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Microbial degradation of illinois no. 6 coal by means of co metabolism with phenols
Coal Science J.A. Pajares and J.M.D. Tasc6n (Editors) 9 1995 Elsevier Science B.V. All rights reserved.
Microbial Degradation of Illinois N o . 6 Coal with Phenols
1753
by Means
of C o m e t a b o l i s m
Y.Kabe, T.Furuta, M.Takai, K.Higashi, S.Katoh and T.Kojima Faculty of Agriculture, Tmamagawa University, Tokyo 194,
Japan
1. Introductio n
Since the phenomenon of microbial solublization of weathered lignite was found by Cohen et.al. 1~, microbial degradation of coal has been studied actively, and various mechanisms have been proposed. Two different approaches have been made to elucidate the mechanism of microbial degradation of coal. One of them is the research of biodegradation of condensed multi ring aromatic hydrocarbon such as anthracene and phenanthrene as model compounds, which has been made as earier as 1950s2>.3>. Another approach is lowering the molecular weight of coal, which is comparatively new. In order to lower the molecular weght of coal, either ether or methylene bridged linkage must be cleaved. This second approach could be subdivided into the following three, 1)depolymerization of lignite4>,5~, 2)cleavage of ether linkage of lignin dimer% of which structue is close to that of coal, 3)cleavage of ether linkage of diphenylether as a model compond of coal by Pfeifer et..al. 7>.8). Thus, there is a fair prospect of biodegradation of coal, but in order to deverope practically, furthermore many sided research must be made. Therfore, in this study for the purpose of the beneficial utilization of coal, many sided research have been carried out to examine simultaneous abilities of cleavage of aromatic ring, degradation of lignin and water soluble coal with four Aspergillus species, of which two species were isolated as coal assimilative microbes and another two species were isolated as lignin assimilative microbes. Because lignin and water soluble coal are macromolecule having complex structure, it is considerd that biodegradation of these substances are very difficult. For this reason, cometabolism with phenols was adopted to accelerate microbial degrdation of lignin and coal.
2. Experimental Microorganisms used were four Asprgillus species, of which two strains, FKS1 and FKS7 were isolated from weathered pine tree sample and another two strains, FHS3 and FHS5 were isolated from soil containing coal. Of these four strains, FHS3 and FHS5 were identified as As. versicolor and As. fumigatus, respectively. These funji were cultivated in the media containing coal or the related substances as a sole source of carbon and energy and mineral solution(NHnNO3:2.5g, NaHPO4:I.0g, KH2PO4:0.5g, MgSO4:0.24g, MnC12:0. 13g, a slight amounts of CaC12, ZnSO 4, FeSO 4 and CuSO 4, and distilled water:lL), and 10 ml of media were put in L shaped test tube(i, d.:l 6mm, L:120mm, H:90mm) and reciproshaked in the speed of 120rpm, at 30~
1754 A kind of coal mainly used was Illinois No. 6 coal, in addition, Wandoan coal, Daton coal and Shenyan coal were occasionary used. Coal was ground to particle size of 63-1061.tm and used without further treatment, or used as water soluble coal with treatment of nitric acd oxidation according to the procedure reported by Crawford et. al.6). Water soluble coal were divied three compornents called water soluble Illinois No. 6 coal A, B and C precipitated at 1700 cm-1 of -COOH increased in turn. The following commercial ragents were used as coal related substances without further purification ;lignin, phenol, guaiacol, veratfic acid and acetoveratrone. Growth was expressed by apparent volume of mycelia in L shaped test tube on standing for 1 hr. Concentration of lignin or water soluble coal was mainly measured by UV spevtra using UV spectrophotometer UV-240 (Shimazu Co. ). 3. Res ults and dis cuss ion 3 . 1 . Degradabilitity of lignin and the related c o m p o u n d s . In order to examine whether aromatic ring was cleaved by four Aspergillus strains FKS 1, FKS7,FHS3 and FHS5, cultivations were carried out in the media containing lignin, phenol, guaiacol (2-methoxyphnol), veratric acid (3,4-dimethoxybenzoic acid) and acetoveratrone (3, 4-dimethoxyacetophenone) as a sole source of carbon and energy. Here, guaiacol, veratric acid and acetoveratrone were used considering as intermediates produced in the degradationof lignin, which had been examined by Cespedes et.al.6). As shown in Fig. 1, in the medium containing lignin, all of four strains slightly grew more than in that containing no carbon source, and all of them remarkably grew in media containing phenol, guaiacol and veratric acid. In the medium containing acetoveratrone, FKS 1 and FHS3 significantly grew, whereas FKS7 and FHS5 grew only to the level of no carbon source. Measuring concentration of compounds, phenol, guaiacol and veratric acid were expended
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i
FKS1
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!
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1
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!
1
FHS5
l
I
/
I
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FHS3
NO CARBON SOURCE LIGNIN
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FKS7
/
PHENOL GUAIACOL VERATRIC ACID ACETOVERATRONE
/
i
0
2
I
I
4
I
0
I
2
I
I
4 0
I
I
2
I
I
4 0
I
I
2
I
1
4
MYCELIAL GROWTH(apparent v o l . / m l ) Figure 1. The growth of four A s p e r g l l l u s species in the media containing 11gnln and the related compounds. 10 ml, 11gnin'2000ppm. others'5OOppm, pH7, 30~ 120rpm, 2weeks.
1755
by all of four strains within 2 weeks. In the medium containing acetoveratrone, it was expended almost perfectly with FKS 1, but the color of the culture changed to yellowish brown and a new absorption maximum appered at 300nm, and some new spots also appeared in TLC. Therefore, it was suggested that it was not perfectly decomosed, but converted to some new colored substances. With FHS3, it was expended almost perfectly after 8 days, and the color of the culture changed to slightly pink, however, as neither new absorption maximum nor spot in TLC was obserbed, it was appear that it was perfectly decomposed.
3.2 Deg radabilitity of coal Cohen et. al. 1~ reported that water soluble black substance was produced when some broken piecies of weathered lignite were put on Polyporus versicolor gown thick on the nutrient medium. Saiki eLM. q) also found similar phenomenon using Joban coal(brown coal) oxygenated with hydrogen peroxide. In this study, similar experiment was attempted, in which Wandoan coal(subbituminous coal), Daton (bituminous coal), Shenyan coal (lignite) and Illinois No. 6 coal (subbituminous coal) were used without any treatmenL Then, small pieces of Daton and Illinois No. 6 coal on FKS 1 and FHS3 strains were covered by drops of water, especially around Illinois No. 6 coal, 5-10mm drops of water were formed after a few days. These color were not black like that reported Saiki et. al.9>, but slightly yellowiish brown. Therefore, it was appear that coal were little degradated. However, it was considered that these drops of water were resulted to undergo some actions of both two strains to solubilize and assimilate coal. Taking notice of this phenomenon with Illinois No. 6 coal, cultivation suspending its fine powder as a sole source of carbon were attempted, but remarkable growth of four strains were not observed. Subsequently, degradability of water soluble Illinois No. 6 coal A and C were examined. FKS 1 and FHS3 grew to the level of lignin with A, and grew with C more than with A which was little oxidized by H202.
3.3 Effect of phenol addition As it is considered that assimilation rate of lignin or water soluble coal is not fast because of high molecular substances, addition of phenol to these media was examined to support rapid growth. The result on lignin was in Fig.2. Here, pH was adjusted to 5 which was not well suited for assimilation of phenol(compare of Fig. 1 at pH7), but FKS 1, FKS7 and FHS3 grew well in mixed medium of lignin and phenol, Especially, the effect was notewothy for FKS 1. The effects of phenol addition to water soluble Illinois No. 6 coal on growth of FKS 1 and FKS 7 were also remarkable, data are not sow n here. In Fig.3, UV spectra of cultures containing lignin and water soluble Illinois No. 6 coal C with FKS1 were shown. Spectra drawn with dotted line are those noninoculated and shaked for the same incubation period. In noninoculated media in phenol addition, absorption of phenol at 270nm was observed, it was disappeared in both cultures containing lignin and water soluble coal with FKS 1, and concentration of lignin and water soluble coal in each culture inoculated decreased more than in that noninoculated. Moreover, when exess of mycelia was inoculated, color of lignin in the culture changed to colorless. As above mentioned, both phenol and lignin or water soluble coal became liable to be degradated by means of cometabolism. Therefore, Aspergillus FKS1 strain would be available to treat waste containing water soluble coal.
1756
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i
FKS7
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FHS5
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-TO
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5
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10
-
0
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'
'
I
5
10 0 5 10 0 5 10 15 INC. PERIOD/D Figure 2. Effect of phenol addition to media containing lignin on the growth of four Asperglllus species. O:lignin 2000ppm, O:phenol 500ppm, @:lignin + phenol 10ml, pHS, 30~ 120rpm.
2
2
La
'1
COAL
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.o 1
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09
d
!
0
200
300
400
500
600
~
200
300
400
"
500
"'."m
600
A/nm A/nm Figure 3. Effect of phenol addition on the decrease of lignin and water soluble coal concentration with the growth of Aspergillus FKSI. a:lignin(noninoculum), b:lignin + phenol(noninoculum) a':coal ( ,, ), b':coal + ,, ( ,, ) c:lignin(inoculum), d:lignin + phenol(inoculum) c':coal ( o ), d':coal + o ( o ) Incubation:pH5(lignin),pH7(coal), 30"C, 120rpm, 2weeks. Dilution:l/25 with water adjusted to pH2(lignin) and pH9(coal). Refferences
1) 2) 3) 4) 5) 6) 7) 8) 9)
Cohen, M.S., et.al., Appl. Environ., vol.44, p.23, 1982. Rogoff, M.H., et.al., J. Bactriol., vol.74, p. 108, 1957. Rogoff, M.H., et.al., J. Bactriol., vol.74, p.264, 1957. Gupta, R.K., et. al., Appl.Biochem. Biotech., vol.24/25, p.899, 1990. Crawford, D.L., et.al., Fuel, vol.70, p.577, 1991. Cespeds, R., et. al., Arch. Microbiol., vol. 158, p., 1992. Pfeifer, F., et. al., Arch. Microbiol., vol. 152, p.519, 1989. Pfeifer, E, et. al., Arch. Microbiol., vol. 159, p.323, 1993. Saiki, H.eLal., Proc. 1st Conf. Coal Utiliz. Tec., p. 264, 1991.
Microbial Degradation of Illinois N o . 6 Coal with Phenols
1753
by Means
of C o m e t a b o l i s m
Y.Kabe, T.Furuta, M.Takai, K.Higashi, S.Katoh and T.Kojima Faculty of Agriculture, Tmamagawa University, Tokyo 194,
Japan
1. Introductio n
Since the phenomenon of microbial solublization of weathered lignite was found by Cohen et.al. 1~, microbial degradation of coal has been studied actively, and various mechanisms have been proposed. Two different approaches have been made to elucidate the mechanism of microbial degradation of coal. One of them is the research of biodegradation of condensed multi ring aromatic hydrocarbon such as anthracene and phenanthrene as model compounds, which has been made as earier as 1950s2>.3>. Another approach is lowering the molecular weight of coal, which is comparatively new. In order to lower the molecular weght of coal, either ether or methylene bridged linkage must be cleaved. This second approach could be subdivided into the following three, 1)depolymerization of lignite4>,5~, 2)cleavage of ether linkage of lignin dimer% of which structue is close to that of coal, 3)cleavage of ether linkage of diphenylether as a model compond of coal by Pfeifer et..al. 7>.8). Thus, there is a fair prospect of biodegradation of coal, but in order to deverope practically, furthermore many sided research must be made. Therfore, in this study for the purpose of the beneficial utilization of coal, many sided research have been carried out to examine simultaneous abilities of cleavage of aromatic ring, degradation of lignin and water soluble coal with four Aspergillus species, of which two species were isolated as coal assimilative microbes and another two species were isolated as lignin assimilative microbes. Because lignin and water soluble coal are macromolecule having complex structure, it is considerd that biodegradation of these substances are very difficult. For this reason, cometabolism with phenols was adopted to accelerate microbial degrdation of lignin and coal.
2. Experimental Microorganisms used were four Asprgillus species, of which two strains, FKS1 and FKS7 were isolated from weathered pine tree sample and another two strains, FHS3 and FHS5 were isolated from soil containing coal. Of these four strains, FHS3 and FHS5 were identified as As. versicolor and As. fumigatus, respectively. These funji were cultivated in the media containing coal or the related substances as a sole source of carbon and energy and mineral solution(NHnNO3:2.5g, NaHPO4:I.0g, KH2PO4:0.5g, MgSO4:0.24g, MnC12:0. 13g, a slight amounts of CaC12, ZnSO 4, FeSO 4 and CuSO 4, and distilled water:lL), and 10 ml of media were put in L shaped test tube(i, d.:l 6mm, L:120mm, H:90mm) and reciproshaked in the speed of 120rpm, at 30~
1754 A kind of coal mainly used was Illinois No. 6 coal, in addition, Wandoan coal, Daton coal and Shenyan coal were occasionary used. Coal was ground to particle size of 63-1061.tm and used without further treatment, or used as water soluble coal with treatment of nitric acd oxidation according to the procedure reported by Crawford et. al.6). Water soluble coal were divied three compornents called water soluble Illinois No. 6 coal A, B and C precipitated at 1700 cm-1 of -COOH increased in turn. The following commercial ragents were used as coal related substances without further purification ;lignin, phenol, guaiacol, veratfic acid and acetoveratrone. Growth was expressed by apparent volume of mycelia in L shaped test tube on standing for 1 hr. Concentration of lignin or water soluble coal was mainly measured by UV spevtra using UV spectrophotometer UV-240 (Shimazu Co. ). 3. Res ults and dis cuss ion 3 . 1 . Degradabilitity of lignin and the related c o m p o u n d s . In order to examine whether aromatic ring was cleaved by four Aspergillus strains FKS 1, FKS7,FHS3 and FHS5, cultivations were carried out in the media containing lignin, phenol, guaiacol (2-methoxyphnol), veratric acid (3,4-dimethoxybenzoic acid) and acetoveratrone (3, 4-dimethoxyacetophenone) as a sole source of carbon and energy. Here, guaiacol, veratric acid and acetoveratrone were used considering as intermediates produced in the degradationof lignin, which had been examined by Cespedes et.al.6). As shown in Fig. 1, in the medium containing lignin, all of four strains slightly grew more than in that containing no carbon source, and all of them remarkably grew in media containing phenol, guaiacol and veratric acid. In the medium containing acetoveratrone, FKS 1 and FHS3 significantly grew, whereas FKS7 and FHS5 grew only to the level of no carbon source. Measuring concentration of compounds, phenol, guaiacol and veratric acid were expended
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i
FKS1
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!
1
v
!
1
FHS5
l
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I
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FHS3
NO CARBON SOURCE LIGNIN
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FKS7
/
PHENOL GUAIACOL VERATRIC ACID ACETOVERATRONE
/
i
0
2
I
I
4
I
0
I
2
I
I
4 0
I
I
2
I
I
4 0
I
I
2
I
1
4
MYCELIAL GROWTH(apparent v o l . / m l ) Figure 1. The growth of four A s p e r g l l l u s species in the media containing 11gnln and the related compounds. 10 ml, 11gnin'2000ppm. others'5OOppm, pH7, 30~ 120rpm, 2weeks.
1755
by all of four strains within 2 weeks. In the medium containing acetoveratrone, it was expended almost perfectly with FKS 1, but the color of the culture changed to yellowish brown and a new absorption maximum appered at 300nm, and some new spots also appeared in TLC. Therefore, it was suggested that it was not perfectly decomosed, but converted to some new colored substances. With FHS3, it was expended almost perfectly after 8 days, and the color of the culture changed to slightly pink, however, as neither new absorption maximum nor spot in TLC was obserbed, it was appear that it was perfectly decomposed.
3.2 Deg radabilitity of coal Cohen et. al. 1~ reported that water soluble black substance was produced when some broken piecies of weathered lignite were put on Polyporus versicolor gown thick on the nutrient medium. Saiki eLM. q) also found similar phenomenon using Joban coal(brown coal) oxygenated with hydrogen peroxide. In this study, similar experiment was attempted, in which Wandoan coal(subbituminous coal), Daton (bituminous coal), Shenyan coal (lignite) and Illinois No. 6 coal (subbituminous coal) were used without any treatmenL Then, small pieces of Daton and Illinois No. 6 coal on FKS 1 and FHS3 strains were covered by drops of water, especially around Illinois No. 6 coal, 5-10mm drops of water were formed after a few days. These color were not black like that reported Saiki et. al.9>, but slightly yellowiish brown. Therefore, it was appear that coal were little degradated. However, it was considered that these drops of water were resulted to undergo some actions of both two strains to solubilize and assimilate coal. Taking notice of this phenomenon with Illinois No. 6 coal, cultivation suspending its fine powder as a sole source of carbon were attempted, but remarkable growth of four strains were not observed. Subsequently, degradability of water soluble Illinois No. 6 coal A and C were examined. FKS 1 and FHS3 grew to the level of lignin with A, and grew with C more than with A which was little oxidized by H202.
3.3 Effect of phenol addition As it is considered that assimilation rate of lignin or water soluble coal is not fast because of high molecular substances, addition of phenol to these media was examined to support rapid growth. The result on lignin was in Fig.2. Here, pH was adjusted to 5 which was not well suited for assimilation of phenol(compare of Fig. 1 at pH7), but FKS 1, FKS7 and FHS3 grew well in mixed medium of lignin and phenol, Especially, the effect was notewothy for FKS 1. The effects of phenol addition to water soluble Illinois No. 6 coal on growth of FKS 1 and FKS 7 were also remarkable, data are not sow n here. In Fig.3, UV spectra of cultures containing lignin and water soluble Illinois No. 6 coal C with FKS1 were shown. Spectra drawn with dotted line are those noninoculated and shaked for the same incubation period. In noninoculated media in phenol addition, absorption of phenol at 270nm was observed, it was disappeared in both cultures containing lignin and water soluble coal with FKS 1, and concentration of lignin and water soluble coal in each culture inoculated decreased more than in that noninoculated. Moreover, when exess of mycelia was inoculated, color of lignin in the culture changed to colorless. As above mentioned, both phenol and lignin or water soluble coal became liable to be degradated by means of cometabolism. Therefore, Aspergillus FKS1 strain would be available to treat waste containing water soluble coal.
1756
!
i
i
!
i
FKS7
E
\
i
FHS5
O
I--(D 113 n Ix3
-TO
r
~
5
0
10
-
0
G
'
'
I
5
10 0 5 10 0 5 10 15 INC. PERIOD/D Figure 2. Effect of phenol addition to media containing lignin on the growth of four Asperglllus species. O:lignin 2000ppm, O:phenol 500ppm, @:lignin + phenol 10ml, pHS, 30~ 120rpm.
2
2
La
'1
COAL
!
.o 1
~,~.,
09
d
!
0
200
300
400
500
600
~
200
300
400
"
500
"'."m
600
A/nm A/nm Figure 3. Effect of phenol addition on the decrease of lignin and water soluble coal concentration with the growth of Aspergillus FKSI. a:lignin(noninoculum), b:lignin + phenol(noninoculum) a':coal ( ,, ), b':coal + ,, ( ,, ) c:lignin(inoculum), d:lignin + phenol(inoculum) c':coal ( o ), d':coal + o ( o ) Incubation:pH5(lignin),pH7(coal), 30"C, 120rpm, 2weeks. Dilution:l/25 with water adjusted to pH2(lignin) and pH9(coal). Refferences
1) 2) 3) 4) 5) 6) 7) 8) 9)
Cohen, M.S., et.al., Appl. Environ., vol.44, p.23, 1982. Rogoff, M.H., et.al., J. Bactriol., vol.74, p. 108, 1957. Rogoff, M.H., et.al., J. Bactriol., vol.74, p.264, 1957. Gupta, R.K., et. al., Appl.Biochem. Biotech., vol.24/25, p.899, 1990. Crawford, D.L., et.al., Fuel, vol.70, p.577, 1991. Cespeds, R., et. al., Arch. Microbiol., vol. 158, p., 1992. Pfeifer, F., et. al., Arch. Microbiol., vol. 152, p.519, 1989. Pfeifer, E, et. al., Arch. Microbiol., vol. 159, p.323, 1993. Saiki, H.eLal., Proc. 1st Conf. Coal Utiliz. Tec., p. 264, 1991.