Journal of Chemical Ecology, Vol. 21, No, 8, 1995

COMPARISON OF ANTIALGAL ACTIVITY OF BROWN-ROTTED AND WHITE-ROTTED WOOD AND IN SITU ANALYSIS OF LIGNIN

J.M. P I L L I N G E R , 1'2'* I. G I L M O U R , 2 and I. RIDGE I ~Department of Biology 2Department of Earth Sciences Open Univers#y, Walton Hall Milton Keynes, MK7 6AA. U.K.

(Received August 1 1994; accepted March 15 t995) Abstract--Brown-rotted wood has been used as a source of lignin to investigate further the antialgal effects of lignocellutosic materials such as decomposing barley straw. The antialgal activity of brown-rotted and white-rotted wood has been determined in a laboratory bioassay. Using pyrolysis gas chromatography-mass spectrometry, the lignin of the rotted wood samples has been compared and the significance of the structure of the lignin in antialgal activity is discussed. Key Words--Antialgal compounds, brown-rotted wood, white-rotted wood, lignin, macromolecular analysis, pyrolysis GC-MS, Chlorella. Microcystis.

INTRODUCTION

Barley straw rotting, submerged and well-aerated, in water has been shown to release substances that inhibit the growth of a range of algae and cyanobacteria (Gibson et al., 1990; Newman and Barrett, 1993). Indeed, this effect is being exploited widely for the control of nuisance algae in waterbodies. We have suggested that the antialgal factor(s) is derived directly from the straw per se (Pillinger et al., 1992) and may be phenolic, particularly polyphenolic, in origin (Pillinger et al., 1994). Straw contains some 15 % by weight of the polyphenolic structural material lignin. We report the effect of lignin on the growth of an alga and a cyanobacterium in a laboratory bioassay. Unfortunately, no method is available that permits the extraction of total *To whom correspondence should be addressed.

1113 [X)98-0331/95/0800-1113507.50/0 '~.~ 1995 Picnum Publishing Corporation

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PILL1NOER ET AL.

lignin (Lewis and Yamamoto, 1990); different forms of lignin reflect the method by which they have been isolated. The lignin extract that best reflects unchanged native lignocellulose is "enzymatically liberated lignin" (Ander and Eriksson, 1975), formed by the action of brown-rot fungi on lignocellulose, whereby cellulose is preferentially utilized leaving a brown powder highly enriched in lignin (Kirk and Adler, 1970). Brown-rotted wood is readily available in nature, and for this reason we have investigated initially the effect of lignin derived from wood rather than from straw. Although brown-rot lignin is representative of the original lignin of the wood, minor chemical changes cannot be ruled out (Kirk and Adler, 1970). On the other hand, a group of specialized fungi that are able to effect white-rotting of lignocellulose substrates do so by metabolizing lignin. The action of fungi on wood has been reviewed by Levy (1987). Mulder et al. (1991) demonstrated that a thoroughly white-rotted wood did not contain lignin moieties, only those of cellulose. We thus chose to use white-rotted wood in antialgal tests as a control to the lignin-enriched brown-rotted wood. We have used on-line pyrolysis gas chromatography-mass spectrometry (Py-GC-MS) to compare the lignin in each of the rotted wood samples. This technique increasingly is being employed to study lignocellulose (e.g., Scheijen and Boon, 1991; Galletti and Reeves, 199I) and was recently summarized by Boon (1989). Macromolecular lignin-rich material can be analyzed directly avoiding selective (Lapierre et al., 1989) and time-consuming chemical extractions. The solid sample is thermally disrupted in the absence of air, the subcomponents separated by conventional gas chromatography, and identified by their characteristic mass spectra. M E T H O D S AND M A T E R I A L S

Algal Toxici~ Experiments. A unicellular green alga, Chlorella vulgaris Beijerinck (CCAP 211/12), and the cyanobacterium (blue-green alga) Microcystis aeruginosa Kutz, emend Elenkin 1924 (CCAP 1450/6), were maintained in liquid culture as below with weekly subculturing. The growth of either organism in the absence (control) and presence of rotted wood (see Table 1 below for details) in algal culture medium was assessed using a laboratory bioassay (Pillinger, 1993). Briefly, an aliquot (1 ml) of stock culture in the exponential phase of growth was added to 50 ml modified Jaworski's medium, buffered with 20 mM HEPES at pH 8.2, in a conical flask and incubated at random in continuous light at 20°C with daily shaking to maintain aeration. All controls and treatments were replicated five times. A comparison of the growth of the organisms between the controls and treatments was obtained by quantifying the chlorophyll a content after three to four days of incubation. The absorbance due to chlorophyll a of a methanolic extract (10 ml) of the dry biomass retained on

ANTIALGAL ACTIVITY OF ROTTED WOOD

1 1 15

a glass-fiber filter (pore size 1.2/~m) was measured at 665 nm (less a background reading taken at 750 nm). In some experiments the growth of Chlorella was measured by counting algal cells using a hemacytometer. Preliminary data indicated a linear correlation between the two methods. All data are reported as percentage of the control growth, allowing comparison between experiments conducted under slightly varying conditions, and, where quoted, standard errors of the mean of the normalized values. The significance of the treatment value (N = 5) as compared to the respective control (N = 5) was determined by an unpaired t test. Analytical Pyrolysis Gas Chromatography-Mass Spectrometry.. A sample of dried, pulverized wood of between 1 and 2 mg was weighed accurately into a Pelletiser (Scientific Glass Engineering, SGE, Milton Keynes, U.K.) and gently compressed. The pellet was introduced into the quartz liner of the furnace (SGE Pyrolyser) maintained at a preset temperature (500°C). A slight positive pressure of helium in the furnace keeps pyrolysis time constant and reduces secondary pyrolysis; pyrolysis products were thus swept onto the column of the gas chromatograph (GC). The GC (Hewlett Packard HP5890 Series II) was fitted with a WCOT 50-m x 0.32-mm capillary column column coated with 0.5/~m film of 5% phenyl methyl silicone. The initial oven temperature was set at 50°C and programmed to rise at 10°C/rain to 100°C after a l-rain hold, then at 5°C/min to 300°C, after which it was isothermal; the complete run lasted for 60 min. Pyrolysis subcomponents were detected using a mass selective detector (HP 5971 series MSD); electron impact ionizing voltage was 70eV with continuous scanning from 50 to 500 mass units. Identification was based on computer matching of mass spectra. The experimental conditions, including the temperature of pyrolysis, gave an acceptable separation of the subcomponents. Blank runs confirmed that memory effects of the pyrolyzer system were negligible. Preliminary analyses indicated a good measure of reproducibility with a kraft lignin standard, Indulin AT (Sigma), and replicate analyses of a white-rotted wood sample (Pillinger, 1993). Pyrolysis is not a highly quantitative method (Faix et al., 1987). However, where similar weights of comparable samples are pyrolyzed under identical conditions within a short time period to reduce effects of instrumental variability, then a quantitative estimate of the various pyrolysis fragments can be made. This was achieved by determining the computer-integrated peak areas of interest as a ratio of a chosen peak that occurred in each sample.

RESULTS

Antialgal Effect of Brown- and White-Rotted Wood. Both brown-rotted (BRW) and white-rotted (WRW) sycamore wood were significantly (t test, P

1116

PtLLINGER

ET AL.

120 l l brown-rotted l [] white-rotted

I

I00-

8O -

6O 4O

2

2

2

2*

5

5

5*

10

10

10"

wt of wood (g/l) FIG. 1. Effect of brown- and white-rotted sycamore wood on growth of Chlorella in vitro. Algal growth quantified by cell counts. The asterisk denotes that the wood was autoclaved in the algal medium prior to the bioassay, otherwise wood was tested without autoclaving. All treatments (N = 2) significantly (t test, P < 0.001) inhibitory compared to controls (N = 2).

< 0.001) inhibitory to the growth of Chlorella, although it can be seen from Figure 1 that BRW was consistently more inhibitory than WRW. The results shown in Figure t represent the data from duplicate experiments in which wood was included in the algal bioassay at a dose equivalent to 2, 5, and I0 g/liter (g/dm 3) respectively. In a third experiment (denoted by an asterisk in Figure 1), similar dose rates of wood were used, but the wood was autoclaved in the algal bioassay medium prior to the experiment. Autoclaving increased the antialgal activity of both types of wood, discernible only at the lower dose rates. Microscopic examination of the W R W (sycamore) indicated that the sample was not homogeneously rotted. When compared to the data for sycamore, a greater antialgal effect of brown- and white-rotted elm was recorded and thus the elm samples were selected for chemical analysis. The latter sample was well rotted and homogeneous on microscopic examination. W R W (elm) was not significantly inhibitory compared to control growth at the three dose rates tested (1, 2, and 4 g/liter whereas BRW (elm) was highly inhibitory to Chlorella (Figure 2). In all cases, the wood was autoclaved in the medium previously shown (Figure 1) to result in greater algal toxicity than with wood not exposed to the period of hot aqueous extraction afforded by autoclaving.

1l 17

ANTIALGAL ACTIVITY OF ROTTED WOOD

120 mR brown-rotted ] f"l white-rotted 100-

..c A

8(I -

60-

8 40-

2(t

0 i

2

4

wt of wood (g/l)

FIC. 2. Effect of brown- and white-rotted elm wood on growth of Chlorella in vitro. Algal growth quantified by chlorophyll a estimation.

A heartwood sample from the white-rotted elm trunk (Table 1) was quite brown in color, although the wood had the texture of WRW. The lack of antialgal activity of the elm heartwood sample was similar to that of the whiterotted fraction, namely 1 g/liter wood resulted in 140% (SE 4-14.7) of the control growth; 2 g/liter in 130% (12.6); and 4 g/liter in 98% (6.0). On autoclaving the elm heartwood sample in algal medium prior to the bioassay, a brown coloration was imparted to the medium as occurred also with BRW. The resulting inactivity of the white-rotted, but brown-colored, elm wood towards Chlorella indicated that reduced light penetration was not responsible for the algal toxicity observed with BRW. Two further white-rotted wood samples, birch and hazel, were not inhibitory to Chlorella (Table 2). However, both samples were inhibitory to the growth of the cyanobacterium Microcystis (Table 2). The data in Table 2 also show the results of the bioassay of W R W (elm) against Microcvstis; it can be seen that this white-rotted sample, which was not toxic towards Chlorella (Figure 2), significantly inhibited the growth of Micocystis at the same three dose rates. Pyrolysis GC-MS of Wood. Quantitative and qualitative differences in the lignin of brown- and white-rotted woods were determined using pyrolysis GCMS. Figure 3 shows the total ion current (TIC) chromatogram, or pyrogram, generated by the mass spectrometer in response to the intensity of ions of the

PILLINGER ET AL.

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TABLE 1. PROVENANCE AND DESCRIPTION OF WOOD SAMPLES a

Wood sample Sycamore, Acer pseud~olatamus L. Elm, Ulmus minor Miller

Location, UK

Type of rot

old woodland, Lancashire old woodland, Lancashire

brown white (partial)

old woodland, Cambridgeshire old farmland hedge, Cambridgeshire old farmland hedge, Cambridgeshire

brown white (well-rotted) heartwood (brown) from white (well-rotted) log white (well-rotted). Coriolus (Trametes) versicolor fruiting on surface white (well-rotted) under bark, Merulius corium fruiting on surface

Birch, Betula sp.

parkland, Berkshire

Hazel, Cor3.,lus avellana L.

twig from parkland, Berkshire

"All samples were air dried, homogenized in a laboratory blender, and sieved to < 2 ram. Samples for pyrolysis GC-MS were further sieved to < 125 #m and stored in glass vials in a desiccator.

TABLE 2. EFFECT OF WHITE-ROTTED WOOD ON GROWTH OF Chlorella AND Microcvstis

Wood

Weight of wood (g/liter) in algal medium

Elm (brown-colored heartwood of sample above)

1 2 4 I 2 4 1 2 4 I 2 4

Birch

Hazel

Elm

% of control growth ( ±SE)" Microeystis

Chlore|la 102 109 10t 174 nd nd 90 t' > 120~' 103 ~' 140 130 98

(8.2) (3.9) (9.5) (12.3)

(3.4) (5.8) (6.6) (14.7) (12,6) (6.0)

14 8 8 9 5 I 60 I0 2 nd' nd nd

(3.7) (1.5) (2.4) (3.21 (1.4) (0.7) (4.8) (3.8) (0.2)

"Algal growth quantified by chlorophyll a estimation. Chlorella, no significant inhibition (P > 0.05); Microo,stis, all data are significantly different from controls (P < 0,001), bData shown in Figure 2. "nd = not determined.

3,

and

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mg.

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50.00

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of WRW

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Weight

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Brown-rotted wood

refer to Figure

elm

35.00

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White-rotted wood

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white-rotted

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1 t20

PILLINGER ET AL.

various pyrolysis subcomponents of the white-rotted and brown-rotted elm samples. The trace for the W R W (elm) shows pyrolytically produced subcomponents of short retention time, which, together with their mass spectra, indicates they were derived from polysaccharide (Boon, 1989; Kivaisi et al., 1990). Such peaks were typically lacking from the BRW (elm) pyrogram. In BRW (elm) and W R W (elm), the area of the peak due to the simplest monomethoxy fragment, methoxy phenol or guaiacol, was remarkably similar (peak number 2 on Figure 3). Thus the integrated areas of other peaks for which spectra were obtained were normalized to that of methoxy phenol. Figure 4 graphically shows the normalized distribution of the 16 major peaks in the BRW (elm) sample compared to the normalized values of the peaks of the same (as determined by their mass spectra) fragments where present in WRW (elm). The tentative identification of the peaks is given in Table 3. The convention of describing lignin subcomponents as either G (guaiacyl unit, with one methoxy group) or S (syringyl unit, dimethoxylated) has been adopted. It can be seen from Figure 4 that peak 6 (2,6-dimethoxyphenol) was some six times more abundant in BRW than WRW. A greater abundance of dimethoxy to monomethoxy substitution was reflected in the overall pattern of the BRW compared to WRW. The ratio of S/G lignin subcomponents in BRW (elm) was

peak number

9" 8'~ 7 ' ~r

~ f

5' 3: 2" 1

r I

0

i 100

j 20(1

3(X)

estimation of peak area (%}

FJc. 4. Comparison of peak areas obtained by pyrolysis GC-MS of brown-rotted and white-rotted elm wood. Computer-integrated peak areas are normalized to the area of peak number 2, identified as methoxyphenol, arbitrarily set at 100%. Peaks 1-16 refer to the pyrograms shown in Figure 3 and identification is shown in Table 3.

ANTIALGAL ACTIVITY OF ROTTED WOOD

1121

TABLE 3. IDENTIFICATION OF PEAKS 1N PYROGRAM OF B R W ELM

Retention time (rain)

M +''

Base peak ~'

2 3 4 5 6 7 8 9 10 1I

5.08 13,76 16+67 19.02 20.00 20.85 22.63 23.52 23.70 24.83 25.57

60 124 138 152 150 154 152 168 164 166 182

109 123 137 150 154 152 168 164 166 182

12 13

26.60 29.18

180 182

180 182

14 15

29.92 30.83

194 196

194 t81

16

31.62

212

167

Peak number 1

Other peak

95 135 139 151 153 149 151 167 165

G or S'

G G G G S G S G G S S S S S

181

S

Identification or partial identification a unidentified methoxyphenol 2 - m e t h o x y ~ - m e t h y l phenol G + 2 m or G + e G + v 2,6-dimethoxypheno[ G + 2 morG + e S + m" or 3 0 M e 2 -methoxy~--( 1-propenyl) phenol G + ( 2 m + e) o r G + 3 m 4-hydroxy-3,5-dimethoxy benzaldehyde S + v 4-hydroxy-3,5+dimethoxy benzaldehyde 2,6-dimethoxy-4-(2-propenyl) phenol l-(4-hydroxy-3,5-dimethoxyphenyl) ethanone unidentified

" M + = molecular ion, equivalent to molecular weight ~'Base peak = the peak of greatest intensity in the mass spectrum+ other significant peaks aid interpretation ' G , guaiacyl: S, syringyl. '~m = methyl (adds 14 mass units to G or S), e = ethyl (adds 28 mass units to G or SL h = hydroxyl (adds 16 mass units to G or S L v = vinyl (adds 26 mass units to G or S), p = propenyl (adds 40 mass units to G or S). "Computer matched as 1,2,3-trimethoxy benzene (93% certainty).

calculated as 1.8, whereas in the WRW (elm) it was 0.3. Both hazel and birch WRW samples had S/G ratios of less than 0+5. The heartwood sample of whiterotted elm, which was ineffective against Chlorella, had a pyrogram typical of the WRW (elm). The S/G ratio of the elm heartwood sample was calculated to be 0.1, consistent with depletion of syringyl lignin following white-rotting, irrespective of the brown coloration typical of BRW. DISCUSSION

Brown-rotted wood, elm or sycamore, was consistently more inhibitory to the growth of Chlorella than was white-rotted wood. The partial inhibitory

1122

PILLINGER ET AL.

effects of sycamore WRW may be explained by the poor degree of homogeneity of the white-rotting as noted on microscopic examination. Extensively whiterotted wood (three species) did not prevent the growth of Chlorella. Based solely on the response of Chlorella, it is tempting to conclude that the lignin enrichment of BRW is responsible for antialgal activity. These data support our hypothesis that, of the lignocellulose content of straw, it is indeed the lignin portion that has antialgal potential. White-rotted wood (three species), however, significantly prevented the growth of Microcystis. Cyanobacteria are reportedly more sensitive than green algae to barley straw (Newman and Barrett, 1993), although the possibility that the prokaryote and eukaryote are adversely affected by different factors cannot be ignored. Pyrolysis GC-MS has allowed us to compare the lignin from a range of rotted wood samples. In general, components with short retention times and mass spectral identification indicating a polysaccharide origin were recorded only in the WRW samples, consistent with the typical action of white-rot fungi, which deplete lignin while preserving holocellulose. Lignin-derived subcomponents were observed in both BRW and WRW; the latter results are in contradiction to those reported by Mulder et al. (1991). A measure of the extent of white-rotting is obviously critical for the interpretation of analytical data relating to the persistence of ligninaceous material. Analysis by pyrolysis GC-MS does not allow us to quantify the total phenolic macromolecular material remaining in the two types of rotted wood, so we cannot discount the possibility that antialgal activity is dependent only on the amount of total lignin, the more resistant Chlorella being unaffected by lower levels of lignin in WRW. However, it is also possible that the structural difference in lignin as identified by Py GC-MS is a significant factor in determining antialgal activity. The pyrograms of the elm wood, rotted in the two distinct fashions, allows a comparison of the nature of the lignin in each. BRW [regarded as closely repesentative of the native (unrotted) wood lignin} shows a high proportion of syringyl to guaiacyl lignin subcomponents, presumably reflecting the distribution of S to G lignin prior to pyrolysis. The action of brown-rot fungi in modifying the native lignin (Mulder et al., 1991; Kirk and Alder, 1970) may account partially for an increase in S units reflected in the S/G ratio of BRW. The pyrogvam of WRW, on the other hand, indicates the presence of G units of lignin. Overall the S/G ratio of the two elm samples, supported by the data from other WRW samples, reflects the structurally different lignin resulting from brown- or white-rot processes. The preferential utilization of syringyl lignin during white-rotting, which is limited to specialized fungi, is well documented (Dare et al., 1988). Niemann et al. (1991), however, have suggested that overall reduction in S lignin in some cases may result from the fungal preference for secondary cell walls, which are

ANTIALGAL ACTIVITY OF ROTTED WOOD

1123

higher in S lignin than primary cell walls, rather than specific action on S lignin, Likewise chemical extraction is selective; Scheijen and Boon (1991) cautioned that the Bjorkman method preferentially extracts secondary cell walls high in S lignin units. Such reports underline the value of the in toto analysis afforded by Py-GC-MS. Under alkaline and oxygenated conditions, precisely those that exist in both field sites and laboratory bioassays where the antialgal effect of barley straw has been demonstrated, phenolic groups would readily autoxidize to quinones that are antialgal (Pillinger et al., 1994). We have proposed (Pillinger et al., 1994) that the process of oxidation of phenolic hydroxy groups increases the antialgal activity of phenolic or polyphenolic substrates. Detailed studies of chemical processes associated with wood pulping have shown that syringyl lignin components have a greater tendency to undergo abiotic oxidation than do guaiacyl units (Kemp( and Dence, 1975). Furthermore, the toxicity of a range of phenylpropanoid compounds towards a green alga, Selenastrum capricornutum, increased with increasing methoxy substitution, and the position of the methoxy groups also influenced sensitivity (Della Greca et al., 1992). The digestibility of forage crops containing similar amounts of lignin was found to be greater in those feeds with lower syringyl lignin (Jung and Himmelbach, 1989 and references therein), suggesting that microbial action is reduced by the dimethoxy substitution of the lignin. It is conceivable, therefore, that lignin with increased dimethoxy groups as compared to a high proportion of monomethoxy substitution could be a significant factor in the efficacy of lignin to reduce the growth rate of algae and cyanobacteria. The greater antialgal activity shown by brownrotted wood over white-rotted wood may be accounted tbr, at least partially, not only by the total amount of lignin present, but also by the increased potential for oxidation offered by syringyl lignin compared to guaiacyl lignin, Softwood lignin is typically of the guaiacyl type, with negligible dimethoxy substitution (Sarkanen and Ludwig, 1971); preliminary studies in our laboratory suggest that brown-rotted conifer wood is indeed less toxic towards Chlorella than that of hardwoods.

CONCLUSIONS

Brown-rotted wood inhibits the growth of two freshwater microorganisms, a green alga, Chlorella, and a cyanobacterium, Microcystis. White-rotted wood, however, was not inhibitory to the green alga but significantly inhibited growth of the cyanobacterium. Lignin was still present in the white-rotted samples, detected by on-line pyrolysis followed by combined gas chromatography-mass spectrometry. Quantification of data from pyrolysis studies must be treated with caution, but nevertheless the method provided a valuable comparison of the type

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PILLINGER ET AL.

o f l i g n i n in s i m i l a r s a m p l e s . T h e l i g n i n r e m a i n i n g in t h e w h i t e - r o t t e d s a m p l e s h a d a l o w e r p r o p o r t i o n o f s y r i n g y l ( d i m e t h o x y s u b s t i t u t e d ) to g u a i a c y l l i g n i n t h a n in t h e b r o w n - r o t t e d w o o d . A p p a r e n t l y , l i g n i n rich in s y r i n g y l u n i t s s h o w s greater antialgal activity, possibily as a result of the greater case of phenolic o x i d a t i o n a f f o r d e d by t h e p r e s e n c e o f d i m e t h o x y g r o u p s c o m p a r e d to m o n o methoxy ones. Acknowledgments--Dr. J.A. Cooper is thanked for helpful discussions and mycological advice and Dr. LR. Newman for continuing collaboration and critically reading the manuscript. John Walters and Tina Wardhaugh are thanked For biological technical support. We thank two anonymous reviewers for their thoughtful comments.

REFERENCES

ANDER,P., and ERIKSSON,K.E. 1975. Influence of carbohydrate on lignin degradation by the whiterot fungus Sporotrichum pulverulentum. Sven. Papperstidn~ 8:643-652. BOON, J,J. 1989. An introduction to pyrolysis mass spectrometry of lignocellulosic material: Case studies on barley straw, corn stem and Agropyron, pp. 25-29, in A. Chesson and E.R. Orksov (eds.). Physicochemical Characterisation of Plant Residues for Industrial and Feed Use. Elsevier Applied Science, London, DARE, P.H., CLARK, T.A., and CHL;-CHou, M. 1988. Consumption of substrate components by the cultivated mushroom Lentinus edodes during growth and fruiting on softwood and hardwood-based media. Process Biochem. October: 156-160. DELLA GRECA, M., MONACO, P., POLLIO, A.. and PREVlTERA~ L, 1992. Structure-activity relationships of phenyipropanoids as growth inhibitors of the green alga Setenastrum capricornuturn. Phytochemist O' 3 t :4119-4123. FAiX, O., MEIER, D., and GROBE, I. 1987. Studies on isolated lignins and lignins in woody materials by pyrolysis-gas chromatography-naass spectrometry and off-line pyrolysis-gas chromatography with flame ionisation detection. J. Anal. Appl. Pyrol. 11:403-416. GALLETn, G.C., and REEVES, J.B.. I11. 1991. Characterisation of beech milted wood Iignin by pyrolysis-gas chromatography-photoionisation mass spectrometry. Anal. Chem. 59:508-513. GIBSON, M,T., WELCH, I.M., BARRE'Wq,P.R.F., and RIDGE, I. 1990. Barley straw as an inhibitor of algal growth II: laboratory studies. J. Appl. Phycol. 2:241-248. JUNG, H.-J.G., and HIMMELSBACH,D.S. 1989. Isolation and characterisation of wheat straw lignin. J. SoL Food Chem. 37:81-87. KEMPF. A.W., and DENCE, C.W. 1975. The reactions of hardwood lignin model compounds with alkaline hydrogen peroxide. Tappi 58: 104-108. KIRK, T,K., and ADLER, E. 1970. Methoxyl-deficient structural elements in lignin of sweetgum decayed by a brown-hot fungus~ A¢'ta. Chem. &'and. 24:3379-3390. KIVAISI, A.K., OP DEN CAMP, H.J.M,, LUBBERDING,H.J., BOON, J.J., and VOGELS, G.D. 1990. Generation of soluble lignin-derived compounds during degradation of barley straw in an artificial rumen reactor. Appl. Microbiol. Biotechnol. 33:93-98. LAPIERRE,C., JOUIN, D., and MONTIES, B. 1989. Lignin characterisation of wheat straw samples as determined by chemical degradation procedures, pp. 118-130, in A. Chesson and E.R. Orksov (eds.) Physicochemical Characterisation of Plant Residues for Industrial and Feed Use. Elsevier Applied Science, London~ LEvy. J.F. 1987. The natural history of the degradation of wood. Phil. Trans. R. Soc, 1.zmdon A 321:423-433.

ANTIALGAL ACTIVITY OF ROTTED WOOD

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LEWIS, N.G., and YAMAMOTO,E. 1990. Lignin: Occurrence, biogenesis and biodegradation. Annu. Rev. Plant Physiol. Plant Mol. Biol. 41:455-496. MULDER, M.M., POREVEEN,J.B.M., BOON, J.J., and MARTINEZ,A,T. 1991. An analytical pyrolysis mass spectrometric study of Euc~.phia eordifolia wood decayed by white-rot and brown-rot fungi. J. Anal. Appl. Pyrol. 19:175-191, NEWMAN, J.R., and BARRE'C'r, P.R.F. 1993. Control of Microcystis aeruginosa by decomposing barley straw. J. Aq. Plant Manage. 31:203-206, NIEMANN, G,J., BOOS, J.J,, PURVEEN. J.B.M_ EIJKEL, G B . , and VAN DER HEIJDEN, E. 1991. A microanalytical approach to plant tissue characterisation: A comparative study of healthy and fungus-infected carnation by pyrolysis-mass spectrometry, J~ Anal. AppL Pyrol. 19:213-236. PtLLISGER, J.M. 1993. Algal control by barley straw: An interdisciplinary study. PhD thesis. Open University, Milton Keynes, U K . PILL1NGER,J.M., COOPER, J.A., RIDGE, I., and BARRET'r, P,R.F. 1992. Bailey straw as an inhibitor of algal growth 1II: The role of fungal decomposition. J. Appl. Phycol. 4:353-355. PIt,LINGER, J.M., COOPER, J,A., and RIDGE, I, 1994. Role of phenolic compounds in the antialgal activity of barley straw. J. Chem. Ecol. 20:1557-1569. SARKANEN,K.V., and LUDWIG, C.H. (eds.). 1971. Lignins: Occurrence, Formation, Structure and Reactions, Wiley Interscience, New York. SCHEIJES, M,A., and Boos, J.J. t991. Microanalytical investigations on lignin in enzyme-digested tobacco lamina and midrib using pyrolysis-mass spectrometry and Curie point pyrolysis-gas chromatography/mass spectromelry. J. Anal. Appl. t~vrol. 19:153-173.

Comparison of antialgal activity of brown-rotted and white-rotted wood andIn situ analysis of lignin.

Brown-rotted wood has been used as a source of lignin to investigate further the antialgal effects of lignocellulosic materials such as decomposing ba...
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