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The biological purification of waste products from olive oil extraction
Bioresource Technology 43 (1993) 215-219
THE BIOLOGICAL PURIFICATION OF WASTE PRODUCTS FROM OLIVE OIL EXTRACTION L. Martinez Nieto," S. E. Garrido Hoyos, a F. C a m a c h o Rubio, a M. P. Garcia Pareja b & A. R a m o s C o r m e n z a n a b "Department of Chemical Engineering, Faculty of Science, bDepartment of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain (Received 4 June 1991; revised version received 15 May 1992; accepted 5 July 1992)
Abstract Aspergillus terreus gave the overall best results in alpechin at approximately 80% concentration, degrading organic material by 53%, expressed as COD, and 67%, expressed as BOD. Degradation of the total phenol content, which included the great majority of phenolic compounds, reached 69%.
osmosis and ultrafiltration (Reimers Sufirez, 1983) and anaerobic microbiological processes (Fiestas Ros de Ursinos et al., 1990). Each solution requires costly investment and maintenance, however, which can be unprofitable for the seasonal oil-extraction industry. Anaerobic purification is the only process which partially repays its own expenses via the production of methane. Researchers seeking alternatives (Garcia Pareja et al., 1987; P~rez Torres, 1988) have isolated lignolitic bacteria and fungi that act upon the general organic content of alpechin, including the antimicrobial phenolic compounds. We have examined the biodegradation of the organic content of alpechin, comparing the results of Bacillus pumilus with those of Aspergillus terreus in an initial 20% alpechin solution under aerobic conditions. We found Aspergillus terreus to be more effective at degrading this waste and so made further studies into the optimum conditions for its activity.
INTRODUCTION "Alpechin' is the Spanish term for the waste produced by the olive-oil extraction industry. Composed of vegetable liquids from the olives and the water used in the extraction process, this slurry results both from the traditional method of pressing and from the newer method of continuous centrifugation. Due to differences between the two methods, and depending upon olive variety and climatic factors, the quantity of alpechin produced per tonne of olives may vary from 0"5 m 3 to more than 1 m 3. This waste is a harmful pollutant which does not readily decompose. Its COD is around 230 000 p.p.m, and its BOD around 150 000 p.p.m. In the Guadalquivir basin alone average production is estimated to be about 1800000 m 3 per year, a volume of pollution comparable to that of a city of 3 500 000 inhabitants. Some 25% of this waste is indiscriminately dumped (Rodrigo Romfin, 1990), while the remaining 75% is left in evaporation pits, which only partially reduce contamination. Consequently, because of its massive scale, and low decomposition rate due to the strong antimicrobial properties of its phenolic compounds (Moreno et al., 1987), alpechin is considered to be one of the major water pollution problems in the Mediterranean basin. To eliminate this pollution various methods have been employed: simple evaporation (Storm, 1989), physical and chemical processes such as inverse
METHODS Alpechin Table 1 shows the principal characteristics of the alpechin used in the experiments. The original material, from Jimena Osuna S.A., had passed through a continuous 'Pieralisi' process, and samples were drawn from the final centrifugation step into sterile flasks and taken to the laboratory. The samples were diluted to concentrations of between 20% and 80%, using Minimum Maintenance Medium (MMM) (Janshekar et al., 1981), the composition of which is described in Table 2. In addition, undiluted samples of alpechin were used. Microorganisms B. pumilus and A. terreus were obtained from the Microbiology Department of the Faculty of Pharmacy of the University of Granada. For test solutions, a culture of B. pumilus was grown on a TSA (tripticasein-soy-agar) slope. For each alpechin concentration, two test tubes (15 ml) were inocu-
Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain 215
216
L. Martinez Nieto, S. E. Garrido Hoyos, F. Camacho Rubio, M. P. Garcia Pareja, A. Ramos Cormenzana
Table 1. Physical and chemical characteristics of the alpechin used
pH COD (g per litre) BOD (g per litre) Total solids (g per litre) Volatile solids (g per litre) Non-volatile solids (g per litre) Settled solids (g per litre) Suspended solids (g per litre) Dissolved solids (g per litre) Total phenolic compounds
5"26 212"25 179"45 55"59 44"46 11"13 7"75 2'44 45"40 O'7%
_
v a c u u m
®
Table 2. Composition of minimal maintenance medium
Bipotassium phosphate Monopotassium phosphate Ammonium sulphate Ammonium nitrate Magnesium sulphate pentahydrate Sodium chloride Ferric chloride hexahydrate Calcium chloride Distilled water
1"6 g 0"50 g 1"25 g 1"0 g 0"50 g 0'25 g 0"025 g 0'010g 1 000 ml
lated using a loop (1-2-3-0× 10 ~ cfu per ml) and incubated at 28°C for 18-24 h under constant shaking. The content of each tube was diluted with MMM to a volume of 150 ml and incubated in two 500-ml flasks in the same manner as above. These two 150-ml samples were combined and added to alpechin to a volume of 3000 ml in the 5-1itre batch reactor and incubated at 28°C for 48 h with constant stirring. A culture of A. terreus, grown on a malt-extract-agar slope, produced spores which were then suspended in a saline solution (1-9 × 10 s viable spores per ml). For each alpechin concentration, two test tubes containing 15 ml were inoculated with 1"5 ml of spore suspension and incubated at 28°C for 72 h with constant shaking. Each tube was diluted with MMM to a volume of 150 ml and incubated in two 500-ml flasks in the same manner as above. These two 150-ml samples were combined and added to alpechin to a volume of 3000 ml in the 5-1itre batch reactor and incubated at 28°C for 6 days. Reactor
The 5-1itre batch reactor (Fig. 1 ) and all its components were sterilised prior to the experiments. A steady flow of air, sterilised by filtration, was maintained at a rate of 720 ml per min and the stirring rate was 200 rpm. Samples were taken at regular intervals to determine the COD, BOD and phenol content. Analytical methods
The chemical oxygen demand (COD) was measured by the methods of Rodier (1985) and Capitfin Vallvey et a/.(1987). The biochemical oxygen demand (BOD) was measured by the methods of Sauter & Stoub (1990).
Fig. 1. Schematic drawing of the batch reactor. 1, Reactor; 2, bath at 28°C; 3, temperature control; 4, filter; 5, stirrer; 6, thermometer; 7, sample port; 8, compressed air; 9, reduction-valve control; 10, flow meter.
The phenol content was measured in a 720 Waters chromatograph, connected to a C1~ column, 30 cm long, measuring two wavelengths (280 and 340 nm) simultaneously for 75 rain (following the technique described by Olano & Hernfindez, 1985). The concentrations of phenolic compounds were calculated using Wavescan EG and PE Nelson software programmes and identified by their retention time and by the ratio between response factors at the two wavelengths. The results were confirmed by TLC and by comparison with commercial phenolic compounds. RESULTS Table 3 shows the results obtained for B. pumilus and A. terreus, in a 20% alpechin solution, the percentage of degradation being calculated on COD and BOD. The time for maximum degradation for A. terreus was 4 days, while for B. pumilus it was 2 days (Table 3). The decomposition of phenolic compounds by A. terreus exceeded that of B. pumilus, because after a period of 48 h the bacteria began metabolically to produce new phenolic compounds. A. terreus, however, produced a negligible quantity of phenolic compounds, and less than the bacteria even after the optimum period of 4 days. B. pumilus degraded 62"1% of the total phenol content over 2 days and A. terreus 68"9% over 4 days. Table 4 summarises the phenolic compounds identified by chromatography in relation to the microorganisms and to the alpechin. Eleven phenol compounds (91% of the total phenolic content of the alpechin) were identified by HPLC, of which seven showed significant effects after fungal treatment (protocatechuic acid + hydroxytirosol, p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, vanillyl alcohol and syringaldehyde). B. pumilus affected only six of these compounds and caused a rise in vanillic acid by its metabolic activity.
217
Purification o f olive oil waste
Table 3. Degradation of alpechin (20% dilution) by Bacillus pumilus and Aspergillus terreus in batch reactors Microorganisms
t
pH~
2 4
Bacillus pumih4s Aspergillus terreus
7.00 5-26
pHt
CODf (mg per litre)
6"94 6'04
BODf (mg per litre)
20 241 15 899
19 870 5 225
% Degradation COD
BOD
52.32 62"55
44.64 85'44
i, initial; f, final; t, days of fermentation. COD~, 42 450 mg per litre; BOD~, 35 890 mg per iitre. For fermentation conditions see Methods.
Table 4. Degradation of phenolic compounds by Bacillus pumilus and Aspergillus terreus Compounds
TP"
Protocatechuic acid + hydroxytirosol Vanillyl alcohol p-hydroxybenzoic acid Vanillic acid Caffeic acid p-Vaniilin Syringaldehyde p-Coumaric acid
% Degradation B. pumilus
A. terreus
64-45 -21'57 -94.70 -63.72 93"01
98.68 27"27 78"89 53.40 93"68 62.52 20.72 94.10
36"06 0.32 14.57 4.11 8-45 0.52 1-28 25-62
"% of total phenol compounds in undegraded alpechin.
Table 5. Degradation of alpechin by Aspergillus terreus after 6 days Concentration of alpechin in the reactor 200/0 40% 60% 80% 100%
t (day) 0 6 0 6 0 6 0 6 0 6
COD (mg per litre)
BOD (mg per litre)
42450 19791 84900 36353 127350 58 905 169 800 80052 212250 129710
35 890 4303 71 780 14850 107670 28 740 143 560 47520 179450 78 100
A COD (rag per litre)
A BOD (rag per litrc)
% Degradation COD
BOD
22659
31 587
53"38
88"01
48547
56930
57.18
79.31
68445
78930
53.75
73.31
89 748
96 040
52.86
66"90
82540
101 350
38"89
56-48
COD~, 212 250 mg per litre; BOD~, 179 450 mg per iitre. For fermentation conditions see Methods.
Table 5 records the results for A. terreus kept in the different alpechin solutions in the reactor for 6 days and lists the percentages of degradation for C O D and BOD. Table 6 shows the elimination rates of organic material per day, of C O D and B O D per unit of volume. T h e tables indicate that, as the alpechin concentrations increase, the percentage degradations of C O D and B O D decrease. Nevertheless, the results expressed by time unit and volume show that increasing the concentration of alpechin increases the decomposition until a maximum of about 80% alpechin is reached.
Figure 2 plots C O D and B O D degradations (calculated from Table 5) in relation to the alpechin concentration; the values fit the following equations formulated by polynomial regression: COD: Y = O ' 4 4 7 + O ' 5 4 1 X - O ' 5 8 9 X
2
(1)
BOD: Y = O . 9 3 5 - O . 2 9 5 X - O . O 6 9 X 2
(2)
X = percentage alpechin concentration; Y-- percentage C O D and B O D degradation. Both equations reproduce the experimental values with a deviation of less than 5%.
218
L. Martinez Nieto, S. E. Garrido Hoyos, F. Camacho Rubio, M. P. Garcia Pareja, A. Ramos Cormenzana Table 6. The elimination of organic material with time by Aspergillus terreus
Decrease in organic material
Concentration of alpechin in the reactor ACOD/At (rag per litre per day)
ABOD/At (mg per litre per day)
3777 8091 11408 14958 13757
5265 9488 13155 16007 16892
2O% 40% 60% 80% 100%
% Degradation 100
80
60
40
20
i
0 0
20
I
I
~-
Fig. 2.
I
40 60 80 % Alpechin c o n c e n t r a t i o n COD
-4--
I
1 O0
120
BOD
The percentage degradation compared to the initial concentration of aipechin.
DISCUSSION This study analyses the biodegradation of alpechin by comparing the lignolitic bacterium B. pumilus and the fungus A. terreus in 20% alpechin solutions where constant conditions were maintained. The results indicated that greater efficiency of A. terreus in degrading the alpechin. Results with A. terreus at alpechin concentrations of 20%, 40%, 60%, 80% and 100% (pure alpechin) indicated that the fungus performed most effectively at about 80% (CODi = 212 250 mg per litre and BODi = 179 450 mg per litre). It is important to note that, after 48 h, B. pumilus began to produce new phenolic compounds and thereby raised the COD. With A. terreus this phenomenon occurred only after 6 days, and thus 4 days was taken to be the maximum fermentation period to give optimum degradation. This degradation process might have an industrial application, since, with the reduction in concentration of a large number of phenolic compounds, the remaining effluent would have less antimicrobial capacity. Thus the time needed in anaerobic digesters for the production of methane would be reduced. The biDmass obtained, rich in protein, could be used as livestock feed, and the effluent could be used either as a fertilizer or, in concentrated form, as a supplementary feed additive.
REFERENCES Capitzin Vallvey, L. F., Alonso Hernfindez, E. J. (1987). T6cnicas en anfilisis de aguas. Fondo Social Europeo, Universidad de Granada, pp. 33-5. Fiestas Ros de Ursinos, J. A., Martin Martin, A. & Borja Padilla, R. (1990). Influence of immobilization supports on the kinetic constants of anaerobic purification of olive mill waste water. Biological Wastes, 33, 131-42. Garcia Pareja, Ma P., Monteoliva Sanchez, M., Garc/a de la Paz, A. M., Corominas, E., P6rez, M. L. & Ramos Cormenzana, A. (1987). Characterization of bacteria with phenol-oxidase activity from soil in the La Laguna area (Spain). Chemosphere, 16, 2627-30. Janshekar, H., Brown, C. & Fietcher, A. (1981). Determination of biodegraded lignin by ultraviolet spectrophotometry. Analytica Chimica Acta, 130, 80-91. Moreno, E., P6rez, J., Ramos Cormenzana, A. & Mart/nez, J. (1987). Antimicrobial effect of waste water from olive oil extraction plants selecting soil bacteria after incubation with diluted waste. Microbios, 51,169-74. Diana, A. & Hernfindez, T. (1985). Study of the separation of low molecular weight phenolic compounds by HPLC and GLC. Bull. Liaison Groupe Polyphenols, 12, 591-3. P6rez Tortes, J. (1988). Transformaci6n microbiana de componentes aromfiticos del alpechin. Tesis Doctoral. Departamento de Microbiologia, Facultad de Farmacia, Universidad de Granada, Spain. Reimers Sufirez, G., (1983). Posibilidades de tratamiento del alpechin par ultrafiltraci6n y 6smosis inversa. Instituto de la Grasa y sus Derivados, Sevilla, Spain.
Purification of olive oil waste Rodier, J. (1985). An61isis de las aguas, ed. Omega S. A., Barcelona, Spain, pp. 528-30. Rodrigo Romfin, J. (1990). Situaci6n en Espafia de los alpechines. Jornadas T6cnicas sobre tratamiento de los alpechines. 31 Mayo-1 Junio. C6rdoba, Spain.
219
Sauter, A. & Stoub, K. (1990). AOAC. Official Methods of Analysis, 11,314-16. Storm, J. (1989). Evaporaci6n del alpechin. Jornadas sobre innovaci6n tecnol6gica, medio ambiente y desarrollo. lnstituto de la Grasa y sus Derivados, Sevilla, Spain.
THE BIOLOGICAL PURIFICATION OF WASTE PRODUCTS FROM OLIVE OIL EXTRACTION L. Martinez Nieto," S. E. Garrido Hoyos, a F. C a m a c h o Rubio, a M. P. Garcia Pareja b & A. R a m o s C o r m e n z a n a b "Department of Chemical Engineering, Faculty of Science, bDepartment of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain (Received 4 June 1991; revised version received 15 May 1992; accepted 5 July 1992)
Abstract Aspergillus terreus gave the overall best results in alpechin at approximately 80% concentration, degrading organic material by 53%, expressed as COD, and 67%, expressed as BOD. Degradation of the total phenol content, which included the great majority of phenolic compounds, reached 69%.
osmosis and ultrafiltration (Reimers Sufirez, 1983) and anaerobic microbiological processes (Fiestas Ros de Ursinos et al., 1990). Each solution requires costly investment and maintenance, however, which can be unprofitable for the seasonal oil-extraction industry. Anaerobic purification is the only process which partially repays its own expenses via the production of methane. Researchers seeking alternatives (Garcia Pareja et al., 1987; P~rez Torres, 1988) have isolated lignolitic bacteria and fungi that act upon the general organic content of alpechin, including the antimicrobial phenolic compounds. We have examined the biodegradation of the organic content of alpechin, comparing the results of Bacillus pumilus with those of Aspergillus terreus in an initial 20% alpechin solution under aerobic conditions. We found Aspergillus terreus to be more effective at degrading this waste and so made further studies into the optimum conditions for its activity.
INTRODUCTION "Alpechin' is the Spanish term for the waste produced by the olive-oil extraction industry. Composed of vegetable liquids from the olives and the water used in the extraction process, this slurry results both from the traditional method of pressing and from the newer method of continuous centrifugation. Due to differences between the two methods, and depending upon olive variety and climatic factors, the quantity of alpechin produced per tonne of olives may vary from 0"5 m 3 to more than 1 m 3. This waste is a harmful pollutant which does not readily decompose. Its COD is around 230 000 p.p.m, and its BOD around 150 000 p.p.m. In the Guadalquivir basin alone average production is estimated to be about 1800000 m 3 per year, a volume of pollution comparable to that of a city of 3 500 000 inhabitants. Some 25% of this waste is indiscriminately dumped (Rodrigo Romfin, 1990), while the remaining 75% is left in evaporation pits, which only partially reduce contamination. Consequently, because of its massive scale, and low decomposition rate due to the strong antimicrobial properties of its phenolic compounds (Moreno et al., 1987), alpechin is considered to be one of the major water pollution problems in the Mediterranean basin. To eliminate this pollution various methods have been employed: simple evaporation (Storm, 1989), physical and chemical processes such as inverse
METHODS Alpechin Table 1 shows the principal characteristics of the alpechin used in the experiments. The original material, from Jimena Osuna S.A., had passed through a continuous 'Pieralisi' process, and samples were drawn from the final centrifugation step into sterile flasks and taken to the laboratory. The samples were diluted to concentrations of between 20% and 80%, using Minimum Maintenance Medium (MMM) (Janshekar et al., 1981), the composition of which is described in Table 2. In addition, undiluted samples of alpechin were used. Microorganisms B. pumilus and A. terreus were obtained from the Microbiology Department of the Faculty of Pharmacy of the University of Granada. For test solutions, a culture of B. pumilus was grown on a TSA (tripticasein-soy-agar) slope. For each alpechin concentration, two test tubes (15 ml) were inocu-
Bioresource Technology 0960-8524/92/S05.00 © 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain 215
216
L. Martinez Nieto, S. E. Garrido Hoyos, F. Camacho Rubio, M. P. Garcia Pareja, A. Ramos Cormenzana
Table 1. Physical and chemical characteristics of the alpechin used
pH COD (g per litre) BOD (g per litre) Total solids (g per litre) Volatile solids (g per litre) Non-volatile solids (g per litre) Settled solids (g per litre) Suspended solids (g per litre) Dissolved solids (g per litre) Total phenolic compounds
5"26 212"25 179"45 55"59 44"46 11"13 7"75 2'44 45"40 O'7%
_
v a c u u m
®
Table 2. Composition of minimal maintenance medium
Bipotassium phosphate Monopotassium phosphate Ammonium sulphate Ammonium nitrate Magnesium sulphate pentahydrate Sodium chloride Ferric chloride hexahydrate Calcium chloride Distilled water
1"6 g 0"50 g 1"25 g 1"0 g 0"50 g 0'25 g 0"025 g 0'010g 1 000 ml
lated using a loop (1-2-3-0× 10 ~ cfu per ml) and incubated at 28°C for 18-24 h under constant shaking. The content of each tube was diluted with MMM to a volume of 150 ml and incubated in two 500-ml flasks in the same manner as above. These two 150-ml samples were combined and added to alpechin to a volume of 3000 ml in the 5-1itre batch reactor and incubated at 28°C for 48 h with constant stirring. A culture of A. terreus, grown on a malt-extract-agar slope, produced spores which were then suspended in a saline solution (1-9 × 10 s viable spores per ml). For each alpechin concentration, two test tubes containing 15 ml were inoculated with 1"5 ml of spore suspension and incubated at 28°C for 72 h with constant shaking. Each tube was diluted with MMM to a volume of 150 ml and incubated in two 500-ml flasks in the same manner as above. These two 150-ml samples were combined and added to alpechin to a volume of 3000 ml in the 5-1itre batch reactor and incubated at 28°C for 6 days. Reactor
The 5-1itre batch reactor (Fig. 1 ) and all its components were sterilised prior to the experiments. A steady flow of air, sterilised by filtration, was maintained at a rate of 720 ml per min and the stirring rate was 200 rpm. Samples were taken at regular intervals to determine the COD, BOD and phenol content. Analytical methods
The chemical oxygen demand (COD) was measured by the methods of Rodier (1985) and Capitfin Vallvey et a/.(1987). The biochemical oxygen demand (BOD) was measured by the methods of Sauter & Stoub (1990).
Fig. 1. Schematic drawing of the batch reactor. 1, Reactor; 2, bath at 28°C; 3, temperature control; 4, filter; 5, stirrer; 6, thermometer; 7, sample port; 8, compressed air; 9, reduction-valve control; 10, flow meter.
The phenol content was measured in a 720 Waters chromatograph, connected to a C1~ column, 30 cm long, measuring two wavelengths (280 and 340 nm) simultaneously for 75 rain (following the technique described by Olano & Hernfindez, 1985). The concentrations of phenolic compounds were calculated using Wavescan EG and PE Nelson software programmes and identified by their retention time and by the ratio between response factors at the two wavelengths. The results were confirmed by TLC and by comparison with commercial phenolic compounds. RESULTS Table 3 shows the results obtained for B. pumilus and A. terreus, in a 20% alpechin solution, the percentage of degradation being calculated on COD and BOD. The time for maximum degradation for A. terreus was 4 days, while for B. pumilus it was 2 days (Table 3). The decomposition of phenolic compounds by A. terreus exceeded that of B. pumilus, because after a period of 48 h the bacteria began metabolically to produce new phenolic compounds. A. terreus, however, produced a negligible quantity of phenolic compounds, and less than the bacteria even after the optimum period of 4 days. B. pumilus degraded 62"1% of the total phenol content over 2 days and A. terreus 68"9% over 4 days. Table 4 summarises the phenolic compounds identified by chromatography in relation to the microorganisms and to the alpechin. Eleven phenol compounds (91% of the total phenolic content of the alpechin) were identified by HPLC, of which seven showed significant effects after fungal treatment (protocatechuic acid + hydroxytirosol, p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, vanillyl alcohol and syringaldehyde). B. pumilus affected only six of these compounds and caused a rise in vanillic acid by its metabolic activity.
217
Purification o f olive oil waste
Table 3. Degradation of alpechin (20% dilution) by Bacillus pumilus and Aspergillus terreus in batch reactors Microorganisms
t
pH~
2 4
Bacillus pumih4s Aspergillus terreus
7.00 5-26
pHt
CODf (mg per litre)
6"94 6'04
BODf (mg per litre)
20 241 15 899
19 870 5 225
% Degradation COD
BOD
52.32 62"55
44.64 85'44
i, initial; f, final; t, days of fermentation. COD~, 42 450 mg per litre; BOD~, 35 890 mg per iitre. For fermentation conditions see Methods.
Table 4. Degradation of phenolic compounds by Bacillus pumilus and Aspergillus terreus Compounds
TP"
Protocatechuic acid + hydroxytirosol Vanillyl alcohol p-hydroxybenzoic acid Vanillic acid Caffeic acid p-Vaniilin Syringaldehyde p-Coumaric acid
% Degradation B. pumilus
A. terreus
64-45 -21'57 -94.70 -63.72 93"01
98.68 27"27 78"89 53.40 93"68 62.52 20.72 94.10
36"06 0.32 14.57 4.11 8-45 0.52 1-28 25-62
"% of total phenol compounds in undegraded alpechin.
Table 5. Degradation of alpechin by Aspergillus terreus after 6 days Concentration of alpechin in the reactor 200/0 40% 60% 80% 100%
t (day) 0 6 0 6 0 6 0 6 0 6
COD (mg per litre)
BOD (mg per litre)
42450 19791 84900 36353 127350 58 905 169 800 80052 212250 129710
35 890 4303 71 780 14850 107670 28 740 143 560 47520 179450 78 100
A COD (rag per litre)
A BOD (rag per litrc)
% Degradation COD
BOD
22659
31 587
53"38
88"01
48547
56930
57.18
79.31
68445
78930
53.75
73.31
89 748
96 040
52.86
66"90
82540
101 350
38"89
56-48
COD~, 212 250 mg per litre; BOD~, 179 450 mg per iitre. For fermentation conditions see Methods.
Table 5 records the results for A. terreus kept in the different alpechin solutions in the reactor for 6 days and lists the percentages of degradation for C O D and BOD. Table 6 shows the elimination rates of organic material per day, of C O D and B O D per unit of volume. T h e tables indicate that, as the alpechin concentrations increase, the percentage degradations of C O D and B O D decrease. Nevertheless, the results expressed by time unit and volume show that increasing the concentration of alpechin increases the decomposition until a maximum of about 80% alpechin is reached.
Figure 2 plots C O D and B O D degradations (calculated from Table 5) in relation to the alpechin concentration; the values fit the following equations formulated by polynomial regression: COD: Y = O ' 4 4 7 + O ' 5 4 1 X - O ' 5 8 9 X
2
(1)
BOD: Y = O . 9 3 5 - O . 2 9 5 X - O . O 6 9 X 2
(2)
X = percentage alpechin concentration; Y-- percentage C O D and B O D degradation. Both equations reproduce the experimental values with a deviation of less than 5%.
218
L. Martinez Nieto, S. E. Garrido Hoyos, F. Camacho Rubio, M. P. Garcia Pareja, A. Ramos Cormenzana Table 6. The elimination of organic material with time by Aspergillus terreus
Decrease in organic material
Concentration of alpechin in the reactor ACOD/At (rag per litre per day)
ABOD/At (mg per litre per day)
3777 8091 11408 14958 13757
5265 9488 13155 16007 16892
2O% 40% 60% 80% 100%
% Degradation 100
80
60
40
20
i
0 0
20
I
I
~-
Fig. 2.
I
40 60 80 % Alpechin c o n c e n t r a t i o n COD
-4--
I
1 O0
120
BOD
The percentage degradation compared to the initial concentration of aipechin.
DISCUSSION This study analyses the biodegradation of alpechin by comparing the lignolitic bacterium B. pumilus and the fungus A. terreus in 20% alpechin solutions where constant conditions were maintained. The results indicated that greater efficiency of A. terreus in degrading the alpechin. Results with A. terreus at alpechin concentrations of 20%, 40%, 60%, 80% and 100% (pure alpechin) indicated that the fungus performed most effectively at about 80% (CODi = 212 250 mg per litre and BODi = 179 450 mg per litre). It is important to note that, after 48 h, B. pumilus began to produce new phenolic compounds and thereby raised the COD. With A. terreus this phenomenon occurred only after 6 days, and thus 4 days was taken to be the maximum fermentation period to give optimum degradation. This degradation process might have an industrial application, since, with the reduction in concentration of a large number of phenolic compounds, the remaining effluent would have less antimicrobial capacity. Thus the time needed in anaerobic digesters for the production of methane would be reduced. The biDmass obtained, rich in protein, could be used as livestock feed, and the effluent could be used either as a fertilizer or, in concentrated form, as a supplementary feed additive.
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