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JOURNAL OF BIOMEDICAL RESEARCH & CLINICAL  PRACTICE

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Original Article

 

Isolation of Microorganisms Associated with Biodegradation of Household Domestic Wastes for Biogas Production in Niger State, Nigeria

Jiya AG,1 Ijah UJJ,2 Galadima M,2 and Akpan UG.3

1Department of Biological Science, Federal Polytechnic Bida, Niger State Nigeria.1

2Department of Microbiology and 3Department of Chemical Engineering, Federal University of Technology Minna, Niger State Nigeria.

 

 

ABSTRACT

*Corresponding Author: Jiya, A. G, Department of Biological Science, Federal Polytechnic Bida, Niger State Nigeria. Tel: +2347030393603, Email: ganaibro74@yahoo.com

This study focused on the isolation of microorganisms associated with biodegradation of domestic wastes in three rural communities (Gbadagbadzu (A), Ndawangwa (B), and Kuchiworo (C)) in Lavun Local Government Area of Niger State, Nigeria, for biogas production. The biogas was produced by anaerobic microbial degradation of different biodegradable household domestic waste aided by fresh rumen of cow. The anaerobic microbial degradation was carried out in a temperature range of 250C to 320C for a detention time of 39 days for rural biogas production. The results showed the presence of the following bacteria: Bacillus cereus, Sphingobacterium yamdrokense, Clostridium perfringens, Salmonella typhi, Alkaligenes faecalis, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae and Bacillus licheniformis while fungi isolated were Muccor pusillus and Aspergillus flavus. The research therefore shows that household domestic wastes have the potential to produce biogas with or without the addition of inoculum.

Keywords: Biogas, Biodegradation, Domestic waste, Household, Microorganisms.

INTRODUCTION


E

nergy crisis and climate change are among the major problems drawing much attention all over the globe and renewable energy has been identified as one of the solutions.1 Biogas is an alternative source of renewable energy, it is clean and environmentally friendly and often produced from organic materials that are first decomposed by microorganisms in an anaerobic environment.2 A complex microbiological process lies behind the efficient production of biogas.3 Many different species of microorganisms need to be active in order for biogas to form and these organisms have to work closely together. A disturbance of this teamwork results in reduced biogas production. 4 Controlling the biogas process in an efficient manner requires the knowledge of microbiology that results in reducing pressure on wood as fuel source and improves the environment.5 Microorganisms require food (substrate) in order to function and grow. The organic waste pre-treated in the biogas process represents the substrate for various microorganisms. These includes sludge from municipal wastewater treatment plants, slaughterhouse waste, waste from the food and feed industries, source-sorted food waste and manure, grease traps, fryer fat, wastes from the dairy and pharmaceutical industries, grass silage, and domestic household wastes.6,7 Careful removal of agro-industrial/domestic household wastes from the environment and converting them to biogas is a recommended method for development of sustainable healthy environment.  Many local communities especially in developing world have no environmentally friendly ways to dispose such wastes. Generally, large amounts of household and municipal wastes are dumped around human settlements, resulting in disposal problems and methane emissions during its natural decomposition.  Some of these wastes are of low density and easily become air borne pollutants.8 Environmental problems associated with poor wastes management have resulted in increased water borne illness especially typhoid fever, dysentery and diarrhoea.9,10 These challenges have continued to retard public health improvement programmes of governments and private organizations. Several reports indicated that organic wastes which represent 45-65% of the volume of municipal wastes is a key challenge in waste management.11,12,13 The aim of this study was to isolate and identify microorganisms associated with biogas production from domestic wastes generated from rural communities in Niger State.

 

MATERIALS AND METHODS

Collection and processing of samples

The substrates used for this study were domestic household wastes including carbohydrate food wastes (boiled yam, yam peels and products, bread crumps, boiled rice, potato peels, cassava peels, cassava products), maize cobs, groundnut shells, leafy vegetables as well as foods containing proteins (beans and beans products, egg shells, fish crumps). They were collected from three local communities: Gbadagbadzu (A), Ndawangwa (B), and Kuchiworo (C), all in Lavun Local Government Area of Niger State, Nigeria. In each of these communities, ten (10) clean waste bags were distributed to ten (10) household for a period of one month. The waste bags were collected every two days and emptied into two clean waste containers in each of the communities giving a total of six waste containers. All the samples collected were air-dried at room temperature (28 + 2°C) for seven days, pounded using a clean mortar and pestle, kept in air-tight containers.

Analysis of substrates for microbiological properties

The microbiological parameters determined  were : total aerobic heterotrophic bacterial counts, methanogenic/anaerobic bacteria counts, faecal coliform and non-faecal coliform counts, total salmonella-shigella counts and fungi counts using Nutrient agar (NA), Mac Conkey Agar (MCA), Eosin methylene blue (EMB) agar and Sabouraud dextrose agar (SDA)   respectively.14, 15

Determination of total aerobic heterotrophic and methanogenic/anaerobic bacteria counts

Substrate homogenate was prepared by dissolving 1g of substrate in 10 mL of sterile distilled water. This was serially diluted and inoculated on Nutrient agar (NA) plates. The plates were incubated at 37°C for 24 hours while plates for anaerobic counts were incubated anaerobically using anaerobic jars at 37°C for 24 – 48 hours. Plates with 30 – 300 colonies were counted (including pin point colonies) and the mean counts calculated factor. The aerobic and anaerobic colony counts were computed as reported by Kiiyukia14 and is given as

                    (1)

where N is the number of colonies per mL of sample, A is the average count per plate and D is the respective dilution factor

Enumeration of coliforms

Samples were serially diluted and the suspension was inoculated into the respective media using pour plating technique. Colonies that grew on the media were sub-cultured repeatedly on the media used for primary isolation to obtain pure cultures. The pure cultures were maintained on agar slants for further characterization and identification using standard biochemical tests.16

Enumeration of fungi

The fungi were enumerated using standard methods reported by Kiiyukia14 and Asikong et al.17 Serially diluted samples were inoculated into sabouraud dextrose agar plates with two vial of chloramphenicol to inhibit the growth of bacteria. The plates were incubated at room temperature (28 ± 2°C) for 3-5 days. Colonies were counted and expressed as colony forming units per gram of sample (cfu/g). Colonies were subcultured repeatedly on media used for primary isolation to obtain pure cultures. The pure cultures were maintained on SDA slants for further characterization and identification.

Identification and characterization of microbial isolates

The bacterial isolates were Gram stained and subjected to biochemical tests including production of catalase, coagulase, indole, oxidase, hydrogen sulphide, methyl–red Voges-prokauer, starch hydrolysis, citrate utilization, sugar fermentation.15,16 The isolates were identified by comparing their characteristics with those of known taxa using Bergey’s Manual of Systematic Bacteriology.18 The fungal isolates were characterized based on the colony morphology, nature of hyphae, nature of conidia and shape. A portion of the mycelial mat of the fungi was picked with sterile needle and placed on a clean, grease-free slide containing a drop of lacto-phenol cotton blue stain. The mycelial growth was teased gently to allow it mix with the stain, covered with cover slip and was observed under a low to high power objectives (x10 and x40) of the light microscope. The fungi isolates were identified by comparing their characteristics with those of known taxa using the schemes of Jott et al.18

Equipment used for the production of biogas

A biodigester capable of producing biogas from household domestic waste was designed and constructed in order to achieve the study objectives. The digester (20 litres capacity) consisted of anaerobic chamber and gas collecting chamber. In between the two chambers was a narrow passage which allowed the flow of gas from anaerobic chamber to gas chamber. As microbial activities began, the emissions were released and in about 21 days it was ready for harvesting. A short valve of 10 mm diameter conveyed the gas from gas chamber to element for burning. In between the burner and gas chamber was a knob which served to regulate the biogas flow as shown in Plate 1.17

Plate 1: Biogas production design for rural communities

RESULTS

Microbiological properties of organic wastes

The total microbial counts of undigested (UDW) and digested (DGW) wastes respectively, are presented in Table 1. The results revealed that total heterotrophic bacterial counts, total fungi, total faecal coliform and total Salmonella-shigella counts were higher in UDW than DGW samples (Table 1). It was 3.8 x 108, 6.7 x 108TVC, 1.3 x 103, 1.10 x103 TFC, 1.4 x 105, 2.3 x 105 TFCC and 6.0 x 106 TSSC for UDW while DGW had bacterial counts of 4.5 x 104, 2.45 x 106, 1.21 x 102 TVC, 1.2 x 102, 1.0 x 102 TFC, 1.6 x 103, 1.2 x 103 TFCC and 3.6 x 103, 2.5 x 106 TSSC respectively.

In the same vein, anaerobic / methanogenic counts were higher in UDW 1.8 x 106 and 2.10 x 106 than DGW 1.31 x 103 and 1.7 x 103. The sum total of bacterial counts for AN/MB UDW was 3.9 x 1036as against 3.01 x 109DGW respectively (Table 1).

Table 1: Microbial counts of undigested and digested organic waste                                            

                                             Aerobic bacteria (Cfu/g)                                    Anae./Methano.

                                                                                                                         Bacteria (Cfu/g)

Sample

TVC

T.FC

TFCC

TSSC

Anae./Methano.

UAL

3.8 x 108

1.3x103

1.4x105

Nil

Nil

UBL

6.7x109

1.10x103

2.3x105

6.0 x 106

Nil

UAL

Nil

Nil

Nil

Nil

1.8 x 106

UBL

Nil

Nil

Nil

Nil

Nil

DLW(CABCD)

4.5 x 104

1.2 x 102

1.6 x 103

3.6 x 103

Nil

DSW(CABCD)

6.0 x 106

1.0 x 102

1.2 x 103

245.0 x 106

Nil

DLW(RA, RB, RC, RD)

Nil

Nil

Nil

Nil

1.31 x 103

DSW(RA RB RC RD)

Nil

Nil

Nil

Nil

1.7 x 103

 

UAL: Undigested household domestic (organic waste), UBL: Fresh content of the rumen of cow, DGW: Digested organic waste, DSW: Disgested Solid Waste, TVC: Total viable counts, TFC: Total fungi counts, TFCC: Total faecal coliform counts, TSSC: Total Salmonella–Shigella counts, Anae./Methano.: Anaerobic/Methanogenic bacteria, CABCD: Communities AB (D the Control), RA, RB, RC, RD: digesters containing waste used for rural biogas production Cfu/g: Colony forming units per gram  Note: UAL and UBL = UDW, DLW and DSW = DGW.

Identification of microbial isolates and their frequencies of occurrence

Morphological characteristics from digested and undigested organic waste revealed a total of nine (9) bacterial species. The bacteria were Bacillus cereus, Clostridium perfringens, Sphingobacterium yamdrokense, Alkaligenes faecalis, Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella typhi, Bacillus licheniformis. All the bacteria were rods (bacolli) except Staphylococcus epidermidis which was cocci.

Macroscopic and microscopic morphology of fungi isolated from digested and undigested organic wastes revealed the presence of Aspergillus flavus and Mucor pusillus.

Table 2, showed the frequency of occurrence of bacterial isolates in liquid digestate from rural digesters (RA, RB, RC and RD respectively). The highest frequency of occurrence was recorded with Bacillus cereus (35.48%) while Bacillus licheniformis  had the least frequency of occurrence (2.65%) Clostridium perfringens, Sphingobacterium yamdrokense, Alkaligenes faecalis, Pseudomonas aeruginosa, Staphylococcus epidermidis Klebesiella pneumoniae and Salmonella typhi had 6.19%, 15.04, 5.30%, 3.30%, 6.19%, 15.92% and 7.96% respectively. The results also revealed that rural digester RA had the highest total number of isolates and decreased in the order RA (50), RB (32), RC (19) and RD (12) which is the least, with Bacillus cereus, Klebesiella pneumoniae and Salmonella typhi found in all the digesters (Table 2).

Table 2: Frequency of occurrence of bacterial isolates from liquid digestate in rural digesters with or without starter culture

 

                           Rural digesters

 

 

RA

RB

RC

RD*

Total (%)

Bacillus cereus

13(11.50)

11(9.73)

9(7.96)

7(6.19)

40(35.48)

Klebsiela pneumoniae    

6(5.30)

3(2.65)

6(5.30)

3(2.65)

18(15.92)

Sphingobacterium yamdrokense

10(8.55)

7(6.19)

0(0)

0(0)

17(15.04)

Salmonella typhi

2(1.77)

1(0.88)

4(3.54)

2(1.77)

9(7.96)

Clostridium perfringens

5(4.42)

2(1.77)

0(0)

0(0)

7(6.19)

Staphylococcus epidermidis

4(3.54)

3(2.65)

0(0)

0(0)

7(6.19)

Staphylococcus aureus

4(3.54)

2(1.77)

0(0)

0(0)

6(5.30)

Pseudomonas aeruginosa

4(3.54)

2(1.77)

0(0)

0(0)

6(5.30)

Bacillus licheniformis

2(1.77)

1(0.88)

0(0)

0(0)

3(2.65)

Total

50(44.23)

33(28.29)

19(16.74)

12(10.61)

113(100)

RD*: Liquid digestate without starter culture (control) Values obtained are significantly different at (p ≤ 0.05)

 

Biogas production  

Figures 1 and 2 show the rates of biogas production from household domestic wastes with or without starter culture in rural digesters RA, RB, RC and RD.  The results indicated that in (39 detention days) rural digester RA had a biogas volume of 98.14 cm3, rural digester RB had 31.53 cm3, RC gave 6.21 cm3 while RD, (control) that was without starter culture gave 4.72 cm3 within a detention time of 33 days (Fig. 1).  Thus, RA gave the highest yield and the yield fluctuated in other digesters in decreasing order giving the least yield in RD. The total volumes were 10539.39 cm3, 5426.71 cm3, 2275.93 cm3 and 124.04cm3 from rural biogas digesters RA, RB, RC and RD respectively (Figure 2). However, while biogas production fluctuated in the same pattern in RA (98.14) and RB (31.53), the pattern changed slightly for RC and RD with RD (8.12) having higher production than RC (6.21).

Figure 1: Biogas Production from organic waste in locally designed biodigesters (RA,RB RC and RD) 

Figure 2: Rate of Biogas Production in locally designed biodigesters (RA, RB, RC and RD)

DISCUSSION

Microbiological counts of the organic waste

The microbial load appeared to be decreasing significantly after 50 days and 39 days of biogas production from the laboratory and rural digesters. This could be due to the production of toxic materials as the end product of metabolism. This agrees with the findings of Farina et al.14 who reported that ammonia stress during thermophilic digestion of poultry droppings had high contents of ammonia. This raises the pH outside the upper minimum range which resulted in the reduction/inhibition of methanogenic organisms. This decrease can also be attributed to the exhaustion of essential nutrients from the digester due to continuous breakdown of complex material to simple organic compounds or could be from the use of different succession of microorganisms participating in the process.17, 19

The anaerobic bacteria counts range from 1.8 x 106 cfu/g and, 2.10x106 cfu/g for UDG (undigested waste), 1.31x102, 1.7x103 DGW (digested waste) respectively (Table 1). The variation in the microbial counts might be attributed to complete anaerobic process and stability of the condition in the anaerobic digester especially when there is co-digestion of different organic wastes. This is in line with the findings of El-Mashad et al.20  that digestion of more than one substrate in the same digester can establish positive synergism and the added nutrients can support anaerobic bacterial growth. The investigators also reported that during mesophilic anaerobic co-digestion of cattle manure, fruit and vegetable wastes (FVW) in a continuous stirred tank reactor at 350C, increasing the percentage of FVW from 20 0C to 500C leads to increase in methane yield from 230 to 450l/kg. This is also in agreement with Eze and Agbo21 who reported that increase in total anaerobic counts is due to the fact that conditions are favourable for their growth and development. The differences may also have resulted from the activities of anaerobic methanogenic organisms consuming methane sors produced from the initial activity. 22,23

The fungal counts (Table 1) showed a decrease from 1.3x103, 1.10x103 undigested waste (UDW) to 1.2x102, 1.0x102 cfu/g digested waste (DGW) respectively. The presence of fungi in anaerobic biogas process may be based on their ability to adhere and penetrate cell walls through which they open the cells for numerous members of bacterial community and speed up the whole decomposition process, while majority may be there as contaminants and when they die, become substrate nutrients.24 The decrease in fungal counts in the present study is contrary to the finding of  Sirohi et al.21 who reported that increase can be traced to the decomposition of lignocellulosic materials. This decrease in microbial counts is also in line with the report of Asikong et al.17.

Identification of bacteria and their frequency of occurrence from biogas produced in the laboratory

Bacillus cereus, Sphingobacterium yamdrokense and Alkaligenes faecalis were the dominant species. This suggests that the species play a vital role in the production of biogas. The frequency of occurrence of Bacillus cereus after digestion must have resulted from microbial succession in which probably the fungal and cellulolytic organisms produce favourable environment for their rapid growth25,26 or as a result of antagonism that results in the production of secondary metabolites such as antibiotics which inhibited the growth of other microorganisms present in the digester thereby paving way for them to get to the final stage of methanogenesis.22 Species of Clostridium, Alkaligenes and Bacillus secret hydrolytic enzymes capable of decomposing organic waste in anaerobic digestion and can also overlap from one stage to another during biogas production also suggest a succession in species of bacteria during methanogenesis.27 The ability of Bacillus species to overlap during biogas production and to survive in both liquid and solid digestate were probably due to the fact that the organisms can produce spores which help them to withstand high temperatures, dryness and heat that evolved from biogas production or harsh anaerobic conditions.22,28 These findings were also in conformity with that of Oluyega29 and Bagudo et al.30. This frequency was also attributable to the fact that methanogens live in a synthrophic or complementary relationship with other organisms that breakdown the biomass to simple monomers.2 Asikong et al.17 reported that the presence of cyanogenic glycosides in cassava peels and other plant peels as in the present study can induce excess acidic production, Nitrogen deficiency and the release of cyanide which is highly toxic to bacteria.

Identification of fungal isolates

The low frequency of occurrence of fungal species owing to the fact that only Aspergillus flavus and Mucor pusillus were isolated in the present study (Table 3) is contrary to the findings of Getu et al.28 who recorded a high frequency of Aspergillus niger to justify the fact that most Aspergillus blend well with plant material and are beneficial in Agriculture.29 It was however observed that fungi count was slightly higher in undigested organic waste than digested organic waste. This was probably due to the ability of fungi to tolerate acidic condition initially than slightly alkaline condition that was later prevalent in some of the sample components such as cassava and orange peels as a result of cyanogenic acid. Furthermore, the reduction in fungi counts after digestion could be due to the inability of the organism to survive in oxygen free environment. This result agrees with the report of Uzodinma et al.24 who observed a reduction in bacterial and fungi counts from various substrates used for digestion. The presence of fungi isolate in organic wastes is an indication of their geotropic nature and possession of extracellular inducible enzymes such as keratinolytic proteases which are crucial for decomposition of protein keratin material in the organic waste.30

 

CONCLUSION

The following microorganisms: Bacillus cereus, Sphingobacterium yamdrokense, Clostridium perfringens, Salmonella typhi, Alkaligenes Faecalis, Pseudomonas aeruginosa, Staphylococcus epidermidis, Klebsiella pneumoniae and Bacillus licheniformisMuccor pusillus and Aspergillu flavus  were involved in biogas production from domestic wastes. Domestic household wastes from laboratory biogas production had the highest rate and total biogas volume of 183.97 cm3 while that from rural biogas production gave the highest rate and total biogas volume of 10539.39 cm3. This implies that domestic household waste could serve as a suitable substrate for biogas production and that the utilization of this substrate for biogas production could solve its disposable problems thus making way for abundant source of sustainable energy.

RECOMMENDATION

It is recommended that, other household domestic waste not used in this study should be harnessed for biogas production. For pathogens like salmonella species amongst others to have been found to be associated with biogas process and to survive the anaerobic process to the end in this study, may pose a threat on agricultural industry and thus, the use of solid digestate be preferred to liquid digestate as organic fertiliser or measures that can allow their elimination be adopted before application.

REFERENCE

1.   Sunday OO. On energy for sustainable development in Ota, Ogun State, Nigeria. Renewable and Sustainable Energy Review; 2012; (16): 2583–2598

2.   Adekunle KF., Kkolie JA.. A Review of Biochemical Process of Anaerobic Digestion. Adances in Bioscience and Biotechnology; 2015; (6): 205-212.

3.   Schnürer A, Jarvis A. Microbiological Handbook for Biogas Plants Swedish Waste Management U2009:03 Swedish Gas Centre Report 207; 2010

4.   Bolarinwa OA., Ugodi EO. Production of Biogas from Starchy Wastes. Journal of Science Research and Development; 2010; (12): 34–45.

5.   Boy E, Bruce N, Delgado H. Birth weight and exposure to kitchen wood smoke during pregnancy in rural Guatemala. Environmental Health Perspectives; 2002; 110 (1): 109–114.

6.   Nordberg U. Biogas- nuläge och framtida potential, Värmeforsk, projektnummer; 2006; T5-503.

7.   Linné M, Ekstrand A, Engelsson R, Persson E, Björnsson L, Lantz M.  Den svenska biogaspotentailen från inhemska restprodukter. Avfall Sverige, Swedish Biogas Association, Swedish Gas Association, Swedish Water. Lund. In Swedish; 2008

8.   Ginting N. Benefits of Using Biogas Technology in Rural Area; Karo District on Supporting Local action Plan for Green House Gas Emission Reduction of North Sumatera Province 2010-2020. IOP Conference Series. Earth and Environment Science; 2018; 65(1):012007

9.   World Health Organisation. Water for life, making it happen: A decade for action; 2005; 215.Geneva 44 at: http//www who in/waster –sanitation health/water for life. PDF.

10. Osibanjo O, Adie GU. Impact of effluent from Bodijaabattior on the physicochemical parameters of Oshenkaye stream in Ibadan City, Nigeria. African Journal of Biotechnology; 2007; 6(15): 1806-1811.   

11. Agamuthu P. Solid waste: Principle and management. Institute of Biological Sciences. University of Malaya, Kaula Lumpur, Malaysia; 2001; 41-101

12. Adeoye PA, Mohammed AS, Musa JJ, Adebayo SE. Solid waste Management in Minna, North Central Nigeria: present practices and future challenges. Journal of Biodiversity and Environmental Sciences; 2011; 1(6): 1-8.

13. Aragaw T., Mebeaselassie A., Amare G. Codigestion of Cattle Manure with Organic Kitchen Waste to increase Biogas Production Using Rumen Fluid as Inoculums. International Journal of Energy and Environmental Research; 2013; 8(11) 443-450

14. Kiiyukia C. Laboratory Manual of Food Microbiology for Ethiopian Health and Research Institute, Jomo Kenyata University of Agriculture and Technology, Nairobi, Kenya; 2003; 3(436) 11-52.

15. Cheesbrough M. Preparation of Reagents and Culture Media. District Laboratory Practices in Tropical Countries. 2nd Edition Edinburg, Cambridge University Press. United Kingdom; 2003; 62-132.

16. Owuama CI. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) using a Novel Dilution Tube Method, African Journal of Microbiology Research; 2017; 11(23) 977-980.

17. Asikong BE, Epoke J, Eja EM, Antai EE.  Potentials of biogas generation by combination of cassava peels (CP) and Poultry Droppings (PD) in Cross River State Nigeria, Nigerian Journal of Microbiology; 2012; (26): 2540-2549.

18. Jott AG., Kileg NR., Sneatt PHA., Stanely TT., Williams ST. Bergys Manual of Systematic Bacteriology, 9th Edition Williams and Wilkins Co. Baltimore, Maryland; 1994.

19. Elango D, Pulikesi M, Bakaralingam P, Ramammuthi V, Sivanesan S. Production of biogas from municipal solid waste with domestic sewage. Journal of Hazardous materials; 2007; 141: 301-304.

20. El-Mashad HM, Zhang R. Biogas production from co-digesting diary manure and food waste Elsevier Journal of Bio resource Technology; 2010; 101: 4021-4028. Available from www.elevier.com/locate/biortech.

21. Eze JI, Agbo KE. Studies on the Microbial spectrum in anaerobic bio-methanation of cow dung in 10m3 fixed dome biogas digester. International Journal of Physical Sciences; 2010; 5(8):1331-1337.

22. Rabah AB, Baki AS, Hassan LG, Musa M, Ibrahim AD. Production of Biogas using abattoir waste at different retention time in Sokoto, Nigeria, Science World Journal; 2010; 5(4): 23 – 26.

23. Dahunsi SO.  Oranusi U S. Co-digestion of food waste and human excreta for biogas production. British Biotechnology Journal; 2013; 3(4): 485-499.

24. Uzodinma EO, Ofoefule JI, Eze I, Mbaey I, Onwuka ND. Effects of some organic waste on biogas yield from carbonated soft drink sludge:. National center for Energy Research and Development, University of Nsukka, Nigeria.  Academic Journal of Scientific Research and Essay; 2008; 3(9): 401-405.

25. Harold H. Engineer, Diary and Beef Housing Equipment. Ontario Ministry of Agriculture, Food and Rural Affairs. London Swine Conference - today’s challenge... Tomorrow’s opportunities; 2007; 3-4.

26. Iyagba ET, Mangibo TA, Mohammed YS. The study of cow dung as co-substrate with rice husk in biogas production. Science Research Essays; 2009; 4(9): 861-866.

27. Okoroigwe EC, Ibeto CN, Ezema CG. Experimental study of anaerobic digestion of dog waste in Enugu State, Nsukka, Nigeria. Scientific Research and Essays; 2014; 9(6): 121 – 127.

28.  Gupta P, Samant, K, Sahu A. Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential. Integrated Journal of microbiology; 2011; 2(12): 1-5

29. Oluyega IO, Femi-ola TO, Opomu D. Anaerobic digestion of cattle feeds mixed with piggery waste for Biogas production and Bio-fertilizer production.  Journal of Microbiology; 2006;  3:2-7.

30. Bagudo BU, Garbe B, Dangoggo SM, Hassan LG. Comparative study of biogas production from locally sourced substrate materials. Nigeria Journal of Basic and applied sciences; 2008; 16(2): 262-266.