Resources, Conservation and Recycling, 5 ( 1991 ) 245-254 245 Elsevier Science Publishers B.V./Pergamon Press plc

Influence of cultural parameters on the depolymerization of a soluble lignite coal polymer

by Pseudomonas cepacia DLC-071

Don L. Crawford 2 and Rajinder K. Gupta Department of Bacteriology and Biochemistry, Institute for Molecular and Agricultural Genetic

Engineering (IMAGE), University of ldaho, Moscow, ID 83843, USA

(Received January 3, 1990; accepted after revision August 28, 1990)

ABSTRACT

Crawford, D.L. and Gupta, R.K., 1991. Influence of cultural parameters on the depolymerization of a soluble lignite coal polymer by Pseudomonas cepacia DLC-07. Resour. Conserv. Recycl., 5: 245- 254.

A lignite coal-depolymerizing bacterium, Pseudomonas cepacia DLC-07, was grown in liquid media containing water soluble lignite coal. The coal substrate was soluble at pH 5.5, but had not been pre- oxidized with nitric acid. Bacterial coal depolymerization was monitored by high performance liquid chromatography (HPLC). Cells were grown on soluble coal in three media: mineral salts broth, pep- tone broth, and Sabouraud dextrose broth (SDB). In some experiments, media were supplemented with specific coal substructure model compounds, or a mixture of compounds, which served as poten- tial inducers of coal-degradative enzymes. In one experiment, peptone broth contained Kraft lignin instead of coal. Uninoculated controls were included in all experiments. P. cepacia DLC-07 depoly- merized coal in all experiments, but depolymerization was greatest in peptone-coal broth. Often coal substructure models examined, indole and p-coumaric acid measurably affected depolymerization. Their presence in SDB led to the appearance of distinctive peaks in HPLC chromatograms. P. cepacia further polymerized Kraft lignin into a product of higher molecular weight than the starting substrate. Spectrometric analyses showed that coal depolymerization was associated with attack on ester and ether bonds in the coal.

I N T R O D U C T I O N

Pseudomonas cepacia strain DLC-07 has previously been shown to co-me- tabolize a wide variety of coal substructure model compounds, to utilize 17- hydroxy substituted benzoic and cinnamic acids or aldehydes as sole carbon and energy sources, and to reduce the average molecular weight of a nitric acid-pretreated, water soluble coal while utilizing the coal as a carbon and

l Paper of the Idaho Agricultural Experiment Station. 2To whom correspondence should be addressed.

0921-3449/91/$03.50 © 1991 - - Elsevier Science Publishers B.V./Pergamon Press plc

246 D.L. CRAWFORD AND R.K. GUPTA

energy source [ 1 ]. Analyses by FT-IR showed that the depolymerized coal contained fewer carbonyl and carboxyl groups, decreased etheric oxygen, and fewer aromatic and conjugated carbon-carbon double bonds as compared to a control coal. 13C-NMR showed a decrease in not only carbonyls and heter- oaromatic carbons, but also in nitrogen, oxygen, and sulfur, and in unsubsti- tuted aromatic carbons, along with an increase in long chain methylenes in the biotransformed coal. Thus, while the coal was depolymerized by the bac- terium, it was not significantly oxidized during depolymerization.

A major objective of research in this laboratory is the development of pro- cesses for the reductive liquefaction of coal by microorganisms. Such lique- faction would result in the conversion of coal to a high value liquid fuel of equal or higher energy content than the starting coal. The results discussed above indicate that P. cepacia DLC-07 may be useful for depolymerizing lig- nite coals prior to using them as substrates for reductive microorganisms. A water-soluble, lower molecular weight coal polymer should be more readily metabolized by bacteria than unmodified, insoluble lignite coal. It would also be useful to develop a coal bio-liquefaction process that does not utilize nitric acid pre-oxidized coal as a substrate, since preoxidation is expensive and in- troduces both nitrogen and oxygen into the coal. In the present paper, P. ce- pacia DLC-07 was shown to readily depolymerize a water-soluble, unoxi- dized lignite coal polymer. It was also shown that the make-up of the culture medium markedly influenced the extent of coal biotransformation by this bacterium. In addition, selected coal substructure model compounds, when used at low concentration in a coal-containing medium, significantly affected coal depolymerization. Apparently, the models induced model compound degradative enzymes which were able to also attack bonds within the coal macromolecule.

MATERIALS AND METHODS

Microorganism

Pseudomonas cepacia strain DLC-07 [1] was used throughout this re- search. Stock cultures were maintained on Sabouraud dextrose agar (SDA) slants at 4°C after growth for 24-48 hr at 30°C. For maintenance, transfers to fresh media were made every 2-3 weeks.

Coal substrate

A polymeric coal substrate, water soluble at pH 5.5, was prepared from 100 g of Vermont lignite coal [ 2 ]. Powdered coal was soaked in water for 8 h and then dissolved in 1 1 of 1N NaOH. This solution was centrifuged to remove undissolved particles, and the supernatant was adjusted to pH 7.0 with dilute

A LIGNITE COAL-DEPOLYMERIZING BACTERIUM PSEUDOMONAS CEPACL4 247

HC1. The precipitated coal was centrifuged, washed, dried, and powdered (yield: 40 g). This coal was water soluble at pH > 7.0. The supernatant from the above precipitation was acidified to pH 5.5. The resulting precipitate was recovered by centrifugation, washed (pH 5.5 ), dried, and powdered (yield: 15 g). The remaining coal in solution was precipitated at pH 1.5. The precip- itate was recovered by centrifugation, washed (pH 1.5 ), dried, and powdered (yield: 10 g). This latter coal polymer dissolved at pH > 5.5 and was used as the substrate for P. cepacia DLC-07.

Metabolism of coal and lignin in peptone broth with and without supplementation with coal substructure model compounds

A loopful of cells (about 106 cells) from an SDA slant of strain DLC-07 was used to inoculate each 250 ml flask containing 100 ml of 1.0% (w/v) Bacto-Peptone broth (Difco, Detroit, MI), pH 5.5. Inoculated flasks were incubated shaking (250 rpm) at 30°C for 18 h, by which time cell numbers had reached about 107 to 108 cells m1-1. Then 25.0 mg of Vermont lignite

C00. .0 ~ o c .

° o

OH R OClt 3

It0

l R-H 11" R=0H TIT v

"VT

R2"R3" H ; R I "0CH 3 R3 xH; RI" R2" 0CH3

R2:H, R$'CH 3, R I :0CH 3

CH3 T r X - 0

v,,, "r X-S Fig. 1. Structures of the ten coal substructure model compounds used as potential co-substrates or inducers of coal degradative enzymes in Pseudomonas cepacia DLC-07. I =p-coumaric acid; II = Caffeic acid; III = 1- (4-hydroxy-3-methoxyphenyl)-2- ( 2-methoxyphenoxy)-3-hydroxypro- pan-l-one; IV=vani l l in ; V=syringaldehyde; VI=acetovani l lone; VIl=4-methylumbel l i fer - one; VIII = indole; IX = dibenzofuran; X = dibenzothiophene.

248 D.L. CRAWFORD AND R.K. GUPTA

coal polymer in 2.5 ml of water (pH 5.5) was added to each flask and to a corresponding uninoculated control. At this time, 25 mg of coal polymer was also added to another 18 h culture grown in peptone broth that had been sup- plemented with an inducing mixture of 9 coal substructure model com- pounds. This mixture [9 mg dissolved in 100 lxl of dimethylformamide (DMF) ] was prepared by combining 1 mg each of compounds I through X (Fig. 1 ), except compound III, in DMF. In a separate experiment, 25 mg of Kraft lignin (Indulin; Sigma Chemical Co., St. Louis, MO) dissolved in water (pH 7.2 ) was added to a peptone broth culture instead of lignite solution. In all experiments, cultures were incubated shaking (250 rpm) at 30°C for 60 days. Samples for molecular weight determinations by high performance liq- uid chromatography (HPLC) were withdrawn from each flask at days 0, 30 and 60. At day 60, cultures were harvested. The cells were removed by cen- trifugation. Then, acid precipitable lignite or lignin (pH 1.5 ) was recovered by centrifugation, washed, and dried for infrared (IR) analysis.

Metabolism of coal in Sabouraud dextrose broth

Pseudomonas cepacia DLC-07 was grown as above, except that Sabouraud dextrose broth (SDB) was substituted for peptone broth. Also, all of the model compounds, including compound III (Fig. 1 ), were used as potential induc- ers, individually and as a mixture of ten, except that 4 mg instead of 1 mg of each compound were added to each 100 ml of medium.

Growth on coal in minimal medium

Using aseptic technique, cells of DLC-07, previously grown for 18 h in SDB at 30 ° C, were collected by centrifugation, washed with sterile water, and re- suspended in 50 ml of mineral salts solution [ 1 ]. A solution of soluble coal polymer (25 mg in 2.5 ml of water at pH 5.5) was then added to this cell suspension. The suspension and a corresponding uninoculated control were incubated shaking (250 rpm) at 30 °C for 30 days. Samples were then with- drawn for HPLC analysis as described above.

Coal and lignin molecular weight determinations

Coal and lignin samples withdrawn from the various uninoculated control and P. cepacia DLC-07-inoculated cultures were characterized for their mo- lecular weight distributions by HPLC as described previously [ 1 ].

A LIGNITE COAL-DEPOLYMERIZING BACTERIUM PSEUDOMONAS CEPACIA 249

Infrared spectroscopy

FT-IR spectra of lignin or coal samples were recorded using a Perkin-Elmer 1600 spectrometer. KBr pellets, which had been well dried and stored under vacuum, were used for all analyses. Spectra were obtained using 2 mg ofl ignin or coal polymer and 150 mg of KBr. Scans were signal averaged to obtain a pr imary spectrum in wave numbers 4400 to 450 c m - ~.

RESULTS AND DISCUSSION

In a previous paper, P. cepacia DLC-07 was shown to grown in an acidic minimal med ium using a nitric-acid-pretreated, water-soluble lignite coal polymer as a sole source of carbon and energy [ 1 ]. During growth the bacter- ium partially depolymerized the coal and altered its chemistry. The bacter- ium also metabolized or co-metabolized a wide range of low molecular weight aromatic coal substructure model compounds. The present study extended

[40- /~ 11.489 (100,939)

120- I / V. Lignite

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E 60-

40-

20- A

Time (rain)

50- (96,172) 11604 / 12.042 (79,988)

y V. Lignite (I noculoted)

D 20-

B o ~ 4 ; 8 ,b ,) ,;~ ,; ,'8

Time (min)

Fig. 2. High performance liquid chromatograms showing molecular weight distribution patterns of coal samples from 60 day peptone-coal broth control and P. cepacia DLC-07-inoculated cul- tures. A= Control coal sample. B =P. cepacia DLC-07 inoculated coal sample (mAU = milliabsorbence units).

250 D.L. CRAWFORD AND R.K. GUPTA

this research to examine the effects of basal growth medium composition on coal depolymerization by DLC-07. In addition, the soluble coal polymer used as a substrate in the present study had not been pre-treated with nitric acid.

HPLC showed no changes in molecular weight distribution patterns for any of the coal samples taken from uninoculated controls. Figure 2A shows an HPLC chromatogram of a coal sample from a typical uninoculated control (60 day, peptone-coal broth). It shows a single, distinctive peak at 11.489 min, representing an approximate molecular weight of 100,939, as deter- mined from a standard curve (described previously [ 1 ] ). Molecular weight was calculated using the equation:

loglo (M.W.) = (7 .1031-0 .1827xRT)

where RT = retention time [ 1 ]. In contrast to the lack of depolymerization in control cultures, coal was

depolymerized in all of the inoculated cultures. However, with the inoculated cultures, the extent of coal depolymerization was not significantly greater than previously observed in a nitric acid-pretreated coal containing minimal me- dium [1 ], except when DLC-07 was grown in peptone-coal broth. In this case, depolymerization was significant after 30 days (data not shown) and extensive after 60 days incubation. HPLC chromatograms of 60 day coal samples from P. cepacia inoculated coal-peptone broth cultures (Fig. 2B) show that the 11.489 min peak of controls was replaced by a major peak at 12.042 min (M.W.=79,489 D) with a shoulder at 11.604 min (96.172 D). Also, a major lower molecular weight shoulder was present at 13.738 min (M.W. = 39,179 D ). These data show that P. cepacia DLC-07 extensively de- creased the average molecular weight of the lignite coal polymer when utiliz- ing peptone as a principal carbon, nitrogen, and energy source for growth.

The FT-IR spectrum of a coal sample from a 60-day inoculated coal-pep- tone broth culture is compared to that from an uninoculated peptone-coal broth control in Fig. 3. The difference spectrum (Fig. 3; A - B ) showed very intense bands at 1760, 1600 (sh), 1595, 1425, 1331 and 1260 cm -1, indicat- ing a decrease in carbonyl content (ester and carboxylate groups), aromatic and conjugated C=C double bonds, methylene bridges, and etheric oxygen [3,4]. These chemical modifications are similar to the changes observed in previous work [ 1 ].

When P. cepacia inoculated liquid media were supplemented with specific low molecular weight coal substructure model compounds (Fig. 1, com- pounds I, II and IV through X) for peptone broth; compounds I through X for SDB), no increases were seen in the level of coal depolymerization as compared to cultures growing in unsupplemented peptone or SDB. However, for SDB-coal broth cultures supplemented with compound I (p-coumaric acid ) or compound VIII (indole), HPLC chromatograms of coal samples did differ from those obtained from unsupplemented medium. With p-coumaric

A LIGNITE COAL-DEPOLYMERIZING BACTERIUM PSEUDOMONAS CEPACIA 2 51

V. Lignite

B

C

'~ (A-B) I --

3589 I ~ 14251135~

1595

4obo 35bo 3obo 2~oo zdoo ,~oo ,o'oo ~m-' ~bo Wavenumbers

Fig. 3. Fourier transform infrared FT-IR) spectra of coal samples from 60-day peptone-coal broth cultures. A = Uninoculated control; B = P. cepacia DLC-07 inoculated. A - B = Control minus inoculated difference spectrum. Y-axis intensity measurement is in units of percent transmittance.

acid supplementation, total depolymerization of the coal was less that ob- served in peptone-coal broth; however, a distinct lower molecular weight peak appeared at a retention time ( 15.209 min ) corresponding to 21,106 D. With indole supplementation, a significant shoulder peak appeared at 11.195 min (M.W. = 114,228 D), but the major peak eluted at 11.89 min (M.W. = 85.270 D ). These data show that the two model compounds influenced the chemistry of coal metabolism by P. cepacia DLC-07; however, the basis of the observed changes remains to be determined. Both p-coumaric acid and indole are to- tally degraded by P. cepacia DLC-07 [ 1 ]. Induction of catabolic enzymes for their degradation possibly led to some unique reactions as the enzymes co- metabolized coal.

In one experiment, Kraft lignin (Indulin) was substituted for coal polymer as a substrate for P. cepacia in the peptone broth. This experiment was carried out to learn if Kraft lignin, an industrially modified lignin that is chemically similar but structurally much simpler than lignite coal, was also significantly depolymerized by the bacterium. As shown in Fig. 4, the HPLC chromato- gram of the lignin from a 60 day uninoculated control showed a peak at 11.908

252 D.L. CRAWFORD AND R.K. GUPTA

500-

< 200- E

I00-

140-

120-

I 00 - ,¢

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6 0 -

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0

(137,744)

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(540,600) 8.598

I 1.908 (84,627)

Indulin (Control)

~ II (36,43e) A

Time (min)

/

8 ,b ,~ Time (min)

11915 (84,57e)

Indul in ( Inoculated)

Fig. 4. High performance liquid chromatograms showing molecular weight distribution patterns of Kraft lignin (Indulin) samples from 60 day peptone broth control and P. cepacia DLC-07 inoculated cultures. A--Control lignin sample. B = P. cepacia DLC-07 inoculated lignin sample (mAU = milliabsorbence units).

min, which corresponds to 84,627 D molecular weight, shoulder peaks were present at 10.75 min ( (M.W.= 137,744 D) and 13.911 min (M.W.=36,438 D) ). Interestingly, the chromatogram from the 60-day P. cepacia inoculated culture showed a major peak at 8.598 min, corresponding to a molecular weight of 340,600 D. The control peak ( 11.915 min; M.W. = 84.378 ) was still present as well. In addition, a small shoulder peak was present at 15.105 min. These data are indicative of polymerization of the lignin by DLC-07 to a product of considerably higher molecular weight than the starting substrate. It remains to be determined whether or not depolymerization reactions oc- curred prior to repolymerization of the lignin. Figure 5 shows the FT-IR spec- tra of the lignin samples. The difference spectrum (Fig. 5; A - B ) showed bands at 1590, 1507, 1457, 1260 and 1025 cm -I , indicating that chemical changes in the lignin as a result ofP. cepacia metabolism were similar to those observed with the lignite coal. However, in contrast to the coal, which was significantly depolymerized, polymerization reactions dominated with the lignin. The basis for this significant difference remains to be elucidated.

A LIGNITE COAL-DEPOLYMERIZING BACTERIUM PSEUDOMONAS CEPACIA 253

8 C m .=_

I-"

Indulin

~ B ~A-B

~ 8 5 5 b 1815

1566 1590

, ~848 , ,'o25 1132

1507 1208 5 4 4 8

.obo ~ o o 3doo z5'oo 2doo 1500 I 0 0 0 cm -t 5()O

Wavenumbers

Fig. 5. Fourier transform infrared (FT-IR) spectra of lignin samples from 60-day peptone broth- lignin cultures. A = Uninoculated control. B = P. cepacia DLC-07 inoculated. A - B = Control minus inoculated difference spectrum. Y-axis intensity is in measurements of percent transmittance.

The results show that the cultural conditions employed, as well as the pres- ence or absence of selected co-metabolizable low molecular weight aromatic compounds, markedly influence the extent to which P. cepacia DLC-07 me- tabolizes the soluble lignite coal. The conditions thus far found optimal for depolymerization of the lignite involve growing the cells in peptone broth to which soluble coal polymer has been added. Under the conditions employed, depolymerization was substantial and appeared not to be highly oxidative. Depolymerization probably resulted from cleavage and /o r other modifica- tion of internal bonds such as ester and ether linkages, aromatic nuclei, and internal carbon-carbon bonds. It was clearly not the result of changes in cul- ture pH, as has been observed in some cases for the biosolubilization of coal [2 ]. While the pH values in inoculated cultures rose to about pH 7.6 over the course of 60-day incubations (whereas controls remained unchanged at pH 5.5 ), a set of experiments (data not shown ) where soluble coal polymer was

254 D.L. CRAWFORD AND R.K. GUPTA

incubated in media of varying pH showed that medium pH did not effect the molecular weight of the coal polymer.

Future research will be concentrated on elucidating the chemistry and en- zymology of depolymerization. The effects of co-substrate such as p-coumaric acid and indole should also be examined, and the data obtained may shed light on the enzymatic mechanisms involved. It will also be interesting to de- termine why the coal polymer was depolymerized by P. cepacia DLC-07 while Kraft lignin, in contrast, was further polymerized. Overall, the results indi- cate that this pseudomonad is a promising bacterial strain for use in the non- oxidative biotransformation of coal to lower molecular weight polymers. Such polymers may be useful for further biological conversion to liquids using an- aerobic, reductive microorganisms.

ACKNOWLEDGEMENTS

This research was supported by grant DE-FG22-88PC88919 from the US Department of Energy, Pittsburgh Energy Technology Center, by a subcon- tract from EG&G Idaho, Inc., funded by the US Department of Energy under subcontract DE-AC07-76ID01570, and by the Idaho Agricultural Experi- ment Station.

REFERENCES

1 Gupta, R.K., Deobald, L.A. and Crawford, D.L., 1990. Depolymerization and chemical modification of lignite coal by Pseudomonas cepacia DLC-07. Appl. Biochem. Biotechnol., 24/25:899-911.

2 Gupta, R.K., Spiker, J.K. and Crawford, D.L., 1988. Biotransformation of coal by ligninol- ytic Streptomyces. Can. J. Microbiol., 34: 667-674.

3 Dryden, I.G.C., 1963. In: H.H. Lowry (Editor), Chemistry of Coal Utilization, Suppl. Vol. Wiley, New York, p. 232.

4 Evans, D.G. and Hooper, R.J., 1981. Deduction of the structure of brown coal by reaction with phenol. In: M.L. Gorbaty and K. Ouchi (Editor), Coal Structure. Amer. Chem. Soc., Washington, DC, p. 195.