Potential of microbial consortium for biological treatment of the effluent from cassava flour production E. L. Vera Cruz, R. Albuquerque Lima, L. Asfora Sarubbo, G.M. Campos Takaki and A. Amorim Salgueiro 1Núcleo
de Pesquisas em Ciências Ambientais, Centro de Ciências e Tecnologia, Universidade Católica de Pernambuco, Rua do Príncipe, 526, Boa Vista, 50050-900, Recife, PE, Brasil. The aim of this work was to obtain a microbial consortium from the effluent of cassava flour production and to investigate its potential for biological treatment of this effluent. Experiments were carried out in a bioreactor with the effluent in the presence of ammonium sulfate at 28 – 30 oC, 1 vvm and 200 rpm. Three pulses of the effluent were weekly added to the bioreactor after seven days of cultivation. The nutrient concentrations of the effluent stimulated the growth of the autochthon microorganisms. The maximum growth of bacteria in the microbial consortium reached 108 CFU/mL at the stationary growth phase. Maximum activity of amylases, cellulases, lipases and proteases were detected after seven days of cultivation. The biodegradation of organic material by the microbial consortium was confirmed by the reduction of the chemical oxygen demand (80 %) and cyanide (28 %) during the biological treatment of the effluent. The microbial consortium obtained from the effluent from cassava flour production has potential on the biodegradation of the industrial effluent. Keywords microbial consortium; cassava flour production; biodegradation
1. Introduction The wastewaters from the production of cassava flour contain high organic material and hydrogen cyanide. These polullants need to be degraded before being launched in the water resources. These effluents are composed by “manipueira” which is the liquid extracted from the cassava during the manufacture process and from water of the washings of this root and the starch, a by product of this industry. The chemical composition of “manipueira” depends on the type of cassava and on the conditions of soil and climate. One tonne of the cassava root contains an average of 600 L of water (60 % moisture) as a constituent of cell juice. Twenty to thirty percent of this water is eliminated in the pressing operation. The “manipueira” contains starch - sedimented or in colloidal suspension, proteins, glucose, hydrogen cyanide and other nutrients [1]. The cassava may contain high concentrations of glycosides with the cyanide ion which releases the hydrogen cyanide by enzymatic hydrolysis process. The ion cyanide is potentially toxic for all the animals; it inhibits the electrons carrier chain. This ion causes the death of aerobic organisms by inhibition of the energy production, necessary for maintenance and cell growth. In the self-depuration of water, which is favored at temperatures higher than 35 °C, the cyanide ion is biodegraded by bacteria [2]. The aerobic biological treatments occur in the presence of oxygen by agitation or by increasing the surface of the liquid effluent to provide an extension area of direct contact with the air. Another possibility of aerobic processes is to introduce bubbles of air in the effluent. Experiments with “manipueira” under aerobic treatment favored reductions of biochemical oxygen demand (BOD) of 98 % and the concentration of cyanide ion by 90 %. The big drawback in the aerobic treatments is the energy cost to promote aeration in the material to be biodegraded [3]. An effluent from cassava flour production presented more than 9,500 mg O2/L of chemical oxygen demand (COD) and 4,000 mg O2/L of BOD. After a treatment in polishing ponds, the organic matter was reduced by more than 90 % for both COD as BOD while the phosphorus and solids did not vary significantly. In this treatment, the ammonia nitrogen, the concentration of chloride and the electrical conductivity were reduced [4]. The stabilization of the effluent from cassava flour production can be obtained by a process of oxidation. In this case, the microbial populations influenced the effectiveness of the treatment. The activated sludge system in the aerobic biological treatment of this wastewater was maintained by appropriate organic refills and by the addition of phosphorus. The efficiency of the system was verified by microscopic control and loss of BOD [5]. In biological processes, large volumes of wastewater are treated by microorganisms that biodegraded the pollutants with relatively low cost. The use of microbial consortia favors the reduction of the degradation time by the action of unidentified species that act in synergism [6]. Microbial populations physiologically adapted to industrial effluents in competition for nutrients through synergistic interactions have been used in the biodegradation of pollutants. A sample of oil was cultivated in a batch bioreactor aiming the biostimulation of indigenous microorganisms. The cells in synergy showed oxidases activities whose enzymes were able to biodegrade aromatic compounds present in petroleum [7]. Corresponding author: e-mail:
[email protected]. Phone: +55 81 21194015
The aim of this work was to obtain a microbial consortium from the effluent of cassava flour production and to investigate its potential for biological treatment of this effluent.
2. Materials and Methods 2.1. Sampling The effluent of the production of cassava flour was collected in an equalization tank of an industry, located in the city of Pombos, Northeast of Brazil. 2.2 Physical and chemical analysis The physico-chemical composition of the effluent was determined for proteins, lipids, carbohydrates and ash [8]. 2.3. Cultivation of the microbial consortium A bioreactor was inoculated with 1500 mL of the effluent from cassava flour production in the presence of ammonium sulfate 0.8 %, at 28 – 30 oC, 1 vvm and 200 rpm, during 28 days (Figure 1). Samples of the effluent supplemented with the nutrient (150 mL) were weekly added to the bioreactor after seven days of cultivation. Samples were taken out from the reactor to evaluate the growth and the biotechnology potential of the microbial consortium. Three independent experiments were carried out.
Fig. 1. Cultivation of the microbial consortium in a batch bioreactor
2.4. Evaluation of microbial growth The microbial growth was determined by aerobic plate count, according to the Standard Methods for the Examination of Water and Wastewater: (i) heterotrophic bacteria at 35 °C after 48 h, in the presence of the culture medium tryptone-glucose-yeast extract agar; (ii) filamentous fungi and yeast at 20 °C after 5 - 7 days, in the presence of malt agar medium and chloranphenicol [8]. 2.5. Detection of enzymatic activities The enzymatic activities of cellulases, amylases, lipases and proteases were investigated in the samples of the microbial consortia. The agar diffusion technique was used by inoculating 10 L of the sample in each specific culture medium, distributed in Petri dishes in the presence of the substrates: carboximethylcellulose for cellulases; starch for amylases; Tween-20 for lipases and gelatin for proteases. The enzymatic activities were revealed after activation at 45 °C during 30 min and the areas of the substrates degraded by each enzyme were measured in centimeters [9]. 2.6. Biological treatment of the effluent
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The effluent samples were treated at 28 °C during 48 h in 500 mL Erlermeyer flasks, containing a working volume of 200 mL (180 mL of the effluent and 20 mL of the microbial consortium) under orbital shaking at 150 rpm. 2.7. Analytical determinations The samples of the microbial consortia and the treated effluent were centrifuged and the supernatants were used to determine pH, COD and cyanide [8].
3. Results and discussions The treatment of wastewater from cassava flour production aims to reduce the organic matter and the toxicity of the effluent so as to achieve the quality standards established by official bodies that control the environmental pollution. The effluent used in this work contained the “manipueira” (liquid extracted from cassava), water from washing processes and sanitary sewage. The concentration of carbohydrates (2.6 %), proteins (1.8 %) and lipids (0.4 %) in the effluent stimulated the growth of the autochthon microorganisms, under experimental conditions. The effluent investigated had an acid pH (4.3) and presented a microbial population formed mainly by bacteria (102 CFU/mL), as well as yeasts (10 CFU/mL) and filamentous fungi (10 - 100 CFU/mL). Table 1 illustrates the results of the bacteria counts of the three consortia of microorganisms obtained from the effluent of the cassava flour production. In the first stage of the microbial consortium growth, until the seventh day, the number of the cells increased from 10 2 to 107 CFU/mL of bacteria. The maximum biomass reached 108 CFU/mL of bacteria at the end of the experiment after the three pulses of the effluent with ammonium sulfate. Table 1 Bacteria counts of the microbial consortia obtained from the effluent of cassava flour production Cultivation time (day)
Standard counting of bacteria (CFU/mL) Consortium A Consortium B Consortium C
0 1 2 3 6 7* 14* 21* 28
5 x 102 3 x 102 2 x 102 2 2 4 x 10 3 x 10 3 x 102 3 x 102 3 x 102 4 x 102 3 x 102 3 x 103 1 x 103 1 x 103 2 x 105 1 x 106 7 7 1 x 10 5 x 10 5 x 107 7 7 1 x 10 5 x 10 2 x 108 7 8 9 x 10 1 x 10 2 x 108 8 8 1 x 10 1 x 10 1 x 108 * Three pulses of the effluent were carried out after 7, 14 and 21 days of cultivation
Figure 2 illustrates the growth curve of microorganisms for the microbial consortium (Consortium C) by bacterial counts. There was a lag phase before the growth rate of the biomass reaches its maximum value. The duration of this lag was two days of cultivation. The absence of inoculum in the submerged cultures could be responsible for this initial lag phase. The specific growth rate was determined in the exponential growth phase, reaching 0.11 cells/h. The maximum stationary growth phase was achieved after the addition of the first pulse of the effluent. The microbial growth was limited (108 CFU/mL), although more than one pulse of the effluent was added. The low mass transfer in batch bioreactor in the presence of higher cell concentrations justified the limited growth. The increase of pH of the culture medium may have also limited the cell growth of the microbial consortia [10]. The variation of pH during the cultivation of the consortia studied is illustrated in figure 2. In the experiments, the initial pH was 4.3; it increased gradually and reached the value of 8.1 in the maximum stationary phase of cell growth. At the end of the experiment, the pH decreased reaching 7.2. This parameter directly influences the growth of microorganisms by changing the enzyme activities, justifying the behavior of microorganisms in the microbial consortia [11]. The nutritional and environmental conditions in the bioreactor did not favor the growth of yeasts and filamentous fungi in the cultivation of microbial consortia. In all the three consortia studied during the exponential growth phase, filamentous fungi were not detected while colonies of yeasts were observed, reaching values from 6 to 53 CFU/mL in the stationary growth phase.
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Fig. 2 Growth curve of bacteria and changes in pH in the cultivation of a microbial consortium obtained from the effluent of cassava flour production. The arrows indicate the addition of the pulses.
The four enzymes investigated (amylases, cellulases, proteases and lipases) showed activities in all the consortia of microorganisms (Table 2). The maximum values of the areas of the substrates degraded by the enzyme were 0.7 – 0.9 cm. It was observed an increase intensity of staining in the activities from the sixth day of cultivation of the three microbial consortia. The agar diffusion technique detects qualitatively the enzyme activities. The areas of the substrate degraded depend on the mass transfer in the solidified culture medium. The results depended on the components and the thickness of the culture medium in Petri dishes, as well as the enzyme migration in the medium. Table 2 Enzymatic activities of the microbial consortia
Cultivation time (days) 1 2 3 6 7 14 21 28
Amylases 0.6 ± 0.06 0.6 ± 0.06 0.7 ± 0.15 0.8 ± 0.10 0.8 ± 0.23 0.7 ± 0.06 0.8 ± 0.10 0.9 ± 0.12
Enzyme activities (cm) Cellulases Proteases 0.6 ± 0.06 0.6 ± 0.10 0.6 ± 0.12 0.7 ± 0.12 0.7 ± 0.06 0.8 ± 0.12 0.6 ± 0.10 0.8 ± 0.26 0.6 ± 0.06 0.9 ± 0.17 0.7 ± 0.12 0.9 ± 0.06 0.7 ± 0.12 0.9 ± 0.10 0.7 ± 0.15 0.9 ± 0.10
Lipases 0.6 ± 0.10 0.6 ± 0.12 0.7 ± 0.12 0.8 ± 0.26 0.9 ± 0.17 0.9 ± 0.06 0.9 ± 0.10 0.9 ± 0.10
The effluent showed a high content of organic matter, expressed as COD, 21,978 mg O2/L and the cyanide concentration of 0.75 mgCN-1/L. During the cultivation of the microbial consortia, a gradual decrease of the COD was observed although three pulses of the effluent were added (Table 3). After seven days of cultivation, the COD reduced 62 % and after 28 days, this reduction reached 90 %. Table 3 Results of COD of the samples of microbial consortia obtained from the effluent of cassava flour production.
Cultivation time (days) 0 7 14 21 28
Consortium A 26,214 7,954 3,059 1,836 1,224
COD (mg O2/L) Consortium B Consortium C 16,063 23,656 10,502 7,034 3,089 11,058 2,471 6,394 1,853 3,197
Media 21,978 8,497 5,735 3,567 2,091
The microbial consortia showed potential on biodegradation of the effluent from cassava flour production. In an aerobic biological treatment, high efficiencies (80 % COD reduction) were obtained when the autochthon microorganisms were inoculated in the effluent. In addition, reduction of cyanide ion reached 28 % under the same experimental conditions, after 48 h, at 30 oC, under orbital agitation. Promising results were also determined with microbial consortia obtained from an industrial textile effluent. Costa et al. [12] investigated the biological treatment of the effluent from a laundry and dyeing industries by a 4
microbial consortium. The indigenous microorganisms of the effluent were stimulated in the presence of glucose 4 g/L and ammonium sulfate 1 g/L under agitation, aeration and controlled temperature. In synergism, these microorganisms showed the ability to decolorize the effluent. A microbial consortium at 5 % v/v was inoculated in the effluent and the biological treatment was carried out at 100 rpm and 0.5 vvm, during 48 h in Erlenmeyer flasks. The maximum discoloration reached 85 % when compared to the raw effluent.
4. Conclusion The microbial consortium from the effluent of cassava flour production is effective in reducing the COD by biodegradation of organic matter by the action of the following enzymes: cellulases, amylases, lipases and proteases. This consortium has the potential to be applied in aerobic biological treatment of the industrial effluent. Acknowledgements The authors acknowledge the UNICAP and the Brazilian agencies: CNPq and FACEPE for the financial support to research and for the scholarships of the students.
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