculation of potential bacterial isolates. The measurement was carried on 0, 3rd, 6th, 9th, and 12th days for all parameters. After the measurement of the selected brewery wastewater parameters, pollutants reduction capabilities of bacterial isolates (singly or in combination) were evaluated. Percentage of pollutants removal was compared against the control (without bacterial inoculation).
Before combination, compatibility test was performed among the three selected potential bacteria. Each of the three selected isolates was grown at room temperature and subsequently tested by the cross-streaking method at 30°C and at 37°C [
Pure colonies of selected potential bacterial isolates were characterized using morphological and biochemical tests. The colony characteristics such as size, shape, color, margin, and elevation were recorded with their biochemical tests.
The morphological characteristics used for identification were colony size, surface (smooth, rough, granular, and papillate), color (colorless, pink, black, red, and bluish‐green), margin (entire, wavy, lobate, and filiform), elevation (flat, raised, low convex, and dome shaped), and their shape such as bacilli/rod, cocci/spherical, and spiral under a microscope. Moreover, cell arrangement (single, chain, pair, diploid, tetrad, and cluster), gram staining, and spore forming test were considered for morphological characterization of the selected isolates. The biochemical tests performed in this study were KOH, catalase, oxidase, and Oxidation Fermentation (O/F) tests.
Pollutant concentration of each treatment sample was analyzed for selected pollutant parameters following standard methods [
Electrical conductivity (EC) was measured with a conductivity meter (Oyster conductivity meter). The pH of the samples was measured with a portable pH meter (Model HI9024, HANNA Instrument) [
BOD was determined by respirometer method by using BOD Trak II™ instrument (HACH Company, Loveland, CO, USA) [
The chemical oxygen demand was measured by closed reflux method using strong chemical oxidant [
Total nitrogen was determined by using persulfate digestion method by oxidation of all nitrogenous compounds to nitrate [
To measure TP vanadomolybdophosphoric acid colorimetric method was used [
Total solids, total suspended solids, and total dissolved solids were determined by gravimetric method at temperature of 103–105°C [
Total dissolved solid (TDS) was analyzed by a well-mixed sample filtered through a standard glass fiber filter, and the filtrate was evaporated to dryness in a preweighed dish
Pollutants removal was confirmed by germination test of wastewater treated with individual and combination of the bacterial isolates. For this test, seeds of beet root
Different parameters like germination percentage, mean germination time (MGT), and seedling length were recorded at different time intervals of plant growth. First recording was done after 12 hrs of incubation and subsequent recordings were done at a day interval till the 6th day of incubation. The Petri dishes were rearranged at random on every one day to ensure no systematic effects due to positioning within the incubator.
Germination in each experimental set was recorded and total germination was calculated and expressed in percentage [
The mean germination time was calculated using the daily counts for each lot [
The shoot length was measured from the base of the primary leaf to the base of the hypocotyl and the mean shoot length was expressed in centimeter. Root length was measured from the tip of the primary root to the base of hypocotyl and mean root length was expressed in centimeter. By adding the root length and shoot length, seedling length was calculated and expressed in centimeter.
Statistical analysis was performed using SPSS program (SPSS; Version 20.0). The data were analyzed through one-way analysis of variance (ANOVA) at 95% confidence level to compare the performance efficiency of each individual and combination treatments. Means separation was done following Duncan’s test.
A total of 40 different bacterial isolates with diverse morphological characteristics were retrieved from brewery effluent, sludge, and soil contaminated with brewery wastewater. Out of 40 isolates, the best three bacteria were selected as potential organisms for waste treatment technology.
Based on the cultural characteristics, morphological tests, and biochemical tests (Table
Morphological and biochemical characteristics of selected bacterial isolates.
Test | EF-01 | SL-10 | SO-25 |
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Surface | Smooth | Smooth | Rough |
Colony shape | Circular | Irregular | Irregular |
Color | Greenish yellow | white | White |
Margin | Entire edge | Filamentous | Lobate |
Elevation | Flat | Convex | Raised |
Cell shape | Rod | Rod | Rod |
Gram stain | − | − | + |
KOH test | + | + | − |
Endospore | − | − | + |
Catalase test | + | + | + |
Oxidase test | + | + | + |
O/F test | F+ | O+ | − |
Suggested genus |
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O: oxidative, F: fermentative, +: positive, −: negative, SL: isolated from sludge, EF: isolated from effluent, and SO: isolated from soil.
The appearance of brewery wastes before and after treatment is shown in Figure
Wastewater samples before (a) and after (b) treatment.
The pH and EC values were slightly increased. The pollutant parameter values of before and after treatment are indicated in Table
Effluent analysis before and after treatment for 12 days.
Number | Treatment | Parameters | |||||||||
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pH | EC mS/cm | BOD (mg/l) | COD (mg/l) | TN (mg/l) | TP (mg/l) | TSS (mg/l) | TS (mg/l) | TDS (mg/l) | |||
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After treatment |
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EF-01(A) |
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SL-10(C) |
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SO-25(D) |
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Comb 1 |
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Comb 2 |
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Control |
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Pollutant removal efficiency (%) of these three individual isolates and their combination is presented in Table
Pollutant removal efficiency (%) of three selected isolates and their combination after 12 days of incubation.
Treatment | Parameters and their removal efficiency (%) | ||||||||
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pH | EC (mS/cm) | BOD | COD | TN | TP | TSS | TS | TDS | |
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SL-10(C) |
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SO-25(D) |
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Comb 1(CD) |
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Comb 2(ACD) |
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Control |
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The results of the study demonstrated that pH values increased in all the treatments (Figure
pH values of individual and combination treatments. A:
A study conducted by Choudhary et al. [
Similar to pH, the EC values of all the treatments were slightly increased in all the isolates (Figure
EC (mS/cm) values of individual and combination treatments. A:
The conductivity of a solution depends on the concentration of all the ions present; the greater their concentrations, the greater the conductivity. The rise in EC values is related to the increment observed in pH values (i.e., an increase in OH−/H+ ions) that ultimately posed increase in EC values. For an acidic solution, the lower the pH is, that is, the higher the H+ concentration, the greater the conductivity will be. So, strongly acidic or strongly basic solution will have high conductivity [
The results of the current study showed a decline in the values of BOD and COD in the three individual isolates and their two mixed combinations (Table
The Ethiopian standard limits for BOD and COD emission of brewery waste are 60 mg/l and 250 mg/l, respectively. From all the treatments, only samples treated with three combinations (comb 2) met the Ethiopian standard limit for COD values. In the case of BOD, none of them met the Ethiopian standard limit. However, their removal percent/efficiency was acceptable.
Figures
BOD (a) COD (b) removal efficiency (%) of individual and combination treatments. A:
In both BOD and COD, the maximum removal efficiency was recorded by three mixed combinations with values of 94.85% and 93.25%, respectively. The findings indicate that the reduction of BOD and COD increases in effluent inoculated with bacterial isolates with the extension of incubation period. Similarly, Metcalf and Eddy [
According to Hidayah and Shovitri [
Further, according to Mongkolthanaruk and Dharmsthiti [
In this study, the results showed that mean TN ranged from 36 mg/l to 41 mg/l with 48.1% to 56.7% removal efficiency for individual isolates. Similarly, for combination treatment, TN values ranged from 18 mg/l to 34 mg/l with removal efficiency of 60.76% to 77.21% (Figure
TN removal efficiency (%) of individual and combination treatments. A:
The highest removal efficiency of TN was in the treatment with three combinations of isolates. The combination (consortium) of bacteria showed significant
The Ethiopian standard limit for TN emission of brewery waste is 40 mg/l. Removal of TN for individual isolates is relatively comparable to the national effluent emission standard limit for brewery wastewater for
Compared to other pollutant removal parameters, TN removal efficiencies of all the isolates were lower than other parameters since most denitrifying heterotrophic bacteria are incomplete denitrifiers, which were only capable of reducing nitrates to nitrites with no further reduction of the nitrites produced. This is because incomplete denitrifying bacteria lack key nitrite reductase enzymes which enable complete denitrifiers to reduce nitrites [
Similarly, among gram-positive bacteria (such as
Another study [
The removal efficiency of TP is indicated in Figure
TP removal efficiency of individual and combination treatments. A:
From all the treatments, the highest TP removal efficiency was in combination 2 which was 78.31% with TP value of 12 mg/l. Concentrations of TP for individual isolates and their combination from the effluents did not meet national effluent emission standard limit (5 mg/l) for TP. This is because the brewery wastes contain high TP concentration (55 mg/l) before the treatment.
As in this study, Krishnaswamy et al. [
Study by Brodisch and Joyner [
The mean effluent removal efficiency of each individual treatment for TSS, TS, and TDS is presented in Figures
TSS (a), TS (b), and TDS (c) removal efficiency (%) of individual isolates and combination treatment. A:
The result reveals that the treatment with three (3) mixed bacteria (comb 2) had the maximum TSS, TS, and TDS removal efficiency with 90.3%, 88.5%, and 88.2%, respectively. This shows that the synergistic effect of bacterial combination treatment brings about enhanced performance for effective biodegradation. As in this study, De Souza et al. [
Likewise, reduction in TSS, TS, and TDS of rubber processing effluent by using
Another interesting observation was that treated brewery wastes enhanced germination of beet seeds compared to the control. Mean comparison of germination percent, seedling length (cm), and mean germination time (MGT) of beet seeds are indicated in Table
Germination percent (%), mean germination time (MGT), and seedling length (cm) after 6th day.
S/N | Treatment | Germination% | MGT (days) | Seedling length (cm) |
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SL-10(C) |
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SO-25(D) |
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Comb 1 (CD) |
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Comb 2 (ACD) |
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Control |
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Before treatment |
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Tap water |
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Mean values (of triplicates) followed by the same letters in each column are not significantly different (
Table
Seeds germination difference in untreated and treated wastes is due to presence of less toxic chemicals in the treated effluent compared to untreated effluent due to the presence of high level of toxic substances in the latter [
The mean germination time (MGT) of all the treatment was 3.1 to 5.2 days (Table
A study conducted by Orhue et al. [
The seedlings length of germinated beet seed after six days of incubation was minimum (2.3 cm) and maximum (6.3 cm). The minimum seedlings length was in seeds germinated in untreated wastewater, followed by control treatment. The maximum germination occurred in three combinations of isolates comb 2 (ACD), followed by comb 1 (CD). This is because untreated wastewater contains high amount of toxic substances such as high amount of total solids in the effluent; as a result germinated seed became dry [
In agreement with the present study, the reduction in seedling (root and shoot) lengths with the elevated amounts of total dissolved solids at higher concentrations has been demonstrated elsewhere [
Based on the results obtained with the brewery wastewater biotreatment experiments,
In this study, the maximum pollutant removal occurred in brewery effluents inoculated with combined bacterial isolates for all parameters indicating their synergistic effect on degradation of wastes. The result also revealed that brewery wastes treated with
Generally, it can be concluded from the treatment performance of this experiment that data generated from this study can give an insight for the use of potent bacterial isolates as an alternative wastewater treatment technology.
Therefore, the development of this experimental system into a large-scale working unit offers an attractive alternative technology for waste treatment.
The authors declare that they have no conflicts of interest.
The authors would like to thank Heineken International Brewery S.C. and Wollega University for funding this study.