RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 10,1869-1872 (1996)

Characterization by Pyrolysis/Gas ChromatographyIMass Spectrometry of the Alkalilignin Obtained from Wheat Straw Soda- cooking Effluents After Treatment with Streptomyces Strains J. Rodriguez: M. Hernhdez: P. Bocchini,” G. C. Galiettib and M. E. Ariasa3 * a Departamento de Microbiologia y Parasitologia. Universidad de Alcal.4. 28871 Alcal.4 de Henares. Madrid, Spain

Cenm di studio per la Conservazione dei Foraggi, Consiglio Nazionale delle Ricerehe, via Filippo Re 8,40126 Bologna, Italy

Alkalilignin samples obtained from untreated paper-mill effluent and eftluent decolorized by Streptomyces UAH 30 and Streptomyces UAH 51 were analyzed by pyrolysidgas chromatography/mass spectrometry. The molecular composition of the samples and the chemical modifications occurring in the lignin units were studied in order to know the mechanism of decolorization carried out by these microorganisms. The pyrolysis data showed a notable reduction in the lignin pyrolysis products of alkalilignins in efiiuent decolorized by Streptomyces strains. Moreover, chemical modifications, such as oxidation and shortening of the side chain of the phenyl-propyl moieties of lignin were also detected.

The discharge into natural waters of alkaline-pulping efff uents, originating from straw soda-cooking processes pose an important environmental problem. The large amount of lignin still remaining in these effluents is responsible for a high percentage of their dark brown color, which is not only aesthetically unacceptable but also produces adverse effects on the aquatic ecosystem,‘ Lig- ninolytic fungi’. and actinomycetes4 have been used for decolorization of these effluents because of their capacity to degrade lignin, to some extent, under laboratory conditions. In spite of data published in this field, the nature of the chemical modifications of the lignin-derived compounds of alkaline effluents, as well as the biological mechanism of color removal by fungi and actinomycetes, remain unclear.

In a previous work carried out in our laboratory, several Srrepromyces strains were selected on the basis of their ability to decolorize a paper-mill effluent obtained after semichemical alkaline pulping of wheat straw. Afterwards, a transformation of the high- and medium-molecular weight fragments of lignin was demonstrated to occur during growth of the strain^.^

Several degradative and non-degradative techniques were applied to analyze the chemical changes produced in lignocellulosic materials as a consequence of the growth of different microorganisms.’~ Pyrolysis (PY) is an interesting degradative technique that can be easily coupled to gas chromatography (GC) and/or mass spectrometry (MS). Moreover, PY/GC/MS is a helpful technique for the analysis of a wide variety of complex polymeric materials and is especially appropriate in studies of the chemical characterization of lignin.’ It requires no sample manipula- tion and provides data for different classes of compounds, e.g. sugars, phenolic derivatives and proteins. Therefore, a generalized depolymerization of lignin with minimal gen- eration of artifacts can be attained by analytical PY8

The application of PY/GC/MS in the characterization of alkaline-cooking effluents from wheat straw after treatment with different fungi has contributed to a better under-

* Author for conespondence.

standing of the chemical changes in the lignin-derived compounds found in this type of effl~ent.~ In the present work, PY/GC/MS was applied to alkalilignin samples obtained from effluents decolorized by two Srrepromyces strains, in order to contribute to a better understanding of the strategy used by actinomycetes to transform the main chromophoric groups in these effluents.

EXPERIMENTAL

Microorganisms and culture conditions Two actinomycete strains, identified as Streptomyces UAH 30 and Streptomyces UAH 51, were selected in our laboratory for their ability to remove the color from a paper- mill effluent.‘ Spore suspensions of each strain kept at - 20 “C were used to inoculate GAE agar plates: After 6-7 days of growth in solid medium, the spores were harvested with 0.01% Tween 80 and 200 pL of a standard spore suspension containing 10’ colony-forming units (cfu) were used as inoculum. Cultures were grown in 250mL flasks containing 5OmL of mineral salt medium’ supplemented with 80% (v/v) total effluent, 1% (w/v) of glycerol and 0.2% (w/v) of ammonium sulfate.‘ Cultures were incubated for 7 days at 37 “C for Streptomyces UAH 30 and at 28 “C for Srreprornyces UAH 51, with shaking at 200rpm. Untreated effluent was used as control.

Effluent characteristics The effluent, provided by a Spanish paperboard manu- facturer, was obtained after semichemical alkaline pulping of wheat straw (soda-cook liquor) and anaerobic and aerobic treatments. The color of the effluent was estimated as 15 000 color units at 465 nm measured according to the procedure previously described. l o

Alkalilignin preparation Untreated and decolorized effluents were acidified with 12 M HCI to pH 1-2 and then centrifuged at 12 00Ox g for 10 min. The alkalilignin obtained after centrifugation was

CCC 095 1-4198/96/15 1869- 0 1996 by John Wiley & Sons, Ltd.

Received 22 August I996 Accepted (revised) 30 Ocrober 1996

1870 PYlGClMS OF WHEAT STRAW ALKALILIGNIN

washed with deionized water and freeze dried in a Christ Alpha 1-4 freeze dryer (B. Braun Biotech International) with LDC- 1M controller. Alkalilignin samples were ana- lyzed by the PY/GC/MS technique.

pYrolysis/gas chromatography/mass spectrometry conditions Alkalilignin samples (0.5 mg) were pyrolyzed at 600 "C for 5 s using a platinum heated-filament pyrolyser with a quartz sample holder (CDS Pyroprobe 1OOO; (Chemical data System, Oxford, PA, USA), interfaced to a GUMS system (interface temperature 200 "C) consisting of a Varian 3400 capillary gas chromatograph (Varian Analytical Instru- ments, Walnut Creek, CA, USA) coupled via a transfer line to a Finnigan MAT Magnum ion trap detector (Finnigan MAT, San Jose, CA, USA). The gas chromatograph column (SPB-5; 30 mx 0.32 mm i.d.; 0.25 p n film thickness; from Supelco, Bellafonte, PA, USA) was programmed from 50 to 300 "C at 5 "C min-' holding the initial temperature for 10 min. The injector temperature was 250 "C. The carrier gas was helium (1 mL min- I ) and the split ratio was set at 1/100. The electron impact mass spectra (1 scan s-I) were obtained at 70 eV and recorded from m/z 40 to 450. Lignin pyrolysis products were identified on the basis of standards from the National Bureau of Standards library of spectra, retention times and spectra reported after PY/GC/MS of lignocellulosic material." The relative percentages of pyr- olysis products were calculated from the relative areas of the peaks.

RESULTS AND DISCUSSION The ability of two Sfrepfomyces strains to decolorize a soda- pulping effluent was demonstrated in previous work with the alkalilignin fraction responsible for most of the color of the eff l~ent .~ The application of PY/GC/MS to analyze this fraction should contribute to a better understanding of the mechanism of decolorization by these microorganisms.

The pyrograms of the alkalilignin obtained from untreated effluent and from effluent decolorized after seven days of growth by S. UAH 30 and S. UAH 51 are presented in Fig. 1 The pyrolysis compounds identified in the samples are shown in Table 1. The pyrolysis products identified in an alkalilignin control were essentially the same as those described for similar samples of the same industrial effl~ent.~

Pyrograms showed a large number of typical compounds, mainly derived from the phenylpropanoid lignin units p - hydroxyphenyl (H; 4-4ydroxyphenyl), guaiacyl (G; 4-hydroxy-3-methoxyphenyl) and syringyl (S; 4-hydroxy- 3,5-dimethoxyphenyl), as well as carbohydrates and proteins. The most volatile products, eluting up to scan 160, were not considered in this analysis, since they constitute the low molecular weight compounds produced by pyrolysis and are not characteristic markers.

Lignin is the main component of the alkalilignin samples (Table 2), so a more exhaustive evaluation of the lignin- derived pyrolysis products was made. Lignin-derived products obtained after treatment of the effluent with the two Streptomyces strains were practically the same as those identified in the untreated effluent alkalilignin. However, differences in the relative amount of these pyrolysis products were observed. A reduction of the relative areas of most of the lignin pyrolysis products after treatment of the effluent with two Strepromyces strains was detected. Such a reduction was found to be similar to that described in the

literature for the same effluent after treatment with the ligninolytic fungi Trametes versicolo? and Phanaerochaere ~hrysoporium.~

Among the lignin related pyrolysis compounds detected in the alkalilignin from the effluent decolorized by the strains, a decrease was observed in the relative amounts of 4-vinylguaiacol (4-vinyl-2-methoxyphenol), 4-propyl- guaiacol (4-propyl-2-methoxyphenol), 4-vinyl-2,6- dimethoxyphenol, 4-allyl-2,6-dimethoxyphenol, and cis- and trans-2,6-dimethoxy-4-propenylphenol, with respect to the control. There is also an important decrease in the relative amount of 4-methylguaiacol (4-methyl-2methoxy- phenol) detected in the alkalilignin from the effluent decolorized by S. UAH 30. On the other hand, an increase in the relative amounts of 2,6-dimethoxyphenol and 4-ethyl- 2,6-dimethoxyphenol was detected after treatment with both S. UAH 30 and S. UAH 5 1. In addition, an increase in the relative amount of guaiacol was also detected in the effluent

20

11 14 I z

63%

h c PI

1100

1 14

21w 1 11 1

Figure 1. F'yrograms of alkalilignin from untreated effluent (a), from effluent decolorized by Streptomyces UAH-30 (b), and from effluent decolorized by Srrepromyces UAH 51 (c). Peak numbers correspond to those listed in Table 1.

PYIGCIMS OF WHEAT STRAW ALKALILIGMN 18

Tablel. Compounds identified by GCIMS from the pyrolysis fragments of alkalilignin samples. The pyrolysis fragment intensities are expressed as

NO

1 2 3 4 5 6 7

8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

37

38 39 40

relative percentages Name

2-Furaldehyde Cyclopent- 1 eneA3,4-dione Styrene 2-Methyl-2-cyclopenten- 1 -one 5-Methyl-2-furaldehyde Phenol 4-Hydroxy-5,6-dihydro(2H)- pyran-Zone 2-Methylphenol 2-(propan-2-one) tetrahydrofuran CMethy lphenol Guaiacol 2,6-Dimethylphenol CEth ylphenol 4-Methylguaiacol CWnylphenol 4-Ethyl-2-methyl phenol 3-Methox ycathecol CEthylguaiacol lndole 4-Viny lguaiacol 2,6-Dimethoxyphenol Eugenol CPropylguaiacol cis-Isoeugenol rrans-Isoeugenol Homovanillin Acetovanillone An anhydroglucopyranose 4-Ethyl-2.6-dimethoxyphenol Guiaiac ylacetone 4-Vinyl-2,6-dimethoxyphenol Propiovanillone 4-Allyl-2,6-dimethoxyphenol cis-2,6 Dirnethoxy-4-propenylphenol S yringaldehyde 1-(3,5-Dimethoxy-4- hydroxypheny1)propyne rruns-2,6-Dimethoxy-4- propen y lphenol Acetosyringone Syringylacetone Propios y ringone

Scan MW Originb

163 96 C 225 96 C 237 104 P 262 96 C 394 110 c 494 94 L-HIP 510 114 C

782 108 L-HIP 844 128 C 853 108 L-HIP 859 124 L-G

1039 122 L-H 1087 122 L-H 1124 138 L-G 1201 120 L-H 1232 136 L-H 1266 140 1300 152 L-G 1324 117 P 1364 150 L-G 1432 154 L-S 1442 164 L-G 1459 166 L-G 1533 164 L-G 1593 164 L-G 1604 166 L-G 1648 166 L-G 1666 162 C

1716 180 L-G 1713 182 L-S

1770 180 L-S 1792 180 L-G 1826 194 L-S 1895 194 L-S 1902 182 L-S 1922 192 L-S

1960 194 L-S

2001 196 L-S 2054 210 L-S 2123 210 L-S

AL-C AL3W AL-SI'

0.92 0.91 0.53 0.06 0.05 0.li 1.08 1.18 1.87 0.17 0.25 0.25 0.38 0.27 0 4.25 6.62 9.08 0.29 0 0.21

0.84 0.9 1.12 0.27 0.09 0.21 3.41 4.11 8.55 7.46 7.35 8.7 0.87 0.72 1.02 2.15 2.66 3.25 6.2 3.7 5.87 4.29 5.08 4.23 0.58 0.44 0.37 3.12 3.5 2.73 3.61 3.69 3.46 0.45 2.13 3.35

16.46 12.48 11.87 5.05 7.24 7.48 0.5 0.33 0.29 0.53 0.39 0.44 0.01 0.05 0.01 3 1.72 1.74 0.03 0 0.01 1.18 2.32 2.06 0.03 2.81 2.25 1.03 1.57 1.31

4.25 3.71 4.01

0.61 0.32 0.32 0.68 0.29 0.29 0.81 0.36 0.24 0.2 0.2 0.22

8.51 4.38 3.34

2.45 0.82 0.79

3.72 1.56 1.45

6.36 4.77 3.7 2.29 9.78 2.4 1.9 1.18 0.87

a Molecular weight (MW). Origin. L: Lignin; C: carbohydrates; P protein; H: p-hydroxyphenyl; G; guaiacyl; S: syringyl. AL-C: alkalilignin from untreated effluent. AL-30: alkalilignin from effluent decolorized by Srrepromyces UAH 30 ' AL-51: alkalilignin from effluent decolorized by Srreptomyces UAH 51

Table 2. Percentage of carbohydrate, protein and lignin-derived pyrolysis products from afka- lilignin samples Carbohydrate Protein Lignin H:G:S' S/G

Sample products pducts products ratio mu0

bAL-C 2.12 1.53 93.19 18:53:29 0.53 'AL-30 3.89 3.31 89.23 23:42:35 0.83 dAL-51 3.37 5.22 88.67 31:44:25 0.56

a p-hydroxyphenyl : guaiacyl : syringyl

' AL-30 alkalilignin from efff hent decolorized by Sfrepfomyces UAH 30.

AL-5 1: alkalilignin from effluent decolorized by Srrepromyces UAH 51.

AL-C: alkalilignin from untreated effluent.

1872 PYIGCiMS OF WHEAT STRAW ALKALILIGNIN

alkalilignin obtained with S. UAH 5 1. These modifications could be attributed 10 the alteration of the side-chain phenyl-propyl (c6-c3) mc:<ties of the lignin, which are transformed into phenyl-ethyl-type (C6-C2), phenyl- methyl-type (c&[) and phenyl-type (c6) products. A similar pattern of break down of the side-chain of the c3-c6 moieties has been reported with ligninolytic fungi.3

It should also be emphasized that an oxidation of the C3- alkyl chain of the G- and S-units took place during the decolorization process. This statement can be supported by the increase in the relative amounts of syringylacetone ([4-hydroxy-3,5-dimethoxyphenyl]propanone) and acetova- nillone ([4-hydroxy-3-methoxyphenyl] methyl ketone) in the alkalilignin from the effluent decolorized by the strain S. UAH 30 and acetovanillone in the alkalilignin from S. UAH 51. In addition, the decrease in the relative amounts of guaiacylacetone ([4-hydroxy-3-methoxyphenyl]propa- none), acetosyringone ([4-hydroxy-3,5-dimethoxy]methyl ketone), syringylaldehyde (4-hydroxy-3S-dimethoxy ben- zaldehyde), and propiosyringone ([4-hydroxy-3,5-di- methoxyphenyllethyl ketone) could indicate the possible degradation of these compounds by both strains.

The H:G:S ratio of the alkalilignin from the treated effluent is shown in Table 2. A decrease in the G-units content of the alkalilignin effluents, decolorized by the strains S. UAH 30 and S. UAH 51 and a decrease in the S- units in the effluent, decolorized by the strain S. UAH 51, was observed. This result seemed to indicate that S. UAH 30 is, to some extent, able to degrade the G-units, in spite of their high resistance to degradation, and that S. UAH 5 1 can degrade G- and S-units. The chemical modifications in the alkalilignin fraction from paper-mill effluent as demon- strated by PY/GC/MS could be attributed to the production of oxidative enzymes during the growth of the strains. Recently, it has been demonstrated that Streptomyces UAH 30 produces several peroxidase activities,'* but data con- cerning the role of these enzymes in the decolorization process have not been provided yet. However, it is known that ligninolytic enzymes, such as peroxidases and laccases, are involved in the decolorization of paper mill effluents by white rot fungi.13. I4 Additional studies should be performed with Streptomyces selected strains to determine the possible involvement of oxidative enzymes in the decolorization of paper-mill effluents.

In conclusion. PY/GC/MS was found to be a suitable

technique to analyze the transformation produced by the biological action of Strepfomyces on alkaline paper-mill effluent derived from lignocellulosic residues. The PY/GC/ MS results obtained after treatment of the paper-mill effluent with Streptomyces strains showed an alteration of the lignin-derived compounds compared to the untreated effluent. The decolorization process carried out by the strains S. UAH 30 and S. UAH 51 could be mainly attributed to a significant reduction in the alkalilignin content and a concomitant chemical modification of the residual lignin.

Acknowledgements This research was in part supported by the EU Project ECLAIR, AGRE- CT90-0044. A grant for collaboration between CNR (Italy) and CIB (Spain) funded the stay of J.R.B. in Centro di Studio per la Conservazione dei Foraggi, CNR Bologna to perform PYiGClMS analyses and that of P.B. in the CIB to process the results. We also wish to thank to Dr. A. Gonzhlez from CIB for his invaluable collaboration.

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