ASSESSMENT OF SOME BACTERIAL AND FUNGAL
STRAINS FOR DAIRY WASTEWATER TREATMENT
Rehab G. Hassan ; Mohamed Ali El-Said and Lameas A. Mohamed
Housing and Building National Research Center, P.O. Box 1770, Cairo, Egypt
Key Words: Biodegradation, Dairy wastewater, Bacterial strains, fungi
ABSTRACT
Microorganisms of the effluents from dairy factories located in 6th
October industrial city (Cairo, Egypt) were isolated and screened for their
ability to reduce the pollutants found in the dairy wastewater effluent.
Five bacterial strains (Staphylococcus aureus, Pseudomonas aeruginosa
Streptococcus thermophilus, Bacillus supergene and Lactobacillus
fermentum) and three fungal strains (Aspergillus sp., Cladosporium sp.
and Fusarium sp) were isolated to be used in biodegradation. A two
stage reactor (incubation then filtration) was used during this study. Two
mixtures were prepared (Mix 1: five bacterial isolated strains and Mix 2:
three fungal isolated strains). The results showed that bacterial and fungal
strains have high efficiency for organic pollutants reduction as the
percentage of reduction reached 79.8 % and 72.5 % respectively. TSS for
both bacterial and fungal mixtures shows high reduction efficiency with
99.5% and 98.9% respectively, However after incubation the reduction
efficiency was not high for both bacterial and fungal mixtures as it
increased after filtration Bacterial mixtures was slightly more effective
than fungal mixtures during the treatment process. Using activated
carbon and sand for filtration after incubation enhances the treatment
efficiency of the pollutants present in dairy wastewater.
INTRODUCTION
The wastewater of dairy industries is increasing rapidly due to the
increase in production rate. At the last few years industrial wastes
generally discharged on lands or into different water sources results in
release of toxic substances into the environment, creating health hazards
(porwal et al., 2015).
In the recent years Egypt ministry of environment has paid a
considerable attention to the industrial wastes as a general protocol to
protect the environment from pollutants. The most common methods
used for treating industrial wastes are physicochemical and biological.
Physiochemical methods usually include partial treatment, higher
quantity of solids, higher cost and use of chemical agents that’s why
biological methods are more preferred for the reduction of wastewater
Egypt. J. of Appl. Sci., 35 (12) 2020 272-283
pollutants. Recently there is a growing interest in the use of biological
methods to treat waste water of dairy industries in due to their advances
over physicochemical methods (Rodrigues et al., 2008).
Many types of physicochemical treatment techniques have been
studied for their applicability in treatment of wastewaters (Rodrigues et
al., 2008). These types of treatments include sedimentation, screening,
aeration, filtration, flotation, degassification, chlorination, ozonation,
neutralization, coagulation, sorption, ion exchange, etc. Several
limitations of physicochemical methods including partial treatment,
higher cost, and generation of secondary pollutants, higher quantity
solids and use of chemicals agents make the biological methods a
favorable alternative for the reduction of pollutants. Pollutants associated
with the food industry including the wastes generated by the dairy
industry namely sludge, heavy organic matter, fats, oil & grease, fatty
acids, nitrogenous compounds are notables (Healy et al.,1995). Of all
industrial activities, the food sector has one of the highest consumptions
of water and is one of the biggest producers of effluents per unit of
production; in addition, they generate a large volume of sludge in
biological treatment (Ramjeawon, 2000).
One of the highest industries in consumption of water and
production of effluents per unit of production is food industry. Also, the
food industries produce large volumes of sludge through the biological
treatment process. Sludge production in aerobic systems is about 0.5 kg
per kg of removed chemical oxygen demand (COD), while in the
anaerobic system; sludge production is about 0.1 kg (Kaur & Chaman
2014). As for milk processing industries, the high load of pollutants in
dairy wastewater led to the discharge of partially treated or untreated
wastewater which in turn caused serious environmental and public health
problems (Kaur & Chaman 2014).Since water is the major component
in the dairy industry, then the safe disposal of the significant effluent
volumes that are frequently generated is a real challenge. Dairy industries
generate, on average, about 6 to 10 L of wastewater per liter of processed
milk (Kolhe & Pawar 2011).
The process of seeding inoculation of microorganisms for
degrading waste materials on streams, rivers and treatment tanks has
been rapidly increasing practice in many countries because it is
economical and the application is uncomplicated. Bioremediation is any
process that uses living microorganisms or their enzymes, to return a
polluted environment to its original condition. As such, it uses relatively
273 Egypt. J. of Appl. Sci., 35 (12) 2020
low-cost, low-technology techniques, such as using environment friendly
microorganisms which generally have a high public acceptance and can
be used on the site (Ojo, 2006). It constitutes the use of natural biota and
their processes for pollution reduction and the end products are nonhazardous
(Ahmedna et al., 2004). The process of biodegradation is a
well- established and powerful technique for treating domestic and
industrial effluents. The performance of a biological process is often
enhanced through bioaugmentation of one or more species of specialized
microorganisms (Sermany et al., 2012). Microbial populations have an
amazing and extensive capacity to degrade variety of organic
compounds. Naturally occurring microorganisms thrive on many of the
complex compounds contained in wastewater. Small size, high surface
area-to-volume ratio and large contact interfaces with their surrounding
environment, are some of the ideal features of microorganisms as
bioindicators of chemical pollutants The microorganisms may be
indigenous to a contaminated area or they may be isolated from
elsewhere and brought to the contaminated site. To get an efficient
biological wastewater treatment it is very important to know the
wastewater microbiota composition and biochemical properties
correlated to the origin of pollutants, as well as the optimum metabolic
activity and physical-chemical conditions (Janczukowicz et al., 2007).
In this study a model was designed to study the ability of a mixture
of bacterial and fungal isolates, which are isolated from dairy
wastewater, to degrade the organic nutrients found in dairy wastewater
and also improving the quality of dairy wastewater. The model was
supplemented with a natural filtration media sand and activated carbon
for a better treatment.
MATERIALS AND METHODS
Fresh Samples were collected from different dairy effluents
treatment factories located in industrial zone of 6th October city in Cairo.
The samples were collected in 5 L polyethylene plastic sterilized
containers. The samples were transferred to the laboratory immediately
and stored at 4˚C to avoid any physicochemical changes in the dairy
wastewater effluent.
Isolation of microorganisms
Serial dilution for the dairy effluent samples was done (10 -1 to 10-
5) then in Erlenmeyer flasks containing enrichment cultural media (sterile
Nutrient Broth and Sabouraud's Broth) 1ml from each dilution was
inoculated respectively. The flasks were kept on rotary shaker at 100 rpm
Egypt. J. of Appl. Sci., 35 (12) 2020 274
at room temperature for 24–96 h. Then a loopful of enriched sample from
Nutrient Broth flasks was streaked on Nutrient Agar petri dishes and
another loopful from Sabouraud's Broth was streaked on Sabouraud's
Agar petri-dishes. Nutrient Agar petri dishes were incubated for 24 h at
35 °c, while the Sabouraud's Agar plates were incubated for 7 days at
28 °c. Triplicate plating was done for each medium.
Well grown individual bacterial colonies on the surface of nutrient
agar petri dishes were picked up and inoculated into 250 ml Erlenmeyer
flasks containing milk broth (peptone 5gm, yeast extract 3 gm and fresh
milk 10 ml). After inoculation flasks were incubated at 35 °C on a rotary
shaker for 24-48 h. After that a loopful was streaked on milk agar petri
dishes and incubated at 35 °C for 24 h. After incubation single pure
colonies were suspended in nutrient broth containing 10% (ʋ/ʋ) glycerol
and stored at – 80 °C for identification and further experiment. The same
was done for fungal colonies that was selected and then inoculated at 28
°C for 7 days. After that single pure colonies were inoculated into
Sabouraud's Broth containing 10% (ʋ/ʋ) glycerol and stored at – 80 °C
for identification and further experiment.
For the fourteen bacterial isolates identification was done by using
biology system BiologTm microplate identification system (BiologTm Gen
Ш, USA), while for the six fungal isolates identification was done
depending on colony morphology and microscopic examination by
lactophenol cotton blue staining method (kaur & Chaman 2014).
Inoculums preparation
A suspension from each microbial isolate of 0.1 ml was inoculated
in 100 ml inoculum medium. The flasks were kept on rotary shaker at
150 rpm for 24 h at 35 °C. also, 0.1 ml of suspension of the fungal isolate
was inoculated in 100 ml inoculum medium and kept on rotary shaker at
120 rpm for 5 days at 28 °C. These suspensions were done to study the
biodegradation efficiency of the microbial isolates; the activity growing
culture of each isolate was then washed with sterile deionized water and
centrifuged at 10,000 rpm for 10 minutes to get a wet pellet for each
isolate. The pellet was then resuspended in sterile deionized water till
turbidity reaches at or above that of McFarland 0.5 standards (Wayne,
2009).
Experimental setup and working
Two models of two stages were set up for the experimental
treatment of the dairy wastewater effluent (Fig. 1) one for bacterial
isolates and the other for fungal isolates. The design of this model was
275 Egypt. J. of Appl. Sci., 35 (12) 2020
obtained from the model suggested by RamaKrishna and Ligy, (2005)
and porwal et al.,(2015) and also from the model suggested by
Arumugam and Sabarethinam,(2008) for treatment of dairy
wastewater. For filtration sand and activated carbon were used during the
treatment process.
The tank of the treatment were washed with alcohol to make it
sterile and then rinsed with sterile distilled water. In the first model the
tankwas fed with 1 L autoclaved untreated dairy wastewater. The
autoclaved effluent was cooled to room temperature and then added to
the reactor. Then 10 ml of each identified bacterial isolate (bacterial
mixture [Mix 1]) was added to the effluent (the same was done in the
second model but 10 ml of identified fungal isolates were added [fungal
mixture [Mix 2]). An aerator was inserted into the reactor and the open
top portion of the reactor was covered. The aerator used maintained the
desired level of dissolved oxygen ˂ 5 mg/L in the effluent and to support
the survival and growth of the aerobic microorganisms used in this study.
The incubation was provided for a period of 48 h. The effluent was given
a retention time of 48 h in the primary tank where the microorganisms
were allowed to carry out degradation. After 48 h, the aeration was
stopped and effluents was allowed to stand for 1 h to allow settling of the
sludge formed after that the treated effluent from primary tank was then
allowed to flow into the filtration tanks. The filtration was carried out in
two tanks one tank contains sand and followed by another tank
containing activated carbon.
Analytical methods
All analytical methods used during this study conformed to the
“APHA; 2017”. The colorimetric technique was used for the
determination of Chemical oxygen demand (COD), Biological oxygen
demand (BOD).The Total Suspended Solids (TSS) was measured by
filtration, drying at 105 °C and then combusting at 550 °C.
Fig. (1): shape of the model used in biodegradation process.
Egypt. J. of Appl. Sci., 35 (12) 2020 276
RESULTS AND DISCUSSION
Table 1 shows the results obtained of the physicochemical analysis
for untreated dairy wastewater. The results of raw dairy wastewater were
in accordance with the findings of porwal et al. (2015). Passeggi et al.
(2009) has reported that the pH of dairy effluents depending on the
nature of the end-product and can range from 4.7 to 12.2. In our study the
influent of dairy wastewater was slightly acidic (6.01 ± 0.17). The acidic
pH is attributed to the breakdown of milk lactose into lactic acid as
mentioned by Slavov, (2017).TSS is one of the main parameters of water
which used to evaluate and determine the efficiency of treatment
processes of wastewater. The dairy wastewater showed high
concentrations of TSS (636 ± 11.21). Porwal et al. (2015), also reported
the same high concentrations of TSS (626.6 ± 8.79 and 601.6 ± 3.46).
BOD is one of the most widely used indicators of water quality. The
influent dairy wastewater showed high concentrations of BOD (1,221 ±
13.01 mg/L). The dairy wastewater are characterized by high levels of
BOD due to the presence of lactose, casein, fats, nutrients, sanitizing
agents and inorganic salts (Kolhe et al. 2009).
Table (1): Characterization of dairy wastewater:
parameters Average ± standard deviation Unit
Color milky -
pH 6.01 ±0.17 -
Turbidity 1133 ±15.33 NTU
Electrical conductivity 442 ±9.06 μS/cm
TSS 636 ±11.21 mg/l
TDS 1790 ±8.22 mg/l
COD 2288 ±18.16 mg/l
BOD 1221 ±13.01 mg/l
O&G 153 ±4.52 mg/l
Identification of isolated microorganisms:
Fourteen bacterial isolates were identified by using BiologTM Gen
III. The identified bacterial isolates showed some repeats and finally, five
bacterial strains were identified as Staphylococcus aureus, Pseudomonas
aeruginosa Streptococcus thermophilus, Bacillus supergene and
Lactobacillus fermentum. Also, six isolated fungal isolates were subject
to identification based on colony morphology and microscopic
examination. Also some repeats were detected. Three fungal strains were
identified as Aspergillus sp., Cladosporium sp. and Fusarium sp.
Characterization of the final dairy wastewater after
biodegradtion:
A mixture of the identified five bacterial species (Staphylococcus
aureus, Pseudomonas aeruginosa Streptococcus thermophilus, Bacillus
277 Egypt. J. of Appl. Sci., 35 (12) 2020
supergene and Lactobacillus fermentum) was prepared with equal
percent and used for the treatment process and named as the bacterial
mixture (Mix 1). The same was carried out separately using three fungal
strains (Aspergillus sp., Cladosporium sp. and Fusarium sp) that were
mixed and used for the treatment process and named as the fungal
mixture (Mix 2). Table 2 shows the values of physicochemical
parameters of treated dairy effluent after aeration stage (primary tank)
and filtration stage (secondary tank) and also the total reduction
percentage. Results showed that the color of dairy wastewater has been
improved, as it was milky and after the treatment process, it was clear.
This improvement may be due to degradation of organic materials by
bacterial and fungal mixtures. Also, using sand and activated carbon as
filter media led to removing more suspended particles and consequently
color improvement (Verma & Madam- war 2002
Table (2): Treatment of Dairy Effluent by using a mixture of
bacterial & fungal isolates:
Parameter
Mix 1 (Bacterial isolate mixture) Mix 2 (Fungal isolates mixture)
Unit
After
incubation
Reduction
(%)
After
filtration
Total
reduction
(%)
After
incubation
reduction
(%)
After
filtration
Total
reduction
(%)
Color
Creamy
white
- clear -
Creamy
white
- clear - -
pH
6.6
±0.12
-
7.3
±0.11
-
6.5
±0.1
-
7.1
±0.05
- -
Turbidity
623
±3.9
45.0
9.0
±0.23
99.2
734
±5.3
35.2
11.0
±0.7
99.0 NTU
EC
220
±2.5
50.2
61
±0.72
86.1
264
±4.5
40.3
78
±1.67
82.4
μS/
cm
TSS
428
±6.5
32.7
3.0
±0.5
99.5
444
±5.5
30.1
7.0
±0.54
98.9 mg/l
TDS
1343
±18.5
24.9
351
±2.21
80.4
1487
±11.6
16.9
398
±4.8
77.8 mg/l
COD
660
±4.2
71.2
493
±3.4
78.5
712
±5.5
68.9
523
±4.5
77.1 mg/l
BOD
328
±5.0
73.1
247
±3.7
79.8
361
±4.6
70.4
336
±5.5
72.5 mg/l
O&G
87
±2.61
43.1
4
±0.31
97.4
91
±3.13
40.5
6
±0.51
96.1 mg/l
For pH value results shown in Table 2 that the pH values moved
towards the neutrality in both bacterial and fungal mixtures. Also it was
clear that both aeration stage and filtration stage have the same effect on the
changes of pH values. Porwal et al. (2015), studied the biodegradation of
dairy wastewater using microbial isolates obtained from activated sludge
and they found the same changes in pH values. The change in pH values
may be attributed to the ability of microorganisms to accumulate organic
acids after the biodegradation process (Kowsalya et al. 2010).
As shown in Table 2 the efficiency of reduction for the turbidity after
incubation was 45% and 35.2% for both bacterial mixture and fungal
mixture respectively. Turbidity decreased due to consumption of organic
Egypt. J. of Appl. Sci., 35 (12) 2020 278
materials and suspended particles by bacteria and fungi through growth and
survival. In addition, after filtration, the increase in removal efficiencies was
significantly observed. The total reduction percent was 99.2% and 99 % for
bacterial mixture and fungal mixture respectively. This observed decrease in
turbidity values after filtration stage was a result of using filter materials
(sand and activated carbon) which absorbed more substances this result was
in correspondence with Porwal el al., 2015).
Electric conductivity is considered as an important parameter which
can be used for quantitative measurement of dissolved ionic constituents in
water and detection of impurities, which are necessary for cooling water and
boiler feed water systems. It can be seen that after filtration stage, a great
reduction in EC values was detected. The bacterial mixture (Mix 1) showed
EC reduction 86.1% while the fungal mixture shows 82.4 %reduction.
Reduction efficiency of EC may be attributed to consumption of ions by
bacteria and fungi for their growth and other metabolic activities (Porwal et
al. 2015). In addition, the removal efficiency of EC was improved after
filtration; this may be due to the adsorption of ions on the activated carbon
layer.
Concerning TSS for both bacterial and fungal mixtures shows high
reduction efficiency with 99.5% and 98.9% respectively as shown in table 2.
However after incubation the reduction efficiency was not high for both
bacterial and fungal mixtures as it increased after filtration and this results
was in correspondence with Porwal et al., 2015 and Shruthi et al., 2012.
TDS reduction efficiency was 80.4% and 77.8% for bacterial and
fungal mixtures respectively as shown in table 2. Gaikwad et al., 2014 had
also reported a maximum of 74.36% reduction in TDS by using microbial
consortia of various bacterial species namely Pseudomonas, Actinomycetes,
Bacillus, Staphylococcus and Streptomyces. The presence of high level of
total suspended solids and total dissolved solids is due to organic and
inorganic matter present in the effluent. A large number of solids are found
dissolved in natural waters, the common ones are bicarbonates, carbonate,
phosphates, chlorides, sulfates and nitrates of calcium, magnesium, sodium,
potassium, iron, magnesium etc. A high content of TDS reduces the ability
to reuse this water for drinking, irrigation and industrial purposes.
For COD the bacterial and fungal mixtures have obtained high
reduction efficiency was 71.2% and 68.9% respectively after incubation
while after filtration the total reduction efficiency was 78.5% and 77.1%
respectively as shown in table 2. Our results are in correspondence with the
reduction in COD seen by Guillen-Jimenez et al., (2000). Chatterjee and
Pugaht (2013) had also reported 67.1% and 48.3% reduction in COD of
diary wastewater with use of two bacterial strains namely Neisseria sp.
and Citrobacter sp. The reduction in COD values might be due to more
279 Egypt. J. of Appl. Sci., 35 (12) 2020
amounts of nutrients present in the form of dissolved and organic nature
which is used by microorganisms for their growth.
Concerning BOD reduction efficiency for both bacterial and
fungal mixtures high reduction efficiency was observed after incubation.
BOD is widely used as an indication of water quality. The significant
decrease in BOD values could be associated with consumption of
organic material by microorganisms as a source of food. The
reduction percentage was 73.1% and 70.4% after incubation for
bacterial and fungal mixtures respectively and increased to reach 79.8%
and 72.5% respectively after filtration. These results were in
correspondence with Porwal et el (2015) and Das & Santra (2010).
Oil & grease reduction efficiencies for both bacterial and fungal
mixtures was high after filtration as it was 97.4% and 96.1 %
respectively while after incubation it was 43.1% and 40.5% respectively
as shown in table 2. These results are with correspondence with Porwal
et el 2015. The presence of sand and activated carbon filter increased
reduction percentage of O&G due to their adsorption abilities. Also,
lower reduction percentage during incubation stage may be attributed to
the difference in degradation power of microorganisms depending on
their lipase system and physicochemical properties of substrate
(Wakelin & Forster 1997).
CONCLUSIONS
From this study we can conclude that the treatment technology of
dairy wastewater by using bacterial and fungal strains was very effective
as we achieved high reduction efficiency in all the tested parameters.
Bacterial mixtures were slightly more effective than fungal
mixtures during the treatment process. Using activated carbon and sand
for filtration after incubation enhances the reduction efficiency of the
pollutants present in dairy wastewater.
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تقييم بعض السلالات البکتيرية والفطرية لمعالجة مياه الصرف
الصحي لمنتجات الألبان
رحاب جمال حسن , محمد عمى السعيد ، لميس احمد محمد
المرکز القومى لبحوث الاسکان و البناء
تمع لمزک الکا نماة الحقمة ال اققمة ممن النةاقماة السما مة ممن م مان ا لبمان التمى تقم فم
م قنممة السمما س مممن أکتموبر ال منالقة رالقمما(رو م ممر واقمماس امم رتعا لمممى تقمقممک الممو مماة
الموجممممممممو و فمممممممم مقمممممممماع ال ممممممممر لمنتجمممممممماة ا لبممممممممان تممممممممع لممممممممزک مممممممممس سمممممميلاة بکتقرقممممممة
Staphylococcus aureus ر
Egypt. J. of Appl. Sci., 35 (12) 2020 282
Bacillus و Pseudomonas aeruginosa, Streptococcus thermophilus
و ممميث سممميلاة ف رقمممة (Lactobacillus fermentum و supergene
لاسمت امعا فم (Fusarium sp و Cladosporium sp. و Aspergillus sp. ر
المعالجة تع است اع مةالک ذو مرحمتقن رالتحضقن ع ترشقح ميک (مذع ال ا رسمة تمع تحضمقر
مق قن رالمزقج 1: مس سيلاة معزولة بکتقرقة و الم زقج 2: يث سيلاة ف رقة معزولة
أوضمحة النتما ج أن السميلاة البکتقرقمة والة رقمة لعما کةماءو لالقمة فم تقمقمک الممو ماة العضموقة
92 ٪ لممى التموال وأرعمرة النتما ج أن السميلاة 97 ٪ و 7 حقث بمغة نسبة تقمقمک از ا زلمة 8
٪78 77 ٪ و 7 البکتقرقمة والة رقمة ذاة کةماءو لالقمة فم ا زلمة الامميا الکمقمع العالقمع بمغمة 7
لمى التوال وم ذلک بع التحضقن لع تکن کةاءو از ا زلة لالقة لکک من ال ممق البکتقمر و
ال ممق الة رقماة حقمث ا ز ة بعم الترشمقح کمما انمع بمة فمى (مذع ال ا رسمة ان ال ممق البکتقمر
أک ر فالمقة امقيً من ال مق الة ر أ ناء لممقة المعالجة و اقضما ن اسمت اع الکربمون المنشم
والرمک لمترشقح بع التحضقن قعزز کةاءو معالجمة الممو ماة الموجمو و فم مقماع ال مر ال مح
لمنتجاة ا لبان
283 Egypt. J. of Appl. Sci., 35 (12) 2020
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