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2022.07.02.55
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Studying the efficiency of the multiple biofilters in the reduction of pollutants from wastewater
Khalid Falih Hassan*1, Ahmed Aidan Al-Hussieny2, Elaff safa Al Deen Hassan3, Ruah Abd Almunim4
1,2,3,4 Water and Environment Directorate /Ministry of Science and Technology / Iraq.  
*Corresponding author: alrubbay@gmail.com
Available from: http://dx.doi.org/10.21931/RB/2022.07.02.55
 

 
ABSTRACT

 
The Microorganisms: yeast Sporobolomyces yunnanensis, Rhodotorula mucilaginosa and Kluyveromyces marxianusin growth of 184× 106 ± 15 cell\ml, and bacteria Pseudomonas aeruginosa and Bacillus cereus in the growth of 9 – 15× 108 ± 5 cell\ml, and algae Chlorella vulgaris in the growth of 45 × 105 ± 5 cell\ ml were selected as a bio filter to reduce organic and nonorganic pollutants from wastewater by COD, TOC, TN and TP concentrations of 455, 151, 9.5 and 31 ppm in treatment period 4, 6 and 18 hr. and pH of 7-8 in 25 Co as a Batch culture system. The yeasts showed ability to reduce COD concentration by 50-64% in 18 hr. treatment period, and the bacteria showed the ability to reduce COD, TOC, TN and TP concentrations by 59-69%, 57-66%, 53-63% and 40-55% in 18 hr. treatment period. At the same time, algae showed efficiency in reducing TN and TP by 81, 82%. The selective microorganisms showed high efficiency as a biofilter to reduce pollutants concentrations from wastewater in three serial steps by a treatment period of 6 hr in each step with a high ability to reduce COD, TOC, TN , and TP concentrations by 83, 91, 100, and 100% in 18 hr. treatment period. Ultimately, this study demonstrated the high efficiency of the multiple biofilters consisting of bacteria, yeasts, and algae in reducing the concentration of pollutants in sewage wastewater.
 
Keyword. Multiple biofilters, Bacteria, Yeast, Algae, COD, TOC, TN.
 
 
 
INTRODUCTION

 
Industrial, agricultural and residential wastewater is one of the main sources of pollution of water bodies after being discharged into rivers without adequate treatment or partial treatment. These wastes contain various organic materials and pathogens that lead to a decrease in the status of natural waters and to the alteration of the balance of the aquatic environment. They also lead to pollution, as the characteristics and composition of the biodegradability of a pollutant are affected by the type of resulting industry and the presence of other pollutants. Environmental conditions such as temperature, humidity and the degree of concentration of the pollutant affect the waters. Taking into account the ability of these organisms to consume nitrogen and phosphorus compounds and carbon and methane compounds. 1
 
Wastewater treatment processes have evolved in recent years and have been developed with highly efficient technologies to reduce pollutants, such as Biofilteration, Bioshaft, and Moving Bed Biofilm Reactor (MBBR) technology, as international companies and research centers compete to find low-cost and high-efficiency methods for treating industrial and domestic wastewater and recycling this water for agricultural, industrial and other uses. The bio-filter technology is one of the efficient techniques for reducing pollutant concentrations from wastewater. These techniques depend on the metabolic activity of microorganisms to reduce the organic and inorganic pollutants in wastewater and select a group of efficient species in biological treatments according to the type of pollutants 2. Also, 3 in their study showed that biofilters are highly effective in the biodegradation of organic and inorganic pollutants in wastewater. This effect depends on the type of pollutant and the microorganisms used as biofilters and the ability of these organisms to coexist within specific conditions in the waste sample, and the exploitation of pollutants as a source of energy liberation for its permanence and reproduction.
 
Bacteria are among the efficient microorganisms in the biological treatment of wastewater, as they can secrete a wide range of decomposing enzymes that break down organic and hydrocarbon materials and convert them into more explicit materials and adsorb many heavy elements on their cell wall; in addition to their spread throughout the water body and their rapid growth. Bacillus, Pseudomonas, and some Micrococcus strains are highly efficient in breaking down dissolved organic matter by their decomposing enzymes 3, and 4; in their study, it was proved that the ability of Chromatium and Bacillus bacteria to consume sulfates significantly in addition to nitrate and nitrite from polluted water and reduce their concentrations significantly. 5 their study showed the ability of bacteria to reduce the concentration level of phosphates, nitrates, and nitrites from polluted water and convert them into food granules stored inside their cells.
 
Bacteria are divided into several classes according to their metabolism and breakdown abilities: (1) hydrolytic bacteria that secrete a group of enzymes that break down polysaccharides, fats, and proteins and convert them into organic acids, amino acids, and other simpler compounds, and (2) fermentative bacteria that work on converting these compounds into alcohol and fatty acids. Consequently, acetogenic bacteria convert it into acetate and hydrogen, then degradable bacteria break down these compounds and convert them into carbon dioxide and water 6.
 
Yeasts are among the efficient microorganisms in the biodegradation of organic pollutants in wastewater; as 7 showed in their study, the efficiency of Candida and Rhodotorula yeasts in reducing pollutant concentrations and using this yeast within a biofilter with an efficiency of more than 40% in reducing pollutants. As well as 8 demonstrated the efficiency of some species of black yeast isolated from domestic sewage wastewater in breaking down organic pollutants because they possess an effective and highly efficient enzymatic system to break down a wide range of hydrocarbons, aromatic and aliphatic substances.
 
The current study aims to use the yeast Sporobolomyces yunnanensis, Rhodotorula mucilaginosa and Kluyveromyces marxianus, Bacillus cereus, Pseudomonas aeruginosa bacteria and Chlorella Vulgaris as biofilters for multiple and sequentially to reduce the concentrations of organic and inorganic pollutants from wastewater.
 

MATERIALS AND METHODS

 
Collection and preparation of samples
 
Wastewater samples were collected from the secondary sedimentation stage of the Al-Rustumiyah wastewater treatment plant using polyethylene plastic bottles (5 liters) and transferred to the laboratory. Other wastewater samples were collected using sterile glass bottles (100ml) to isolate and identify bacteria and yeasts.
 

Isolation, culturing, and identification of yeasts
 
Yeasts were isolated from sewage water. Potato dextrose agar (PDA) medium was prepared with the addition of the antibiotic chloramphenicol prepared by dissolving 250 mg of chloramphenicol in 250 ml of distilled water,  one ml of the sample placed in sterile glass plates, and adding the sterilized solid media then was shaken well, and the plates were incubated at  27 °C for 48 hr. A smear was taken from each colony using a sterile loop and transferred to a new sterile plate containing PDA media by streak plate method and incubated at  27 °C for  48 hr, after which the resulting yeasts were diagnosed by conducting biochemical tests and based on the key of taxonomy  9, Sporobolomyces yunnanensis, Rhodotorula mucilaginosa and Kluyveromyces marxianus were diagnosed.
 

Isolation and identification of bacteria
 
One milliliter of sewage was taken from a sewage water sample and planted by spreading a nutrient medium on the plates. The plates were incubated at 37 °C for 24 hours. A smear from each bacterial colony was transferred to the diagnostic media (differential and selective) to differentiate between different bacterial species and obtain pure bacterial colonies, and biochemical tests were used to diagnose the bacteria. Bacillus cereus was isolated and identified according to 10, and a pure isolate of Pseudomonas aeruginosa was used, diagnosed by VITEK 2 compact system in the Bacteriological Laboratory/Water Research Center.
 
 
Algae culturing and diagnosis
 
Suitable biomass for Chlorella vulgaris cells was obtained within liquid cultures based on 11 through the availability of nutrients in sufficient quantities and the use of culturing with the continuous movement to eliminate local environments at a temperature of (25 ± 2 C) and a light intensity of (250 microeinstein /m 2 / s), green algae were diagnosed by preparing slides and examining them at a power of 400 X using a compound light microscope 12 and 13.
 

Chemical tests
 
The chemical factors (the chemical oxygen requirement (COD), total organic carbon (TOC), total phosphorous (TP), and total nitrogen (TN)) were measured before and after treatment according to 14.
 

Counting of bacterial cells number
 
Cell numbers of Pseudomonas aeruginosa and Bacillus cereus were calculated by preparing McFarland 0.5 solution according to 15.
 

Counting of yeast and algae cells number
 
The numbers of yeast and algae cells were calculated in 1 ml of broth using the Hemocytometer (Neubauer counting chamber) 16 and depending on the equation:
 
Number (cells/ml) = Number of cells in 4 squares x 4 x 10
 
The multiple biofilters
 
Three isolates of yeast Sporobolomyces yunnanensis, Rhodotorula mucilaginosa and Kluyveromyces marxianus were used as a mixture of the first stage of the biofilter to reduce the concentration of pollutants from sewage wastewater in pH 5-6 and covering the bottles with foil aluminum to prevent the light.   Bacillus cereus and Pseudomonas aeruginosa were used in the second stage as a mixture in pH conditions 7-8, and Chlorella vulgariswas used in the last stage of the biofilter in pH 7-8.
 

Experiments
 
Yeasts Sporobolomyces yunnanensis, Rhodotorula mucilaginosa and Kluyveromyces marxianus were used with a growth density of 184 x 106 ± 15 cells/ml, while Bacillus and Pseudomonas were used at a growth density ranging 9-15 x 108 ± 5 cells/ml and Chlorella vulgarisat a growth density of 45 x 105 ± 5 cells/ml to reduce pollutant concentration in a sample of sewage water in terms factors the chemical requirements for oxygen (COD), total organic carbon (TOC), total phosphorous (TP), and total nitrogen (TN)  using of the glass cylinders, which are contained a sterile wastewater sample (1 liter) each separately, and 20 ml of the broth of the selected microorganisms were added for each cylinder with the period treatment of 4, 6 and 18 hrs.; also it was used a wastewater sample without any addition as the control. The factors COD, TOC, TN, and TP were measured before and after treatment and for each cylinder to know the efficiency of these selected microorganisms in the treatment processes.
 
The selected microorganisms were used as multiple biofilters using graduated glass cylinders containing a sterile sewage wastewater sample ( 1 L), and 20 ml of broth for each yeast were added to them at a treatment period of 6 hrs. The treated samples were taken and filtered with filter paper (0.45 µ)  and added 20 ml of each bacteria to them at a treatment period of 6 hrs. The samples were filtered and 20 ml of broth algae were added at a treatment period of 6 hrs. The factors COD, TOC, TN, and TP were measured before and after the treatment, and a wastewater sample was used as a control.
 

Calculations
 
The percentage of concentration reduction % = initial concentration- final concentration / initial concentration x 100.
 

RESULTS AND DISCUSSION

 
Efficiency of yeasts Sporobolomyces yunnanensis, Rhodotorula mucilaginosa and Kluyveromyces marxianus in reducing pollutants concentration from sewage wastewater:
 
Table (1) shows the reduction of pollutant concentration from sewage wastewater for the control in terms of the factors COD, TOC, TP, and TN at a treatment period of 4, 6, and 18 hrs., the concentration of the factors before treatment was 455, 151, 9.5 and 31 ppm. The concentration of the factors became 454, 151, 9.2 and 29, respectively, after 4 hrs. After 6 hrs. of the treatment period, the concentration of the factors became 452, 144, 8.7 and 29.5 ppm, respectively, and after 18 hrs. The concentration of the factors COD, TOC, TP, and TN became 440, 128, 8.5 and 26 ppm with a reduction percentage of 3, 15, 10 and 16%, respectively.
 


Table 1. Reducing the concentration of pollutants from wastewater for the control in terms of COD, TOC, TP, and TN at a treatment period of 4, 6, and 18 hrs.
 
         

 
Table (2) shows the reduction of pollutants concentration from wastewater in terms of factors COD, TOC, TP, and TN using Sporobolomyces yunnanensis yeast for a treatment period of 4, 6, and 18 hours, as the concentration of factors before treatment was 455, 151, 9.5 and 31 ppm, respectively. Consequently, after 4 hrs. of the treatment period, the concentrations became 451, 138, 9.2, and 29 ppm, respectively, and then after 6 hrs. of the treatment period, the concentration of factors became 377, 128, 9.2, and 28.7 ppm, respectively. While, the concentrations of factors became 225, 92, 7.4 and 26 ppm after 18 hrs. The reduction percentage was also 50.5, 39, 22 and 16%, respectively.
 


Table 2. Reducing the concentration of pollutants from wastewater in terms of factors COD, TOC, TP, and TN using Sporobolomyces yunnanensis yeast for a treatment period of 4, 6, and 18 hours.
 
         

 
Table (3) shows the reduction of pollutants concentration from wastewater in terms of factors COD, TOC, TP, and TN using Rhodotorula mucilaginosa yeast at a treatment period of 4, 6, and 18 hrs., as the concentrations of factors before treatment were 455, 151, 9.5, and 31 ppm, respectively. The concentration became 455, 140, 9.5, and 31 ppm, respectively, after 4 hrs. of treatment also after 6 hrs. of treatment, the concentrations became 365, 126, 8.7, and 29 ppm, respectively, while the concentrations of COD, TOC, TP and TN became 210, 82, 6.6 and 22.5 after 18 hrs. of treatment with the reduction percentage of 54, 46, 30.5 and 27%, respectively.
 


Table 3. Reducing the concentration of pollutants from wastewater in terms of factors COD, TOC, TP and TN using yeast Rhodotorula mucilaginosa for a treatment period of 4, 6 and 18 hours
 
         

 
Table (4) shows the reduction of pollutant concentrations from wastewater in terms of the factors COD, TOC, TP, and TN using Kluyveromyces marxianus yeast for a treatment period of 4, 6, and 18 hrs. The concentrations of the factors before treatment were 455, 151, 9.5 and 31 ppm, respectively. The concentrations became 450, 148, 9, and 30 ppm, respectively, after 4 hrs. of treatment. However, the concentrations of factors became 360, 122, 6.4, and 30 ppm, respectively, after 6 hrs. of treatment. Whilst, the concentrations became 162, 35, 4.6, and 25 ppm after 18 hrs. With the reduction percentage of 64, 77, 32, and 19%, respectively.
 
 

 
Table 4. Reducing the concentration of pollutants from wastewater in terms of factors COD, TOC, TP and TN using Kluyveromyces marxianus yeast for a treatment period of 4, 6 and 18 hours.
 
         

       
 
 
The current study showed the high efficiency of the selected yeasts in reducing the concentration of the chemical oxygen requirement of sewage wastewater with a rate ranging from 50-64% compared to the control during a treatment period of 18 hrs. The results illustrated the high ability of the yeast Kluyveromyces marxianus to reduce the concentration of pollutants from the wastewater in terms of the factors COD, TOC, TP, and TN compared to the control. The results matched with the study 14 where they showed the ability of the yeast Kluyveromyces marxianus on the production of a group of effective enzymes in biodegradation processes such as β-galactosidase, inulinase, β-polygalacturunases, and a group of peroxidase enzymes, and the current study agreed with 15 as they showed the ability of Rhodotorula mucilaginosa to break down the complicated organic compounds associated with heavy metals in the contaminated water due to its effective enzymatic system. A study 16 proved that the use of yeasts in wastewater treatment processes has an influential role in the biodegradation of organic waste. At the same time, its efficiency is less in the process of reducing the concentrations of nitrogen and phosphorous compounds due to its nutrition system and the effectiveness of its enzymes in fermentation.This was proven by the current study results, as the percentage of reduction in the concentration of nitrogen and phosphorous compounds did not exceed 12-32%.
 

Efficiency of Bacillus and Pseudomonas bacteria in reducing pollutants concentration from sewage wastewater:
 
Table (5) shows the reduction of pollutants concentration from wastewater in terms of the factors COD, TOC, TP, and TN using Bacillus cereus at a treatment period of 4, 6, and 18 hours, as the concentrations of the factors before treatment, were 455, 151, 9.5 and 31 ppm, respectively. While the concentrations became 450, 147, 8.5, and 29 ppm, respectively after 4 hrs. of treatment, however after 6 hrs. of treatment, the concentration of COD, TOC, TP, and TN factors became 325, 130, 7.8, and 21 ppm, respectively, and after 18 hrs of treatment, the concentrations became 185, 65, 3.5 and 16.5 ppm with a reduction rate of 59, 57, 63 and 46%, respectively.
 
 

 
Table 5. Reducing the concentration of pollutants from wastewater in terms of COD, TOC, TP, and TN using Bacillus cereus for a treatment period of 4, 6, and 18 hours.
 
         

 
Table (6) shows the reduction of pollutants concentration from wastewater in terms of factors COD, TOC, TP, and TN using Pseudomonas aeruginosa for a treatment period of 4, 6, and 18 hrs., as the concentrations of factors before treatment were 455, 151, 9.5, and 31 ppm, respectively. After 4 hours of treatment, they became 435, 150, 9.5, and 28.7 ppm, respectively; however, the concentrations became 320, 122, 6.6 and 18 ppm after 6 hours of treatment. At the same time, the concentrations after 18 hrs. of treatment became 140, 52, 4.5, and 14 ppm with a reduction rate of 69, 66, 53, and 55%, respectively.
 


Table 6. Reducing the concentration of pollutants from wastewater in terms of factors COD, TOC, TP, and TN using Pseudomonas aeruginosa for a treatment period of 4, 6 and 18 hours.
 
         

 
The results of the current study illustrated the high efficiency of the selected bacteria in reducing the concentration of pollutants from the sewage wastewater during the treatment period of 18 hours compared to the control, as the reduction percentage of the concentration of the chemical oxygen requirement for Bacillus and Pseudomonas was 59-69%, the total organic carbon was 57-66%, as well as for total nitrogen was 53-63%, and total phosphorous was 40-55% compared to the control. So, the results matched with study 18, where they demonstrated the ability of Bacillus to secrete several enzymes that destroy organic and hydrocarbon compounds with high efficiency and use in treating water contaminated with oil derivatives. Also, the results of the current study are in agreement with the study 19; it was showed the efficiency of microorganisms in the biodegradation of organic pollutants, as they demonstrated the ability of Bacillus bacteria to produce a group of peroxidase enzymes that decompose and destroy organic materials and aliphatic and aromatic compounds with high efficiency. The results of the current study also coincided with study 20, as they showed in their study the high enzymatic efficiency of Pseudomonas bacteria in destroying many organic compounds and hydrocarbon pollutants, including the cytochrome group and the biphenyl dioxygenase group, which have a wide range and high efficiency in the biodegradation of organic pollutants and benzene compounds. The current study's results also agreed with 21 as they showed in their study the ability of bacteria Pseudomonas produces a wide range of enzymes that break down hydrocarbons and benzene compounds and their ability to biodegrade these compounds with high efficiency.
 

Efficiency of Chlorella vulgaris in reducing pollutants concentrations from sewage wastewater:
 
Table (7) shows the reduction of pollutants concentration from wastewater in terms of the factors COD, TOC, TP, and TN using Chlorella vulgaris for a treatment period of 4, 6, and 18 hrs., as the concentrations of factors before treatment, were 455, 151, 9.5 and 31 ppm, respectively, however, the concentrations became 410, 148, 8, and 29 ppm, after 4 hrs. of treatment. While after 6 hrs. of treatment, the concentrations of the factors became 388, 133, 5.2, and 20 ppm, respectively, and after 18 hrs. of treatment, the concentration of the factors COD, TOC, TP, and TN became 270, 95, 1.8 and 5.6 ppm with the reduction rate of 40, 37, 81 and 82%, respectively.
 


Table 7. Reducing the concentration of pollutants from wastewater in terms of factors COD, TOC, TP and TN using Chlorella vulgaris for a treatment period of 4, 6 and 18 hours.
 
         

 
The current study results showed the ability of Chlorella vulgaris to reduce the concentration of nitrogenous and phosphorous compounds from sewage wastewater with a reduction rate of 81 and 82% compared to the control.
 
The current results agreed with 22 in their study to reduce the concentration of nitrogen and phosphorous nutrients from sewage wastewater, as they demonstrated the ability of Chlorella vulgaris to reduce total nitrogen concentrations by 87% and total phosphorous by 98%  to benefit from them in growth and proliferation, as they are essential elements for algae growth. The current results agree with study 23. They clarified the essential and influential role of using this alga in wastewater treatment processes because of its high ability to reduce nitrogen and phosphorous nutrients concentrations and exploit them in growth and proliferation.
 
 
Efficiency of the multiple biofilters in reducing pollutants concentration from sewage wastewater:
 
Table (8) shows the percentage of reducing the concentration of pollutants from wastewater in terms of the factors COD, TOC, TP, and TN for the control treatment by using the multiple biofilters at a treatment period of 18 hrs., as the concentrations of the factors before treatment were 560, 172, 25 and 42 ppm, respectively. As well as, after 18 hours of treatment, distributed in three stages (6 hrs. for each stage). The results showed an increase in COD concentration, which became 820 ppm, while the concentration of TOC, TP, and TN were 168, 13.5, and 39 ppm, with a reduction percentage of 2.10 and 7%, respectively.
 


Table 8. Reducing the concentration of pollutants from wastewater in terms of the factors COD, TOC, TP, and TN for the control treatment using the multiple biofilters for 18 hours.
 
         

 
Table (9) shows the reduction of pollutants concentration from wastewater in terms of the factors COD, TOC, TP, and TN using the multiple biofilters for a treatment period of 18 hours, as the concentration of the factors before treatment were 560, 172, 25 and 42 ppm, respectively. In addition, it was distributed into three phases (6 hrs. for each phase); after 18 hrs. of treatment, the concentration of the factors became 0, 15, 95, and 0 ppm with the reduction percentage of 83, 91, 100, and 100%, respectively.
 


Table 9. Reducing the concentration of pollutants from wastewater in terms of factors COD, TOC, TP, and TN using the multiple biofilters for a treatment period of 18 hours.
 
         

 
The results of the current study showed the high ability of the biofilter used to reduce the concentrations of organic and inorganic pollutants from sewage wastewater and the high efficiency of the microorganisms used within this filter during 18 hrs. of the treatment period, also the nitrogen and phosphorous compounds were removed from the wastewater sample at a rate of 100%, as the results of the current study agreed with 3 in their study demonstrated the ability of biofilters to reduce the concentration of pollutants from wastewater if appropriate microorganisms were used which can biodegrade when the appropriate conditions were provided for the growth of these organisms, and the results of the current study matched with the study 25, as they proved the ability of the biofilter is eliminated of the total nitrogen from sewage waste by 100% and reduced organic pollutants in terms of total organic carbon measurement by 95%. The results also matched with 26 and 27, as they proved in their study that the efficiency of biofilters depends on the selection of the species of microorganisms used to treat industrial and domestic wastewater used as biological filters and appropriate conditions provide for the continuity of their activity. The reduction of pollutants concentration from wastewater is based on these microorganisms' metabolism and degradation activity.
 

CONCLUSION

 
The current study demonstrated the high efficiency of the multiple biofilters consisting of bacteria, yeasts, and algae in reducing the concentration of pollutants in sewage wastewater in terms of COD, TOC, TP, and TN with reduction rates of up to 100%.
 

Conflicts of interest
 
   The authors declare no discrepancy of interest related to this article.
 
Acknowledgments
 
    We are thankful to the Bacteriological Laboratory/Water Research Center/Water and Environment Directorate /Ministry of Science and Technology / Iraq. Also, we did not get the financial source of the study.
 

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Received: 15 March 2022 / Accepted: 8 April  2022 / Published:15 May 2022
 
 
Citation.   Falih Hassan K , Aidan Al-Hussieny A, safa Al Deen Hassan E , Abd Almunim R. Studying the efficiency of the multiple biofilters in the reduction of pollutants from wastewater. Revis Bionatura 2022;7(2) 55. http://dx.doi.org/10.21931/RB/2022.07.02.55
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