Optimization of hemolysin formation in Alcaligenes species isolated from abattoir wastewater samples in Akure, Ondo State, Nigeria

DOI: 10.48129/kjs.9406


  • Olubukola Olayemi Olusola-Makinde Department of Microbiology, Federal University of Technology, Akure, Nigeria
  • Daniel Juwon Arotupin Department of Microbiology Federal University of Technology Akure Nigeria
  • Anthony Ifeanyi Okoh Applied and Environmental Microbiology Research Group University of Fort Hare Alice South Africa




Abattoir wastewater, Alcaligenes spp., haemolysin, pathogenicity


The function of haemolysin is significant during infections, it can also act as toxin; therefore, it has been used as molecular marker for pathogenicity. This study evaluated conditions for optimal haemolysin production by Alcaligenes faecalis strains isolated from a city abattoir wastewater. The conditions investigated for haemolysin production were size of the inoculum, initial pH of production medium, bacterial incubation temperature, agitation speed, and growth media. Thereafter, the effects by various treatments on the haemolytic activity of the produced haemolysin were assessed.  The genus Alcaligenes was assigned to the test organisms after analysis of their 16S rRNA gene sequence with accession numbers: MF498824, MF498825 and MF498827 accordingly. Optimum conditions for haemolysin production for Alcaligenes faecalis strain OS42 were inoculum size 0.5% (v/v), pH 9, 20 oC, 0 rpm and brain heart infusion broth. Then, 77% and 79% haemolytic activities were attained at 20 h for strains OS42 and OS61 respectively. Cholesterol and EDTA did not affect crude haemolysin production. This work revealed haemolysin production by Alcaligenes strains sourced from abattoir wastewater effluent, and demonstrated that this effluent is contaminated with pathogenic Alcaligenes strains. This is a public health risk due to their prospective to cause human and animal infections.


Akinro, A.O., Ologunagba, I.B. and Yahaya, O. (2009). Environmental implication of unhygienic operation of a city abattoir in Akure, Western Nigeria. Journal of Engineering and Applied Science, 4: 61-63.

Alouf, J.E. (1998). Streptococcal toxins (streptolysin 0. streptolysins, erythrogenic toxin), Pharmacology and Therapy, 11: 661-717.

Aniebo, A.O., Wekhe, S.N. and Okoli, I.C. (2011). Abattoir blood waste generation in River state and its environmental implications in the Niger Delta. Toxicology and Environmental Chemistry, 91: 619-625.

Balan, V., Jin, M., Culbertson, A. and Uppugundla, N. (2013). The saccharification step: Trichoderma reseei cellulase hyper producer strains. In Lignocellulose Conversion Enzymatic and Microbial Tools for Bio-ethanol Production (eds. Faraco, V.), Springer: Berlin/Heidelberg, Germany, 65–92.

Beem, J.E., Nesbitt, W.E. and Leung, K.P. (1998). Identification of hemolytic activity in Prevotella intermedia, Oral Microbiology and Immunology, 13: 97–105.

Bhakdi, S., Tranum-Jensen, J. and Sziegoleit, A. (1985). Mechanism of membrane damage by streptolysin-O, Infections and Immunity, 47: 52–60.

Bharali, P., Das, S., Konwar, B.K. and Thakur, A.J. (2011). Crude biosurfactant from thermophilic Alcaligenes faecalis: Feasibility in petro-spill bioremediation. International Biodeterioration and Biodegradation, 65: 682-690.

Chukwu, O., Mustapha, H. I., Abdul-Gafar, H. B. (2008). The effect of Minna waste on surface water quality. Environmental Research Journal, 2: 339-342.

Coker, A.O., Olugasa, B.O. and Adeyemi, A.O. (2001). Abattoir Effluent Quality in South Western Nigeria. Proceedings of the 27th WEDC Conference, Lusaka, Zambia, 20-21, August 2001, 1-4.

Das, R., Sinha, P. and Kar, D. (2016). Characterization of lasB and plcH gene from Pseudomonas aeruginosa isolates in Silchar, Clinical Microbiology, 5: 239-242.

Deshpande, R.G. and Khan, M.B. (1999). Purification and characterization of hemolysin from Porphyromonas gingivalis A7436. FEMS Microbiology Letters, 176: 387-394.

El Samak, M., Solyman, S.M. and Hanora, M. (2018). Antimicrobial activity of bacteria isolated from Red Sea marine invertebrates, Biotechnology Reports, 19: e00275: 1-7.

Fatokun, E.N., Nwodo, U.U. and Okoh, A.I. (2016). Classical Optimization of Cellulase and Xylanase Production by a Marine Streptomyces Species. Applied Science, 6: 1-14.

Funk, P.G., Staats, J.J., Howe, M., Nagaraja, T.G. and Chengappa, M.M. (1996). Identification and partial characterization of an Actinomyces pyogenes haemolysin. Veterinary Microbiology, 50: 129-142.

Gupta, U. and Kar, R. (2008). Optimization and scale up of cellulase free endo xylanase production by solid state fermentation on corn cob and by immobilized cells of a thermotolerant bacterial isolates. Jordan Journal of Biological Science, 1: 129–134.

Hang’ombe, B.M., Kohda, T., Mukamoto, M. and Kozaki, S. (2006). Purification and sensitivity of Clostridium chauvoei haemolysin to various erythrocytes, Comparative Immunology, Microbiology and Infectious Diseases, 29: 263–268.

Hussain, T., Edel-Hermann, V., Roohi, A., Munir, S., Ahmed, I., Khan, J., Yong Kim, K. and Anees, M. (2013). Biochemical characterization and identification of bacterial strains isolated from drinking water sources of Kohat, Pakistan, African Journal of Microbiology Research, 7: 1579-1590.

Jaapar, S.Z., Kalil, M.S., Ali, M. and Anuar, N. (2011). Effects of Age of Inoculum, Size of Inoculum and Headspace on Hydrogen Production using Rhodobacter sphaeroides. Bacteriology Journal, 1: 16-23.

Jacobs, A.A.C., Loefen, P.L.W., Van den Berg, A.J.G. and Storm, P.K. (1994). Identification, purification, and characterization of a thiol-activated hemolysin (suilysin) of Streptococcus suis. Infections and Immunity, 62: 1742-1748.

Jang, H.D. and Chang, K.S. (2005). Thermostable cellulases from Streptomyces sp.: Scale-up production in a 50-l fermenter. Biotechnology Letters, 27: 239–242.

Jiajun, X., Linjun, G., Shipeng, W. and Yitong, L. (2010). Comparative impact of cadmium on two phenanthrene-degrading bacteria isolated from cadmium and phenanthrene co-contaminated soil in China. Journal of Hazardous Materials, 174: 818–823.

Jones, C.M. and Niederweis, M. (2011). Mycobacterium tuberculosis can utilize heme as an iron source. Journal of Bacteriology, 193: 1767-1770.

Ju, S., Lin, J., Zheng, J., Wang, S., Zhou, H. and Sun, M. (2016). Alcaligenes faecalis ZD02, a novel nematicidal bacterium with an extracellular serine protease virulence factor, Applied Environmental Microbiology, 82: 2112–2120.

Juturu, V. and Wu, J.C. (2014). Microbial cellulases: Engineering, production and applications. Renewable and Sustainable Energy Reviews, 33: 188–203.

Kimizuka, R., Miura, T. and Okuda, K. (1996). Characterization of Actinobacillus actinomycetemcomitans hemolysin. Microbiology and Immunology, 40: 717–723.

Ktari, N., Trabelsi, I., Bardaac, S., Trikib, M., Bkhairiaa, I., Ben, R., Salema, S., Nasria, M. and Ben-Salahb, R. (2017). Antioxidant and hemolytic activities, and effects in rat cutaneous wound healing of a novel polysaccharide from fenugreek (Trigonellafoenum-graecum) seeds, International Journal of Biological Macromolecules, 95: 625–634.

Marchlewicz, B.A. and Duncan, J.L. (1982). Lysis of erythrocytes by a hemolysin produced by a Group B Streptococcus sp., Infections and Immunity, 34: 787-794.

Naeem, A.H., Mumtaz, S., Haleem, A., Qazi, M.A., Malik, Z.A., Dasti, J.I. and Ahmed, S. (2017). Isolation and Molecular Characterization of Biosurfactant-Producing Bacterial Diversity of Fimkassar Oil Field, Pakistan, Arabian Journal of Science and Engineering, 42: 2349-2355.

Nduka, O. (2011). Environmental Microbiology of Aquatic and Waste Systems, 1st ed.; Springer Dordrecht Heidelberg, London, New York, 1- 324.

Nwodo, U.U., Rumbold, K., Agunbiade, M.O., Green, E., Nwamadi, M. and Okoh, A.I. (2013). Characterization of an Exopolymeric Flocculant Produced by a Brachybacterium sp. Materials, 6:1237-1254.

Ogbonna, D.N. and Ideriah, T.J.K. (2014). Effect of Abattoir Waste Water on Physico-chemical Characteristics of Soil and Sediment in Southern Nigeria. Journal of Science Research Reports, 3: 1612-1632.

Olson, E.R. (1993). Influence of pH on bacterial gene expression. Molecular Microbiology, 8: 5-14.

Papagianni, M. and Moo-Young, M. (2002). Protease secretion in glucoamylase producer Aspergillus niger cultures: Fungal morphology and inoculum effects. Protease secretion in glucoamylase producer Aspergillus niger cultures: Fungal morphology and inoculum effects. Process Biochemistry, 37: 1271–1278.

Prigent, D. and Alouf, J.E. (1976). Interaction of streptolysin O with sterols. Biochimica et Biophysica Acta (BBA) – Biomembranes, 443: 288-300.

Public Health England, (2015). Health Protection Representative, 9: 1-1221.

Rahman, A., Srivastava, S.S., Sneh, A., Ahmed, N. and Krishnasastry, M.V. (2010). Molecular characterization of tlyA gene product, Rv1694 of Mycobacterium tuberculosis: a non-conventional hemolysin and a ribosomal RNA methyl transferase, BMC Biochemistry, 11: 35-43.

Rajesh, T., Jong-Min, J., Yong-Hyun, K., Hyun-Joong, K., Da Hye, Y., Sung-Hee, P., Kwon-Young, C., Yun-Gon, K., Jaebum, K., Seunho, J. Hyung-Yeon, P. and Yung-Hun, Y. (2013). Functional analysis of the gene SCO1782 encoding Streptomyces hemolysin (S-hemolysin) in Streptomyces coelicolor M145, Toxicology, 71: 159–165.

Rajeshkumar, K and Jayachandran, K. (2004). Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR7. Applied Biochemistry and Biotechnology, 118, 65-72.

Ray, A.K., Bairagi, A., Ghosh, K.S. and Sen, S.K. (2007). Optimization of fermentation conditions for cellulase production by Bacillus subtilis CY5 and Bacillus circulans TP3 isolated from fish gut. Acta Ichthyologica et Piscatoria, 3: 47–53.

Sato, T., Kamaguchi, A. and Nakazawa, F. (2012). Purification and characterization of haemolysin from Prevotella oris, Journal of Oral Bioscience, 54: 113–118.

Shu, C.H. and Lung, M.Y. (2004). Effect of pH on the production molecular and weight distribution of exopolysaccharide Antrodia by camphorate in batch cultures. Process Biochemistry, 39: 931–937.

Silva, T.A., Noronha, F.S.M., Farias, L.D. and Carvalho, M.A.R. (2004). In-vitro activation of the hemolysin in Prevotella nigrescens ATCC 33563 and Prevotella intermedia ATCC 25611, Research in Microbiology, 155: 31-38.

Singh, V., Chaudhary, D., Mani, I., Somvanshi, P., Rathore, G. and Sood, N. (2010). Molecular identification and codon optimization analysis of major virulence encoding genes of Aeromonas hydrophila. African Journal of Microbiology Research, 4: 952-957.

Stipcevic, T., Piljac, T. and Isseroff, R. (2005). Di-rhamnolipid from Pseudomonas aeruginosa displays differential effects on human keratinocyte and fibroblast cultures. Journal of Dermatological Science, 4: 141-143.

Takada, K., Fukatsu, A., Otake, S. and Hirasawa, M. (2008). Isolation and characterization of hemolysin activated by reductant from Prevotella intermedia. FEMS Immunology and Medical Microbiology, 35: 43–47

Tapi, A., Chollet-Imbert, M., Scherens, B. and Jacques, P. (2010). New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction, Applied Microbiology and Biotechnology, 85: 1521–1531.

Techapun, C., Charoenrat, T., Poosaran, N., Watanabe, M. and Sasaki, K. (2002). Thermostable and alkaline-tolerant cellulase-free xylanase produced by thermotolerant Streptomyces sp. Ab106. Journal of Bioscience and Bioengineering, 93: 431–433.

Wongwongsee, W., Chareanpat, P., Pinyakong, O. (2003). Marine Pollution Bulletin, 74: 95–104

Xia, X., Lin, S., Xia, X.X., Cong, F.S. and Zhong, J.J. (2014). Significance of agitation-induced shear stress on mycelium morphology and lavendamycin production by engineered Streptomyces flocculus. Applied Microbiology and Biotechnology, 98: 4399–4407.

Xia, Z. (2013). Effect of Tween 80 on the production of curdlan by Alcaligenes faecalis ATCC 31749. Carbohydrate Polymerase, 98: 178 –180.

Zhang, X. and Austin, B. (2005). Haemolysins in Vibrio species. Journal of Applied Microbiology, 98: 1011-1019.

Zhu, L., Olsen, R.J., Lee, J.D., Porter, A.R., DeLeo, F.R. and Musser, J.M. (2017). Contribution of Secreted NADase and Streptolysin O to the Pathogenesis of Epidemic Serotype M1 Streptococcus pyogenes Infections, The American Journal of Pathology, 187: 1-9.