African Journal of Biotechnology
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African Journal of Biotechnology Vol. 2 (11), pp. 438-443, November 2003 ISSN 1684-5315 © 2003 Academic Journals
Full Length Research Paper
Transformation kinetics of mixed polymeric substrates under transitory conditions by Aspergillus niger
Lacina Coulibaly 1*and Spiros N. Agathos2
1Laboratoire d’Environnement et de Biologie Aquatique, UFR-Sciences et Gestion de l’Environnement, Université d’Abobo-Adjamé 02 BP 801 Abidjan 02, Côte d’Ivoire. 2Unit of Bioengineering, Catholic University of Louvain Place Croix du Sud 2 Bte 19, 1348 Louvain-la-Neuve, Belgium.
*Corresponding author. E-mail: lacina91@hotmail.com. Tel: 07497153.
Accepted 18 October 2003
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A mixture of polymeric substrates (simulating a complex wastewater) was transformed under sewer conditions and aerobiosis by Aspergillus niger in a tanks-in-series reactor at a hydraulic retention time of 14 h. Starch was totally removed after 7 h of incubation. Removal of the protein portion with a molecular weight larger than 2 kDa followed the starch removal and the total proteins were the latest to be removed. Alkaline phosphatase, leucine aminopeptidase, valine aminopeptidase, a-bglucosidase and a-mannosidase were abundantly secreted in the growth medium. This research is the first report on mixed polymeric substrate biodegradation under sewer condition by A. niger, and could be considered as an open window on fungal biomass valorisation in wastewater treatment.
Keywords: Polysaccharide, wastewater, starch, bovine serum albumin, macromolecules, tank-in-series reactor, enzyme.
Abbreviations. HRT: Hydraulic retention time. BSA: Bovine serum albumin. TSA: Tryptic soy agar. Peptides (MW>2000): Peptide with molecular weight larger than 2 kDa.
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The organic composition of wastewater is typically 40-60% proteins, 25-50% carbohydrates and 10% lipids (Metcalf and Eddy, 1991). 50-60% of the dissolved organic carbon has a molecular weight greater than 1 kDa (Grady et al., 1984; Levine et al., 1985; Logan and Jiang, 1990). Dissolved proteinaceous material, defined as peptides larger than 2 kDa, constitutes more than 75% of the protein contents in wastewater (Confer and Logan, 1991). Glucids are abundant in wastewater and include simple sugar, dissolved macromolecules, particulate polysaccharides and largely insoluble and heterogeneous exopolymers produced by bacteria (Pavoni et al., 1972; Tago and Aida, 1977). Macromolecules have small diffusion coefficients that limit their movement to unattached cells and aggregate in suspended growth reactors and in biofilms (Logan et al., 1987ab). Also, bacteria are unable to directly assimilate those molecules unless a first hydrolysis step occurs (Eliosov and Argaman, 1995; Salyers et al., 1996).
Relative to these difficulties, biodegradation of wastewater macromolecular substrates has been studied in conventional treatment systems in order to understand the processes mechanisms and to optimise their biodegradation (Banerji et al., 1966, 1968; Maxham and Maier, 1978; McLoughlin and Crombie-Quilty, 1983; Haldane and Logan, 1994; Confer and Logan, 1997ab; Ubukata, 1997; Hvitved-Jacobsen et al., 1998). In sewers, organic pollutants are removed by physical, chemical and biological processes that take place naturally (Koch and Zandi, 1973; Green et al., 1985; Nielsen et al., 1992; Özer and Kasirga, 1995; Raunkjaer et al., 1995; Warith et al., 1998; Vollertsen and Hvitved-Jacobsen, 1998).
Bioaugmentation has been shown as a promising alternative to remove recalcitrant organic substances from wastewater and to enhance the processes rates (Van Limbergen et al., 1998; Ro et al., 1997). Bioaugmentation of sewer networks with well-selected microorganisms under transitory conditions could be useful in partially or totally releasing assimilated molecules back into solution, and increasing their transportation to the cells. The consequences of this fractionation of macromolecules will be of benefit for enhancing substrate removal in a sewer network by suspended bacteria, biofilm and bacteria adsorbed on sediment. There are potential investments and operating cost savings by reducing the size of treatment systems, especially in environments where the build-up of large wastewater treatment facilities is impossible. The residual biomasses of fungi used in industries to produce enzymes are stocked in landfill nowadays, but they could serve as inoculums for pretreatment.
The aims of this study were (i) to determine the kinetics of the pretreatment under transitory conditions of a synthetic wastewater containing a mixture of starch and Bovine Serum Albumin (BSA) as two model macromolecular substrates, and (ii) to verify the secretion of extracellular enzymes in the growth medium by Aspergillus niger.
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Microorganisms and culture conditions
A. niger MUCL 28817 was obtained from the fungal collection of the Catholic University of Louvain (MUCL). A. niger was cultivated on tryptic soy agar (TSA) from Difco laboratories (Detroit, Mich., USA) in a 260-ml flat bottle (Nunc, Roskilde, Denmark) at 28°C for 7 days. Prior to use in the reactor system, A. niger was precultured in the medium described by (Garcia et al., 1997). The preculture of spores and the recovery of the fungal biomass were proceeded as described previously (Coulibaly et al., 2002). An aliquot of 50 ml of fungal biomass suspended in 100 ml of sterile distilled water was filtered as above and the biomass was used to inoculate the reactor.
Reactor system
The sewer simulating system was the reactor system previously described (Coulibaly et al., 2002). Briefly it is composed by a set of five stirred tanks in series. The system included one membrane pump (Prominent, CfG, Heidelberg, Germany), which fed the first reactor, and four peristaltic pumps (Gilson, manupilus 2, Namur, Belgium) linking each reactor to its neighbouring unit. The reactors and the feeding reservoir were agitated with magnetic stirrers (Ika-Combimag RCO, Namur, Belgium). The reactor system was operated at an overall hydraulic residence time (HRT) of 14 h, which is encountered in long sewer lines (Özer and Kasirga, 1995).
Reactor inoculation and sampling
The reactor system was inoculated and sampled in the same way as described in our previous research (Coulibaly et al., 2002). Briefly, it was filled with 500 ml of synthetic wastewater. Then, the first reactor was inoculated with biomass prepared as indicated above.
Synthetic wastewater composition
The synthetic wastewater was composed of (in mg l-1) KH2PO4, 700; K2HPO4, 1400; (NH4)2SO4, 195; CaCl2, 50; MgSO4.7H2O, 12.5; MnSO4, 5; FeCl3, 5; ZnSO4.7H2O, 5; BSA, 250; and Starch, 250. The pH of the medium was 6.8 after autoclaving. The starch was autoclaved separately to avoid precipitation. BSA was added to the medium after dissolution in sterile water and filtration through a 0.2 µm filter.
Biomass (SS) determination
Fungal biomass in the reactors was determined by dry cell weight. Filtration of mixed liquor was done on Whatman N° 4 filter paper, followed by oven drying at 105°C.
Protein determination
Total protein was determined using the Bicinchoninic acid (BCA) method (Smith et al., 1985). While peptides with molecular weight larger than 2 kDa (MW>2000) were determined using the Coomassie Blue (CB) method (Confer and Logan, 1997a).
Starch Determination
The miniaturised starch iodine complex method of McCready et al. (1950) was used to monitor the starch concentration in the reactors.
Substrate removal calculation
The concentration of substrate removed (DS) in a time point was calculated by subtracting the substrate concentration from that of the feeding.
The initial specific
substrate uptake rate (
Enzyme profiles
Enzyme activities were determined with the API ZYM kit from biomerieux (Marcy-l’Etoile, France) and the manufacturer’s instructions were followed throughout. The API ZYM kit is a standardised semiquantitative micromethod able to detect 19 different types of enzymes. It has previously been used to screen enzymatic profiles in environmental research (McKellar, 1986; Boczar et al., 1992; Cicek et al., 1998; Morgan and Pickup, 1993). All reagents used were of analytical grade.
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Triplicate tests were performed for each assay. The biotransformation of a mixture of polymeric substrates made of starch and Bovine Serum Albumin (BSA) in an equal ratio was performed under transitory conditions. The HRT was maintained to 14 h, at pH 6.8 and the dissolved oxygen concentration was kept above 2 mg O2 l-1, with starch initial specific biodegradation and BSA of 0.03 g (g SS h)-1 and 0.01 g (g SS h)-1, respectively.. The biomass concentration in the reactor R1 at the beginning of the experiment was 1185 mg l-1. The initial substrates concentrations in the feeding were 250 mg l-1 for starch and 260 mg l-1 for BSA. Figure 1 shows the kinetics of polymeric substrates transformation in the reactors R1, R3 and R5. In R1 (Figure 2), one could observe the transitory behaviour of the biomass that was washed out. In this reactor, starch was first completely removed after 7 h of incubation. The starch degradation rate was maintained until 14 h, after what it decreased. The dwindling of the starch degradation rate observed above 14 h, could be explained by the combined effect of the enrichment of the reactor R1 in starch (by dilution) and the biomass washout. In R1, peptides (MW>2000) and total protein were fairly removed. Peptides (MW>2000) were about 20% removed while about 10% of the total proteins were removed. These substrates removal rate decreased rapidly due to the washout of the biomass and the enrichment of the reactor liquor by dilution with fresh medium.
Figure 1. Kinetics of starch and BSA removal by A. niger under transitory conditions in reactors R1, R3 and R5. Average initial synthetic wastewater composition: Starch, 250 mg l-1; BSA, 260 mg l-1. Initial biomass concentration in the reactor R1 was 1185 mg l-1.
Figure 2. Kinetics of A. niger biomass migration trough the reactors R1, R3 et R5 . Initial biomass concentration in the reactor R1 was 1185 mg l-1.
The biomass profile in R3 (Figure 2) could be divided into two steps. In the First instance, the biomass accumulates in the reactor, and thereafter is washed out. The biomass accumulation reached a maximum of 800 mg l-1 after 14 h before being washed out. As observed for R1 (Figure 1), starch was first removed. About 95% of the starch was removed after 7 h of incubation and this rate of degradation was maintained until the end of the experiment. The disappearance of total protein and peptides (MW>2000) increased with time. About 98.5% of the peptides (MW>2000) were removed after 24 h, whereas within the same period, about 48.5% of total protein portion were removed. The removal rate of the peptides (MW>2000) was superior at all time than the total protein.This difference could be explained by the conversion of before being washed out. The macromolecules degradation rate in R5 (Figure 1) was comparable to that of R3. In R5, about 90% of starch was degraded after 7 h of incubation in comparison with 95% in R3. Peptides (MW>2000) were totally removed after 24 h whereas about 38% of total protein was removed at the same period. The starch initial specific biodegradation (0.03 g (g SS h)-1) was three times higher than the BSA (0.01 g (g SS h)-1). The difference observed between the biodegradation rate of starch and protein has previously been observed in natural environment and in laboratory set-up (Confer and Logan, 1997ab; Raunkjaer et al., 1995); suggesting the difficulty to degrade protein in wastewater.
The enzymatic activities in the supernatant of R5 and on the biomass were checked in order to further understand the biotransformation of starch and BSA under transitory conditions by A. niger. Figures 3A and 3B show the enzymatic profiles in the supernatant and on the biomass. Fourteen of the nineteen tested enzymes were secreted in the growth medium by A. niger. For all of the enzymatic activities measured, that adsorbed on the fungal biomass are in general greater than the activities in the growth medium. These enzymes could be grouped into phosphatases (alkaline phosphatase, acid phosphatase, phosphohydrolase), lipases (esterase, esterase-lipase, lipase), proteases (leucine, valine and cystine aminopeptidases, trypsin) and polysaccharide hydrolases (b-galactosidase, a-glucosidase, a-mannosidase, b-glucosaminidase). The most abundantly produced enzymes were alkaline phosphatase, acid phosphatase, phosphohydrolase, esterase-lipase, leucine and valine aminopeptidases, a-glucosidase and b-glucosaminidase. Leucine and valine aminopeptidases were abundantly secreted in the medium and their highest activity were 131.6 nmole (ml h)-1 and 52.6 nmole (ml h)-1 after 24 h. The period (24 h) required to reach the maximum activity of these enzymes was greater than the HRT (14 h) of the reactor system, indicating that they were secreted within a 10 h lag period. This lag period required for A. niger to secrete proteases could be an explanation of the shift in the BSA removal by the fungi. As shown in figure 3, secretion of a-glucosidase was rapid. a-glucosidase had a maximum activity of 43.8 nmole (ml h)-1 at 14 h, which coincides with the HRT of the reactor system (14 h). The coincidence between the HRT of the reactor and the period required to get the maximum activity of a-glucosidase could explain the rapid removal of starch in the reactor system. Henze and Mladenovski (1991) and Raunkjaer et al. (1995) had previously observed some differences between starch removal and protein degradation in a real sewage. But these authors did not explain the differences observed by enzymatic activities. The decrease in protease and amylase activities was concomitant with the biomass washout of the reactor system. The enzymatic profiles observed in this research were similar to those found in wastewater and activated sludge matrices (Morgan and Pickup, 1993; Frolund et al., 1995).
Figure 3. Enzymatics activities accumulated in reactor R5 by A. niger under transitory conditions, when degrading a synthetic wastewater containing starch (250 mg l-1) and BSA (260 mg l-1). The initial biomass concentration in the reactor R1 was 1185 mg l-1.
The mixed polymeric substrates degradation mechanism could implicate both the biomass and the extracellular enzymes. The capability of A. niger to degrade in a realistic period (14 h) and condition (biomass, 1.2 g l-1; HRT, 14 h) a mixture of polymeric substrates under transitory conditions is an interesting finding highlighted in this study. Bioaugmentation with A. niger under transitory condition could be considered for pretreatment wastewater containing polymeric substrates without any enzymes addition. This could result in a cost savings in wastewater treatment and the management of A. niger waste biomass issuing from fermentation industries.
ACKNOWLEDGEMENTS
This research was supported by a fellowship of The Ministry of Higher Education, Scientific Research and Technological innovation of Côte d’Ivoire. We thank Professor A. M. Corbisier (MUCL) for providing the stock culture of A. niger, Dr Seydou TIHO, Dr Pierre Wattiau and Dr Patrick Gerin for their useful discussions.
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Banerji KS, Ewing BB, Engelbrecht RS, Speece ER (1968). Kinetics of starch removal in activated sludge systems. J. Water Pollut. Control Fed. 40: 161-173. [Pubmed]
Banerji KS, Ewing BB, Engelbrecht RS, Speece ER (1966). Mechanism of starch removal in activated sludge process. Proc. 21st Ind. Waste Conf., Purdue Univ., Ext. Sed. 121: 84-102.
Boczar BA, Begley WM, Larson RJ (1992). Characterization of enzyme activity in activated sludge using rapid analyses for specific hydrolases. Water Environ. Res. 64: 792-796.
Cicek N, Franco JP, Suidan MT, Urbain V, Manem J (1998). Characterization and comparison of a membrane bioreactor and a conventional activated sludge system in the treatment of wastewater containing high molecular weight compounds. Water Environ. Res. 71: 64-70.
Confer RD, Logan EB (1997a). Molecular weight distribution of hydrolysis products during the biodegradation of model macromolecules in suspended and biofilm cultures. I. Bovine serum albumin. Water Res. 31: 2127-2136.
Confer RD, Logan EB (1997b). Molecular weight distribution of hydrolysis products during the biodegradation of model macromolecules in suspended and biofilm cultures. II. Dextran and dextrin. Water Res. 31: 2137-2145.
Confer RD, Logan EB (1991). Increased bacterial uptake of macromolecular substrates with fluid shear. Appl. Environ. Microbiol. 57: 3093-3100.
Coulibaly L, Naveau H, Agathos SN (2002). A tanks-in-series bioreactor to simulate macromolecule-laden wastewater pretreatment under sewer conditions by Aspergillus niger. Water Res. 36: 3941-3948. [Pubmed]
Eliosov B, Argaman Y (1995). Hydrolysis of particulate organics in activated sludge systems. Water Res. 29: 155-163.
Frolund B, Griebe T, Nielsen PH (1995). Enzymatic activity in the activated sludge floc matrix. Appl. Microbiol. Biotechnol. 43: 755-761. [Pubmed]
Garcia G, Bonilla VJL, Jiminez PPR, Kirchman L (1997). Biodegradation of phenol compounds in vinasse using Aspergillus terreus and Geotrichum candidum. Water Res. 31: 2005-2011.
Grady CPLJr, Kirsh EJ, Koczwara MK, Trogovcich B, Watts RD (1984). Molecular weight distributions in activated sludge effluents. Water Res. 18: 239-246.
Green M, Shelef G, Messing A (1985). Using the sewerage system main conduits for biological treatment. Greater Tel-Aviv as a conceptual model. Water Res. 19: 1023-1028.
Haldane GM, Logan BE (1994). Molecular size distributions of a macromolecular polysaccharide (dextran) during its biodegradation in batch and continuous cultures. Water Res. 28: 1873-1878.
Henze M, Mladenovski C (1991). Hydrolysis of particulate substrate by activated sludge under aerobic, anoxic and anaerobic conditions. Water Res. 25: 61-64.
Hvitved-Jacobsen T, Vollertsen J, Nielsen PH (1998). A process and model concept for microbial transformation in gravity sewers. Water Sci. Technol. 37: 233-241.
Koch CM, Zandi I (1973). Use of pipelines as aerobic biological reactors. J. Water Poll. Control Fed. 45: 2537-2548. [Pubmed]
Levine DA, Tchobanoglous G, Asano T (1985). Characterization of the size distribution of contaminants in wastewater: Treatment and reuse implication. J. Water Pollut. Control Fed. 57: 805-816.
Logan BE, Jiang Q (1990). A model for determining molecular sizes distributions of DOM. J. Environ. Eng. 116: 1046-1062.
Logan BE, Hermanowicz SW, Parker DS (1987a). Engineering implications of a new trickling filter model. J. Water Pollut. Control Fed. 59: 1017-1028.
Logan BE, Hermanowicz SW, Parker DS (1987b). A fundamental model for trickling filter process design. J. Water Pollut. Control Fed. 59: 1029-1042.
Maxham VJ, Maier JW (1978). Bacterial growth on organic polymers. Biotechnol. Bioeng. 20: 865-898.
McCready RM, Guggolz J, Silviera V, Owens SH (1950). Determination of starch and amylose in vegetables. Application to peas. Anal. Chem. 22: 1156-1158.
McKellar RC (1986). Determination of the extracellular and cell-associated hydrolase profiles of Pseudomonas fluorescens Sp. Using the analytab API ZYM system. J. Dairy Sci. 69: 658-664. [Pubmed]
McLoughlin AJ, Crombie-Quilty MB (1983). The kinetics of protein removal by activated sludge. Water Res. 17: 161-166.
Metcalf and Eddy Inc. (1991). Wastewater engineering : Treatment, Disposal, reuse. Mcgraw-Hill, New York.
Morgan JAW, Pickup RW (1993). Activity of microbial peptidases, oxidases and esterases in lake waters of varying trophic status. Can. J. Microbiol. 39: 795-803.
Nielsen PH, Raunkjaer K, Norsker NH, Jensen NA, Hvitved-Jacobsen T (1992). Transformation of wastewater in sewer systems. A review. Water Sci. Technol. 27: 17-31.
Özer A, Kasirga E (1995). Substrate removal in longer sewer lines. Water Sci. Technol. 31: 213-218.
Pavoni JL, Tenney MW, Echelberger WFJr (1972). Bacterial exocellular polymers and biological flocculation. J. Water Pollut. Control Fed. 44: 414-431. [Pubmed]
Raunkjaer K, Hvitved-Jacobsen T, Nielsen PH (1995). Transformation of organic matter in a gravity sewer. Water Environ. Res. 67: 181-188.
Ro KS, Babcock RW, Stenstrom MK (1997). Demonstration of bioaugmentation in a fluidized-bed process treating 1-naphthylamine. Water Res. 31: 1687-1693.
Salyers AA, Reeves A, Delia J (1996). Solving the problem of how to eat something as big as yourself: Diverse bacterial strategies for degrading polysaccharides. J. Ind. Microbiol. Biotechnol. 17: 470-476.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem., 150: 76-85. [Pubmed]
Tago Y, Aida K (1977). Exocellular mucopolysaccharide closely related to bacterial floc formation. Appl. Environ. Microbiol. 34: 308-314. [Pubmed]
Ubukata Y (1997). Kinetics of polymeric substrate (dextrin or peptone) removal by activated sludge: hydrolysis of polymers to monomers is the rate-determining step. Water Sci. Technol. 36: 159-167.
Van Limbergen H, Top EM, Verstraete W (1998). Bioaugmentation in activated sludge: current features and future perspectives. Appl. Microbiol. Biotechnol. 50: 16-23.
Vollertsen J, Hvitved-Jacobsen T (1998). Aerobic microbial transformations of suspended sediments in combined sewers. A conceptual model. Water Sci. Technol. 37: 69-76.
Warith MA, Kennedy K, Reitsma R (1998). Use of sanitary sewers as wastewater pre-treatment systems. Waste Manag. 18: 235-247.
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) in the reactor R5 was calculated by
dividing the concentration of substrate removed (
