Culture optimization for production and characterization of bioflocculant by Aspergillus flavus grown on chicken viscera hydrolysate

  • Jibrin Ndejiko Mohammed
  • Wan Rosmiza Zana Wan DagangEmail author
Original Paper


The economics of bioflocculant production is coupled with the use of a low-cost substrate at appropriate culture conditions. The use of a waste substrate for this purpose offers an additional treatment measure to mitigate environmental pollution. We investigated the growth of Aspergillus flavus and its bioflocculant yield using chicken viscera hydrolysate as the sole media. The effects of culture conditions including time, pH, shaker speed, temperature and inoculum size on bioflocculant production were all investigated and optimised through response surface method based on the central component design (CCD) package of Design Expert. Next, the purified bioflocculant was physically and chemically characterised. Under optimised culture conditions (incubation time 72 h, pH 7, shaker speed 150 rpm, temperature 35 °C and inoculum 4%), 6.75 g/L yield of crude bioflocculant was recorded. The bioflocculant activity was mostly distributed in the cell-free supernatant with optimum efficiency of 91.8% at a dose of 4 mL/100 mL Kaolin suspension. The purified bioflocculant was a glycoprotein consisting of 23.46% protein and 74.5% sugar, including 46% neutral sugar and 2.01% uronic acid. The X-ray photoelectron spectroscopy fundamental analysis of the purified bioflocculant indicated that the mass proportion of C, O and N, were 63.46%, 27.87% and 8.86%, respectively. The bioflocculant is mainly composed of carbonyl, amino, hydroxyl, and amide functional groups. This study for the first time indicates a high potential of bioflocculant yield from chicken viscera at the appropriate culture conditions.


Bioflocculant Bioflocculation Efficiency Chicken viscera 



The authors would like to thank Universiti Teknologi Malaysia, GUP Tier1 (Q.J130000.2545.13H22) and Demand-Driven Innovation Grant (R.J130000.7845.4L190) for their financial support.

Compliance with ethical standards

Conflict of interest

The authors declare no financial or commercial conflict of interest.


  1. Aljuboori AHR, Idris A, Abdullah N, Mohamad R (2013) Production and characterization of a bioflocculant produced by Aspergillus flavus. Bioresour Technol 127:489–493PubMedCrossRefGoogle Scholar
  2. Aljuboori AHR, Idris A, Al-joubory HHR, Uemura Y, Abubakar BI (2015) Flocculation behaviour and mechanism of bioflocculant produced by Aspergillus flavus. J Environ Manage 150:466–471PubMedCrossRefGoogle Scholar
  3. Ates O (2015) Systems biology of microbial exopolysaccharides production. Front Bioeng Biotechnol 3:200PubMedPubMedCentralCrossRefGoogle Scholar
  4. Bhattacharjee S (2016) DLS and zeta potential–what they are and what they are not? J Control Release 235:337–351PubMedCrossRefGoogle Scholar
  5. Bukhari NA, Loh SK, Nasrin AB, Jahim JM (2018) Enzymatic hydrolysate of palm oil mill effluent as potential substrate for bioflocculant BM-8 production. Waste Biomass Valoriz 256:25. CrossRefGoogle Scholar
  6. Chen H, Zhong C, Berkhouse H, Zhang Y, Lv Y, Lu W, Yang Y, Zhou J (2016) Removal of cadmium by bioflocculant produced by Stenotrophomonas maltophilia using phenol-containing wastewater. Chemosphere 155:163–169PubMedCrossRefGoogle Scholar
  7. Clogston JD, Patri AK (2011). Zeta potential measurement. In Characterization of nanoparticles intended for drug delivery (pp. 63–70): Springer, New YorkCrossRefGoogle Scholar
  8. Coates J (2000) Interpretation of infrared spectra, a practical approach. Encyclopedia of analytical chemistry, Wiley, HobokenGoogle Scholar
  9. Comte S, Guibaud G, Baudu M (2006) Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: part I Comparison of the efficiency of eight EPS extraction methods. Enzyme Microbial Technol 38(1–2):237–245CrossRefGoogle Scholar
  10. Dong S, Ren N, Wang A, Ma F, Zhou D (2008) Production of bioflocculant using the effluent from a hydrogen-producing bioreactor and its capacity of wastewater treatment. Paper presented at the 2nd international conference on bioinformatics and biomedical engineering, 2008. ICBBE 2008, pp 3018–3022Google Scholar
  11. Gomaa EZ (2012) Production and characteristics of a heavy metals removing bioflocculant produced by Pseudomonas aeruginosa. Pol J Microbiol 61(4):281–289Google Scholar
  12. Gong W-X, Wang S-G, Sun X-F, Liu X-W, Yue Q-Y, Gao B-Y (2008) Bioflocculant production by culture of Serratia ficaria and its application in wastewater treatment. Bioresour Technol 99(11):4668–4674PubMedCrossRefGoogle Scholar
  13. Gong Y, Lin L, Shi J, Liu S (2010) Oxidative decarboxylation of levulinic acid by cupric oxides. Molecules 15(11):7946–7960PubMedPubMedCentralCrossRefGoogle Scholar
  14. Guo X, Wang X, Liu J (2016) Composition analysis of fractions of extracellular polymeric substances from an activated sludge culture and identification of dominant forces affecting microbial aggregation. Sci Rep 6:28391PubMedPubMedCentralCrossRefGoogle Scholar
  15. He J, Zou J, Shao Z, Zhang J, Liu Z, Yu Z (2010) Characteristics and flocculating mechanism of a novel bioflocculant HBF-3 produced by deep-sea bacterium mutant Halomonas sp V3a’. World J Microbiol Biotechnol 26(6):1135–1141CrossRefGoogle Scholar
  16. Hefnawy MA, Gharieb MM, Shaaban MT, Soliman AM (2017) Optimization of culture condition for enhanced decolorization of direct blue dye by Aspergillus flavus and Penicillium canescens. J Appl Pharm Sci 7(02):083–092Google Scholar
  17. Jamdar S, Harikumar P (2008) A rapid autolytic method for the preparation of protein hydrolysate from poultry viscera. Bioresour Technol 99(15):6934–6940PubMedCrossRefGoogle Scholar
  18. Kaichev V, Miller A, Prosvirin I, Bukhtiyarov V (2012) In situ XPS and MS study of methanol decomposition and oxidation on Pd (111) under millibar pressure range. Surf Sci 606(3–4):420–425CrossRefGoogle Scholar
  19. Kamsani N (2012) Optimization of oil palm empty fruit bunch degradation using crude cellulase from Aspergillus Niger EFB1 in rotary drum bioreactor. Universiti Teknologi MalaysiaGoogle Scholar
  20. Khattak G, Mekki A, Gondal M (2013) XPS studies of pulsed laser induced surface modification of vanadium phosphate glass samples. J Phys Chem Solids 74(1):13–17CrossRefGoogle Scholar
  21. Kumar CG, Joo H-S, Kavali R, Choi J-W, Chang C-S (2004) Characterization of an extracellular biopolymer flocculant from a haloalkalophilic Bacillus isolate. World J Microbiol Biotechnol 20(8):837–843CrossRefGoogle Scholar
  22. Kurane R, Hatamochi K, Kakuno T, Kiyohara M, Hirano M, Taniguchi Y (1994) Production of a bioflocculant by Rhodococcus erythropolis S-1 grown on alcohols. Biosci Biotechnol Biochem 58:428–429CrossRefGoogle Scholar
  23. Lasekan A, Bakar FA, Hashim D (2013) Potential of chicken by-products as sources of useful biological resources. Waste Manage 33(3):552–565CrossRefGoogle Scholar
  24. Lei X, Chen Y, Shao Z, Chen Z, Li Y, Zhu H et al (2015) Effective harvesting of the microalgae Chlorella vulgaris via flocculation–flotation with bioflocculant. Bioresour Technol 198:922–925PubMedCrossRefGoogle Scholar
  25. Li Z, Chen R-W, Lei H-Y, Shan Z, Bai T, Yu Q et al (2009a) Characterization and flocculating properties of a novel bioflocculant produced by Bacillus circulans. World J Microbiol Biotechnol 25(5):745CrossRefGoogle Scholar
  26. Li Z, Zhong S, Lei H-Y, Chen R-W, Yu Q, Li H-L (2009b) Production of a novel bioflocculant by Bacilluslicheniformis X14 and its application to low temperature drinking water treatment. Bioresour Technol 100(14):3650–3656PubMedCrossRefGoogle Scholar
  27. Li L-X, Xing J, Ma F, Pan T (2015) Introduction of compound bioflocculant and its application in water treatment. Adv J Food Sci Technol 9(9):695–700CrossRefGoogle Scholar
  28. Li Y, Xu Y, Liu L, Li P, Yan Y, Chen T et al (2017) Flocculation mechanism of Aspergillus niger on harvesting of Chlorella vulgaris biomass. Algal Res 25:402–412CrossRefGoogle Scholar
  29. Liu C, Wang K, Jiang J-H, Liu W-J, Wang J-Y (2015) A novel bioflocculant produced by a salt-tolerant, alkaliphilic and biofilm-forming strain Bacillus agaradhaerens C9 and its application in harvesting Chlorella minutissima UTEX2341. Biochem Eng J 93:166–172CrossRefGoogle Scholar
  30. Liu G, Huang Y, Qu X, Xiao J, Yang X, Xu Z (2016a) Understanding the hydrophobic mechanism of 3-hexyl-4-amino-1, 2, 4-triazole-5-thione to malachite by ToF-SIMS, XPS, FTIR, contact angle, zeta potential and micro-flotation. Colloids Surf A 503:34–42CrossRefGoogle Scholar
  31. Liu W, Hao Y, Jiang J, Zhu A, Zhu J, Dong Z (2016b) Production of a bioflocculant from Pseudomonas veronii L918 using the hydrolyzate of peanut hull and its application in the treatment of ash-flushing wastewater generated from coal fired power plant. Bioresour Technol 218:318–325PubMedCrossRefGoogle Scholar
  32. Liu C, Hao Y, Jiang J, Liu W (2017) Valorization of untreated rice bran towards bioflocculant using a lignocellulose-degrading strain and its use in microalgal biomass harvest. Biotechnol Biofuels 10(1):90PubMedPubMedCentralCrossRefGoogle Scholar
  33. Luo Z, Chen L, Chen C, Zhang W, Liu M, Han Y et al (2014) Production and characteristics of a bioflocculant by Klebsiella pneumoniae YZ-6 isolated from human saliva. Appl Biochem Biot 172(3):1282–1292CrossRefGoogle Scholar
  34. Mahapatra S, Banerjee D (2013) Fungal exopolysaccharide: production, composition and applications. Microbiol Insights 6:1PubMedPubMedCentralCrossRefGoogle Scholar
  35. Makapela B, Okaiyeto K, Ntozonke N, Nwodo UU, Green E, Mabinya LV et al (2016) Assessment of Bacillus pumilus isolated from fresh water milieu for bioflocculant production. Appl Sci 6(8):211CrossRefGoogle Scholar
  36. Mohammed JN, Dagang WRZW (2019) Role of cationization in bioflocculant efficiency: a review. Environ Process. CrossRefGoogle Scholar
  37. More T, Yadav J, Yan S, Tyagi R, Surampalli R (2014) Extracellular polymeric substances of bacteria and their potential environmental applications. J Environ Manage 144:1–25PubMedCrossRefGoogle Scholar
  38. Nie M, Yin X, Jia J, Wang Y, Liu S, Shen Q et al (2011) Production of a novel bioflocculant MNXY1 by Klebsiella pneumoniae strain NY1 and application in precipitation of cyanobacteria and municipal wastewater treatment. J Appl Microb 111(3):547–558CrossRefGoogle Scholar
  39. Nouha K, Kumar RS, Tyagi RD (2016) Heavy metals removal from wastewater using extracellular polymeric substances produced by Cloacibacterium normanense in wastewater sludge supplemented with crude glycerol and study of extracellular polymeric substances extraction by different methods. Bioresour Technol 212:120–129PubMedCrossRefGoogle Scholar
  40. Nwodo U, Okoh A (2013) Characterization and flocculation properties of biopolymeric flocculant (Glycosaminoglycan) produced by Cellulomonas sp. Okoh. J Appl Microb 114(5):1325–1337CrossRefGoogle Scholar
  41. Oberoi HS, Sandhu SK, Vadlani PV (2012) Statistical optimization of hydrolysis process for banana peels using cellulolytic and pectinolytic enzymes. Food Bioprod Process 90(2):257–265CrossRefGoogle Scholar
  42. Okaiyeto K (2016) Evaluation of flocculating potentials and characterization of bioflocculants produced by three bacterial isolates from Algoa bay. University of Fort Hare, South AfricaGoogle Scholar
  43. Okaiyeto K, Nwodo U, Mabinya L, Okoh A (2014) Evaluation of the flocculation potential and characterization of bioflocculant produced by Micrococcus sp. Leo. Appl Biochem Microbiol 50(6):601CrossRefGoogle Scholar
  44. Okaiyeto K, Nwodo UU, Okoli AS, Mabinya LV, Okoh AI (2016a) Studies on bioflocculant production by Bacillus sp. AEMREG7. Pol J Environ Stud 25(1):241–250CrossRefGoogle Scholar
  45. Okaiyeto K, Nwodo UU, Okoli SA, Mabinya LV, Okoh AI (2016b) Implications for public health demands alternatives to inorganic and synthetic flocculants: bioflocculants as important candidates. MicrobiologyOpen 5(2):177–211PubMedPubMedCentralCrossRefGoogle Scholar
  46. Park C, Novak JT (2007) Characterization of activated sludge exocellular polymers using several cation-associated extraction methods. Water Res 41(8):1679–1688PubMedCrossRefGoogle Scholar
  47. Patil SV, Salunkhe RB, Patil CD, Patil DM, Salunke BK (2010) Bioflocculant exopolysaccharide production by Azotobacter indicus usingflower extract of Madhuca latifolia L. Appl Biochem Biotechnol 162(4):1095–1108PubMedCrossRefGoogle Scholar
  48. Peng L, Yang C, Zeng G, Wang L, Dai C, Long Z et al (2014) Characterization and application of bioflocculant prepared by Rhodococcus erythropolis using sludge and livestock wastewater as cheap culture media. Appl Microbiol Biot 98(15):6847–6858CrossRefGoogle Scholar
  49. Raza W, Yang W, Jun Y, Shakoor F, Huang Q, Shen Q (2012) Optimization and characterization of a polysaccharide produced by Pseudomonas fluorescens WR-1 and its antioxidant activity. Carbohydr polym 90(2):921–929PubMedCrossRefGoogle Scholar
  50. Ren D, Li H, Pu Y, Yi L (2013) Medium optimization to improve the flocculation rate of a novel compound bioflocculant, CBF-256, using response surface methodology and flocculation characters. Biosci Biot Biochem 77(11):2242–2247CrossRefGoogle Scholar
  51. Richmond A (2017) Cell response to environmental factors. In: Richmond A (ed) Handbook of microalgal mass culture (1986). CRC Press, Boca Raton, pp 69–100Google Scholar
  52. Ruperez P, Leal J (1981) Extracellular galactosaminogalactan from Aspergillus parasiticus. Trans Br Mycol Soc 77(3):621–625CrossRefGoogle Scholar
  53. Salehizadeh H, Shojaosadati S (2001) Extracellular biopolymeric flocculants: recent trends and biotechnological importance. Biotechnol Adv 19(5):371–385PubMedCrossRefGoogle Scholar
  54. Salehizadeh H, Vossoughi M, Alemzadeh I (2000) Some investigations on bioflocculant producing bacteria. Biochem Eng J 5(1):39–44CrossRefGoogle Scholar
  55. Sheng G-P, Yu H-Q, Li X-Y (2010) Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol Adv 28(6):882–894PubMedCrossRefGoogle Scholar
  56. Sun P-F, Lin H, Wang G, Lu L-L, Zhao Y-H (2015a) Preparation of a new-style composite containing a key bioflocculant produced by Pseudomonas aeruginosa ZJU1 and its flocculating effect on harmful algal blooms. J Hazard Mater 284:215–221PubMedCrossRefGoogle Scholar
  57. Sun P, Hui C, Bai N, Yang S, Wan L, Zhang Q et al (2015b) Revealing the characteristics of a novel bioflocculant and its flocculation performance in Microcystis aeruginosa removal. Sci Rep 5:17465PubMedPubMedCentralCrossRefGoogle Scholar
  58. Sun D, She J, Gower JL, Stokes CE, Windham GL, Baird RE et al (2016) Effects of growth parameters on the analysis of Aspergillus flavus volatile metabolites. Separations 3(2):13CrossRefGoogle Scholar
  59. Tan W-X, Lin Z-T, Bu H-T, Tian Y, Jiang G-B (2012) Nano-micelles based on a rosin derivative as potent sorbents and sinking agents with high absorption capabilities for the removal of metal ions. RSC Adv 2(18):7279–7289CrossRefGoogle Scholar
  60. Tang W, Song L, Li D, Qiao J, Zhao T, Zhao H (2014) Production, characterization, and flocculation mechanism of cation independent, pH tolerant, and thermally stable bioflocculant from Enterobacter sp. ETH-2. PLoS ONE 9(12):e114591PubMedPubMedCentralCrossRefGoogle Scholar
  61. Wang L, Ma F, Qu Y, Sun D, Li A, Guo J et al (2011) Characterization of a compound bioflocculant produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6. World J Microbiol Biotechnol 27(11):2559–2565CrossRefGoogle Scholar
  62. Wang Z, Shen L, Zhuang X, Shi J, Wang Y, He N et al (2015) Flocculation characterization of a bioflocculant from Bacillus licheniformis. Ind Eng Chem Res 54(11):2894–2901CrossRefGoogle Scholar
  63. Xiong Y, Wang Y, Yu Y, Li Q, Wang H, Chen R et al (2010) Production and characterization of a novel bioflocculant from Bacillus licheniformis. Appl Environ Microbiol 76(9):2778–2782PubMedPubMedCentralCrossRefGoogle Scholar
  64. Yue L, Ma C, Chi Z (2006) Bioflocculant produced by Klebsiella sp. MYC and its application in the treatment of oil-field produced water. JOUC 5(4):333Google Scholar
  65. Zhang Z-Q, Bo L, Si-qing X, Wang X-J, Yang A-M (2007) Production and application of a novel bioflocculant by multiple-microorganism consortia using brewery wastewater as carbon source. J Environ Sci 19(6):667–673CrossRefGoogle Scholar
  66. Zhao H, Zhong C, Chen H, Yao J, Tan L, Zhang Y et al (2016) Production of bioflocculants prepared from formaldehyde wastewater for the potential removal of arsenic. J Environ Manage 172:71–76PubMedCrossRefGoogle Scholar
  67. Zhong C, Cao G, Rong K, Xia Z, Peng T, Chen H et al (2018) Characterization of a microbial polysaccharide-based bioflocculant and its anti-inflammatory and pro-coagulant activity. Colloids Surf B 161:636–644CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Jibrin Ndejiko Mohammed
    • 1
    • 2
  • Wan Rosmiza Zana Wan Dagang
    • 2
    Email author
  1. 1.Department of MicrobiologyIbrahim Badamasi Babangida UniversityLapaiNigeria
  2. 2.Faculty of ScienceUniversiti Teknologi MalaysiaSkudaiMalaysia

Personalised recommendations