Products of Wheat Straw Biodegradation by Cyathus

19

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on May 14, 2018 | https://pubs.acs.org Publication Date: April 11, 1983 | doi: 10.1021/bk-1983-0214.ch019

Products of Wheat Straw Biodegradation by Cyathus stercoreus T H O M A S P. A B B O T T , C H R I S T O P H E R R O N A L D D. P L A T T N E R

J A M E S , and

U.S. Department of Agriculture, Agricultural Research Service, Northern Regional Research Center, Peoria, IL 61604

The residual biodegradation products of wheat straw lignin attacked by Cyathus stercoreus for thirty days were identified. Low-molecualr-weight nonvolatile materials constituted less than 5% of the total C released from labeled lignin. In this portion, syringic acid, vanillic acid, p-hydroxy benzoic acid, acetic acid, and 2-methoxy-succinic acid were found. The majority of the water-soluble biodegradation products (89%) are lignin-carbohydrate complexes with molecular weights greater than 1000. Analysis of the gases resulting from lignin breakdown and metabolism reveals primarilyCO with smaller amounts of ethanol, methanol, and acetone. The gas phase, flushed out with air and recovered, constitutes 50% of the total lignin breakdown products. 14

2,

This chapter not subject to U.S. copyright. Published 1983, American Chemical Society

Furda; Unconventional Sources of Dietary Fiber ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


268

UNCONVENTIONAL SOURCES OF DIETARY FIBER

In the f i e l d of biomass u t i l i z a t i o n , separating l i g n i n from c e l l u l o s e and h e m i c e l l u l o s e by b i o l o g i c a l means has aroused a great deal of i n t e r e s t . Degradation of l i g n i n during the process of s e p a r a t i o n i s not n e c e s s a r i l y detrimental i f the l i g n i n can be converted to u s e f u l chemicals. To f u l l y understand the b i o l o g i c a l approach to modifying and s o l u b i l i z i n g l i g n i n , the biodégradation products of l i g n i n must be i d e n t i f i e d and quantitated. Cyathus stercoreus (Schw.) de Toni NRRL 6473 degrades l i g n i n p r e f e r e n t i a l l y during a 62-day fermentation of wheat straw, but the biodégradation products have not been c h a r a c t e r i z e d (1). Other workers have i d e n t i f i e d r e s i d u a l phenolics i n biodégradation broths of s e v e r a l woods and s y n t h e t i c or i s o l a t e d s o l u b l e l i g n i n s (2). More recent p u b l i c a t i o n s have i d e n t i f i e d f u n c t i o n a l group changes i n degraded l i g n i n s rather than s p e c i f i c low molecular weight compounds (3). A l l e s s e n t i a l l y i d e n t i f y o x i d i z e d species as a r e s u l t of fungal or b a c t e r i a l degradation. Current knowledge i n l i g n i n biodégradation supports a mechanism of random o x i d a t i o n , generating aromatic compounds with v a r i o u s oxygen-containing s i d e groups, a l i p h a t i c c a r b o x y l i c a c i d s from subsequent r i n g cleavage and u l t i m a t e l y , carbon d i o x i d e (4-7). The purpose of the present work was to i d e n t i f y and q u a n t i t a t e the r e s i d u a l products a f t e r a 30-day fermentation of C^. stercoreus on wheat straw. The approach to t h i s problem i s l i m i t e d by a few, s i g n i f i c a n t c o n s i d e r a t i o n s . I d e a l l y , p r e l i m i n a r y e x t r a c t i o n of the straw e l i m i n a t e s i n t e r f e r i n g substances, such as f a t t y a c i d s and other e x t r a c t a b l e s present i n l a r g e q u a n t i t y , which overwhelm minute amounts of biodégradation products and thereby hinder t h e i r i d e n t i f i c a t i o n . However, the fungus does not grow w e l l when a l l s o l u b l e n u t r i e n t s are removed, as w i l l be shown i n l a t e r s e c t i o n s . A l s o , s t e r i l i z a t i o n by a u t o c l a v i n g may generate s o l u b l e l i g n i n s that cannot be a t t r i b u t e d to fungal a c t i o n , and t h e r e f o r e c o n t r o l s must be run. More s p e c i f i c a l l y , r e p r o d u c i b i l i t y of fermentation on a s o l i d substrate i n o c u l a t e d with a 1 cm plug of slowgrowing stercoreus i s d i f f i c u l t , e s p e c i a l l y because t h i s fungus i s s e n s i t i v e to excess water and w i l l not grow i n submerged c u l t u r e , although a high l e v e l of moisture i s necessary f o r fungal growth. A f t e r the fermentation i s complete, extracted water-soluble degradation products l o s e much of t h e i r water s o l u b i l i t y i f taken to dryness i n a i r or i f heated. Freeze-drying overcomes t h i s problem, but molecular weight d i s t r i b u t i o n s should be determined before f r e e z e - d r y i n g because polymer chains can be f r a c t u r e d by growing solvent c r y s t a l s (8). 2


19.

ABBOTT ET AL.

Wheat Straw Biodégradation

269

Products


M a t e r i a l s and Methods Sample P r e p a r a t i o n . Three wheat straw samples were t e s t e d . One was a l o c a l l y obtained, d r i e d and baled wheat straw; the other two were Red River 68 and Butte HRS, grown i n an environmental chamber. While monitoring the p l a n t growth and l i g n i n production i n the straw as reported by Stone et a l . (9), we grew the p l a n t s f o r 38 days (Butte HRS) or 48 days (Red River 68) and cut them f o r l i g n i n l a b e l i n g j u s t p r i o r to heading out of the g r a i n . In order to l a b e l the l i g n i n , the roots were cut o f f and the f r e s h l y cut s u r f a c e was immediately immersed i n a 10-ml s o l u t i o n of uniformly l a b e l e d C-phenylalanine i n a growth chamber u n t i l the s o l u t i o n was n e a r l y a l l taken up. The cut surface was then immersed 1-2 cm i n d i s t i l l e d water f o r 3 days, d r i e d at 45° C to constant weight and ground to pass through a 1-mm screen. The ground straw was washed with hexane at 35° C and d r i e d , then soaked i n water at 4° C f o r 16 h, washed, and f i n a l l y t r e a t e d with protease at room temperature f o r 3 h, water washed and f r e e z e - d r i e d . The protease used was Subtilopeptidase-A ( B a c t e r i a l , Type VII) from Sigma Chemical Company i n a pH 7 borate b u f f e r at a c o n c e n t r a t i o n of 1 mg/ml. T o t a l C content, as decompositions per minute per mg (DPM/mg), was determined by p y r o l y s i s of the straw i n an oxygen atmosphere to CO2 and trapping of the CO2 i n 10 ml of a s c i n t i l l a t i o n c o c k t a i l c o n s i s t i n g of 1,300 ml toluene, 1,200 ml methanol, 300 ml 2-aminoethanol, 0.0375 g l,4-[2-(4-methyl-5-phenyl-l,3-oxazolyl) benzene and 30.0 g 2,5-diphenyloxazole. Unlabeled wheat straw obtained l o c a l l y was hexane washed, d r i e d , soaked at 4° C i n water that was decanted, and then e i t h e r fermented d i r e c t l y or a f t e r f u r t h e r water washing. A l s o , at the end of the 4° soak the straw was broken up i n a Waring Blendor to prevent t r a p p i n g of water i n s i d e the hollow straw stem. Fermentation was accomplished i n a i r at 25° a f t e r i n o c u l a t i o n with a 1-cm plug of C^. stercoreus that had been grown f o r 1 week on a potato dextrose agar p l a t e . ll+

2

ll+

2

Separation of Biodégradation Products. The scheme devised f o r s e p a r a t i o n and recovery of biodégradation products i s i l l u s t r a t e d i n F i g u r e 1. The ether phase e x t r a c t , l a b e l e d C i n Figure 1, would c o n t a i n a c i d i c m a t e r i a l s such as v a n i l l i n , syringealdehyde and s i m i l a r compounds i f they were present. Some p o l y f u n c t i o n a l a c i d i c compounds remain i n the a c i d i f i e d water phase D. An a l t e r n a t e route using i o n exchange r e s i n i s b e t t e r s u i t e d f o r s e p a r a t i o n and i d e n t i f i c a t i o n of p o l y f u n c t i o n a l a c i d i c compounds, such as hydroxybenzoic a c i d . These compounds w i l l end up i n the water e x t r a c t l a b e l e d Ε i n Figure 1. Unless other s o l u b l e compounds mask t h e i r presence, these a c i d s can a l s o be i d e n t i f i e d i n the o r i g i n a l extracted fermentation broth.



270


40g Unlabeled or 0.5g C Labeled U

Wheat Straw Fermented 30 Days Filtration 1. Whatman 541 paper 2. 0.45/im membrane

A

Retentates

Filtrate

Ethanol

Adjust pH to 10 Ether

Β Ether Phase

F

H2O Phase HCIto pH 4

Insoluble

Soluble (alternate r o u t e ) j

Ether

Ether Phase

o n

e x c n a n g e

| H +

,

" " ^ - ^ Ether

Ether Phase

D H 0 Phase 2

Ε H2O Phase

Dialysis

Dialysate

Figure 1.

Retentate

Separation of biodegraded wheat straw.


19.

ABBOTT ET A L .


Products

C h a r a c t e r i z a t i o n Methods. Labeled m a t e r i a l s i n s o l u b l e e x t r a c t s were q u a n t i t a t e d by counting 100 u l a l i q u o t s of the v a r i o u s s o l u t i o n s i n the same 10 ml s c i n t i l l a t i o n c o c k t a i l used to trap CO2. The q u a n t i t y o f C i n evolved gases was measured by d a i l y 15-min f l u s h e s o f the sealed fermentations with a i r i n t o the same s c i n t i l l a t i o n c o c k t a i l . The gas-phase components were i d e n t i f i e d by t h e i r r e t e n t i o n times on a 5 f t X 1/8 inch Porapak Q column. To trap enough gas f o r i d e n t i f i c a t i o n , the evolved gases from 2 days of fermentation were f l u s h e d with helium i n t o a 1 f t X 1/8 i n c h Porapak Q f i l l e d s t a i n l e s s - s t e e l column f i l l e d with Porapak Q and immersed i n l i q u i d N2. The column e x i t gas passed through a bubbler c o n t a i n i n g s c i n t i l l a t i o n f l u i d . After the 15-min f l u s h the column was capped on the entrance s i d e and a new bubbler was attached. The column was then allowed to warm to room temperature. Trapped CO2 r a p i d l y eluted as the column warmed, and when gas e v o l u t i o n stopped the column was i n s e r t e d i n t o a Tracor Model 560 gas chromatograph (GC); the entrance side of the trap column was attached to the i n j e c t i o n port and the e x i t s i d e to a 4 f t by 1/8 i n c h Poropak Q column. The b a s e l i n e had been e s t a b l i s h e d p r e v i o u s l y but r e s i d u a l CO2 came o f f as helium c a r r i e r gas was turned on and the oven was programmed from 40° to 180° a t 5°/min. Samples from the water phase were t e s t e d f o r v o l a t i l e s other than CO2. They were tested on the same Poropak Q column as gas-phase samples, but the column was programmed from 100 to 170° a t 10°/min and held at 170° f o r 20 min. To ensure that the v o l a t i l e s other than CO2 were from l i g n i n degradation, the same l i q u i d N2 trapping procedure was followed. The column was attached a t room temperature to a GC with a thermoconductivity (TC) detector l e a d i n g to a flowing s c i n t i l l a t i o n f l u i d trap f o r gases as they e l u t e d . R e s u l t s of the a n a l y s i s of v o l a t i l e s by GC were confirmed by high-pressure l i q u i d column chromatography (HPLC) and i n f r a r e d s p e c t r a of the major components. Less v o l a t i l e low-molecular-weight biodégradation products, were c h a r a c t e r i z e d by gas chromatography-mass spectroscopy (GCMS). Higher molecular weight compounds were c h a r a c t e r i z e d by gel permeation chromatography (GPC), membrane f i l t r a t i o n , d i a l y s i s and n u c l e a r magnetic resonance (NMR). The methylated (CH2N2) low-molecular-weight compounds were analyzed on a Kratos MS-30 equipped with a Perkin Elmer Sigma 3 gas chromatograph and a 3 f t X 1/8 inch OV-1 column. Retention times of the sample peaks i d e n t i f i e d by GC-MS were compared to those of known standards on a 6 f t X 1/8 inch OV-1 column on a Tracor model 560 GC f o r c o n f i r m a t i o n . L i g n i n content was measured by the UV method (10). C o n t r o l samples of wheat straw o x i d i z e d by the standard nitrobenzene method (11) and separated by the scheme i l l u s t r a t e d i n F i g u r e 1 were a l s o t e s t e d by the GC-MS method to confirm the v a l i d i t y of the s e p a r a t i o n and recovery techniques. 1I+


271



272


To c h a r a c t e r i z e high-molecular-weight biodégradation products, an Amicon Model 202 D i a f i l t r a t i o n Apparatus was used s e q u e n t i a l l y with XM300, PM10 and UM2 membranes at 10, 17 and 55 p s i N2 pressure, r e s p e c t i v e l y . The samples tested were water-soluble biodégradation products that had never been f r e e z e d r i e d . Concentrations of the v a r i o u s f r a c t i o n s were determined on a l i q u o t s so that GPC curves could be determined on samples that had always been i n s o l u t i o n . High-molecular-weight biodégradation products f o r NMR were i s o l a t e d by d i a l y s i s and membrane f i l t r a t i o n . D i a l y s i s bags of regenerated c e l l u l o s e were c h a r a c t e r i z e d with standard dextrans and r a f f i n o s e . GPC curves of a d e x t r a n - r a f f i n o s e mixture before and a f t e r s e p a r a t i o n by d i a l y s i s i n d i c a t e s a nominal molecular weight of 3,000 or greater was r e t a i n e d f o r NMR a n a l y s i s . High-molecular-weight compounds i s o l a t e d by t h i s method were f r e e z e - d r i e d , and t h e i r ^ C NMR spectrum determined on a Briiker WH 90 i n D2O. Gel permeation chromatography (GPC) curves were determined with a Waters 244 HPLC on 2 columns of E - l i n e a r μ-Bondagel, 7.8 mm X 30 cm each. Water was the solvent at 1 ml/min and ambient temperature. Detection was by r e f r a c t i v e index and UV absorbance at 254 mn. Results and D i s c u s s i o n Recovery of the gas phase of fermented wheat straw and separation of the r e s i d u e by the method i l l u s t r a t e d i n Figure 1 gave a m a t e r i a l balance of the v a r i o u s f r a c t i o n s by weight C content, as shown i n Tables I and I I . Although only 4.6% of the o r i g i n a l 40 g of straw i s e x t r a c t e d as water-soluble m a t e r i a l a f t e r the fermentation and 9.3% of the r a d i o a c t i v e straw by weight, a higher percentage of the o r i g i n a l C (13.3%) i s i n the recovered water-soluble m a t e r i a l (Table I ) . Thus there i s a higher c o n c e n t r a t i o n of l i g n i n degradation products i n the fermentation b r o t h than the c o n c e n t r a t i o n of l i g n i n i n the wheat straw. A u t o c l a v i n g unfermented straw y i e l d s 0.8% of the o r i g i n a l r a d i o a c t i v i t y i n the water phase. C a r e f u l drying to avoid l o s s of moderately v o l a t i l e substances such as v a n i l l i n involved f r e e z e - d r y i n g the water-soluble m a t e r i a l from the 40-g straw fermentations. A d d i t i o n a l l y , -^C-labeled water s o l u b l e s were counted by t e s t i n g a l i q u o t s (100 u l ) i n s c i n t i l l a t i o n f l u i d , both before and a f t e r f r e e z e - d r y i n g f o r 54 h past apparent dryness. Less than 4.3% of the o r i g i n a l C was l o s t on f r e e z e drying and r e c o n s t i t u t i o n , and no C was detected i n the -70° f r e e z e - d r y i n g t r a p . Some l o s s of both weight and C w i l l occur from d i s s o l v e d CO2, methanol and ethanol. From the r e s u l t s i n Tables I and I I i t can a l s o be concluded that the gas phase contains approximately 13% of the o r i g i n a l C. The t o t a l l i g n i n i s degraded 27% a f t e r 30 days, h a l f i n the gas phase and h a l f i n the water phase of the fermentation, before e i t h e r phase i s separated f o r i d e n t i f i c a t i o n . I d e n t i f i c a t i o n of the components of the gas phase by r e t e n t i o n time i s i l l u s t r a t e d i n Figure 2. 1I+

ll4

ll+

ll+

1 4

1I+


19.

ABBOTT ET A L .


273

Products

A t y p i c a l flame i o n i z a t i o n d e t e c t i o n (FID) curve i s shown i n Figure 2 (curve 2). Gases and water-phase v o l a t i l e s i d e n t i f i e d by r e t e n t i o n time are ethanol, methanol, acetone and a c e t i c a c i d . A comparison of counts c o l l e c t e d over 10° i n t e r v a l s i n the programmed chromatograph to the r e t e n t i o n times of peaks detected by both FID and TC i n d i c a t e s that a l l of the gases i n a d d i t i o n to CO2 have at l e a s t part of t h e i r o r i g i n i n the Rel a b e l e d l i g n i n (compare F i g u r e 2, curve 3 to F i g u r e 2, curves 1 and 2). R e s i d u a l ethanol i n the water phase was determined by HPLC to be 0.02% or 0.033 g i n a 30-day, 40-g straw fermentation with f r e e ( f i l t e r e d ) atmospheric gas exchange. CO2 and water were evident i n the i n f r a r e d spectra of fermentation gases, with no evidence of s i g n i f i c a n t amounts of hydrocarbon gases. As i n d i c a t e d by weight and C a n a l y s i s (Table I I ) , few i f any low-molecular-weight aldehydic phenols were i s o l a t e d i n ether phase C. Therefore, the a n a l y s i s scheme (Figure 1) was tested by adding 0.05 g v a n i l l i n to a 30-day fermented wheat straw and c a r r y i n g i t through the e x t r a c t i o n and f r e e z e - d r y i n g procedure. The added v a n i l l i n was e a s i l y determined i n ether e x t r a c t C by GC. Only a t r a c e was found i n e t h a n o l - s o l u b l e e x t r a c t F, but even t h i s t r a c e , 0.1 mg, was detectable, i n c o n t r a s t to the e x t r a c t s of biodegraded wheat straw that contained no a l d e h y d i c phenols. The absence of v a n i l l i n , syringealdehyde, and s e v e r a l other phenols i n the water-soluble biodégradation products was confirmed by t h i n l a y e r chomatography (TLC) i n 95/5 benzene/methanol, u s i n g TJV and radiocarbon d e t e c t i o n . Of a l l the compounds examined, only carboxylated phenols do not migrate away from the o r i g i n i n t h i s s o l v e n t . Ether e x t r a c t i o n procedures have been used a l s o to i s o l a t e aldehydic phenols from a l k a l i n e nitrobenzene o x i d i z e d l i g n i n to c h a r a c t e r i z e the l i g n i n i n v a r i o u s p l a n t s . Fe(NH^)2(S0i+)2 washed ether should be used to prevent peroxide o x i d a t i o n of the aldehyde groups (12) . Using Fe(NHi ) 2(S0if) 2 washed ether throughout, we demonstrated again that aldehydic phenols were not present i n the ether e x t r a c t s of biodegraded wheat straw i n which over 2 g of l i g n i n i s degraded, but these phenols are e a s i l y found and q u a n t i t a t e d when 2 g of wheat straw i s o x i d i z e d by the standard nitrobenzene method and the phenols are recovered as i l l u s t r a t e d i n F i g u r e 1. Table I I I l i s t s the compounds found i n nitrobenzene o x i d i z e d wheat straw, as w e l l as s i m i l a r r e s u l t s f o r e x t r a c t s of the biodégradation products. The absence of low-molecular-weight biodégradation products i n the ethanol e x t r a c t i s confirmed by the o b s e r v a t i o n that the gas chromatographic curve f o r the ethanol s o l u b l e s ( s i l y l a t e d ) of fermented wheat straw i s i d e n t i c a l to the curve f o r s i m i l a r d e r i v a t i z e d e x t r a c t s of unfermented wheat straw c a r r i e d through the same e x t r a c t i o n procedure. To t e s t f o r p o l y s u b s t i t u t e d phenols i n the water s o l u b l e biodégradation b r o t h , the methylated (CH2N2) d e r i v a t i v e of f r e e z e - d r i e d e x t r a c t A (Figure 1) was e a s i l y produced and y i e l d e d interprétable mass s p e c t r a . S e v e r a l phenolic standards were


1

ll+

+


274


Table I.

UNCONVENTIONAL

SOURCES

OF

DIETARY

FIBER

Quantitation of Biodégradation Products

Material Balance by Weight

Starting material

Total Sample

Lignin

Basis

Basis

6.4% H 0, 2.56 g 15.6% lignin, 6.24

100%, 6.24

9

Hexane extract 4° Water extract, decanted

0.4%,

0.16

5.5%,

2.2 g

Total Sample Basis A l l Soluble Nutrients Removed 6.4% H 0, 2.56 g 15.6% l i g n i n , 6.24

g

9

g

0.4%,

-0-

g 4.8%,

g

0.3 g

4° Water extract and water wash Fermented filtered H 0 solubles

0.16

5.6%,

2.25

g

2.6%,

1.02

g

2.3%,

0.939 g

2

4.6%,

1.92

g

7.1%,

0.44

g

0.45 urn f i l t e r e d H 0 solubles 2

EtOH solubles after fermentation

1.3%,

0.53

g

6.4%,

0.3+0.1 g

6

Fermented extracted dry straw

69.5%, 27.8 g

Lost or i n gas phase

12.5%, 5.0 g

65.2%, 4.07

g

16.5%, 1.03 g

78.8%, 31.52

6.3%,

Trapped gases

Average of 5 determinations on 40 g wheat straw ^Lignin determinations by UV method (10) Average of 2 determinations. ^Decomposition per minute. e

Not corrected for fungal mass.


g

2.5 g

g

19.

ABBOTT E T A L .


14

C

Balance


d

Products

6

DPM X10" , Percent

Sample 1

Sample 2

Sample 3

Sample 4

Butte HR

Butte HR

RR 68

RR 68

38.03

25.04

43.88

35.68

5.0, 14.0%

5.47, 14.4%

3.33, 13.3%

5.27, 12.1%

0.25, 0.7%

0.13, 0.3%

25.0, 65.8%

6.2% 5.04, 13.3%

4.72, 13.2%

16.9, 67.3%

24.8, 56.5

12.3%

17.6%

1.27, 5.1%

5.90, 13.5%

12.8, 35.8%

-6.4% 20.2, 56.7%


276


TABLE I I .

Separation

of Water-Soluble Biodégradation Products

by Ether E x t r a c t i o n (Figure 1)


Percent of Water-Soluble Products i n Ether E x t r a c t of Basic H 0

Ether E x t r a c t of A c i d i c H 0

2

By weight 14 By

C label

2

0.41

0.96

0.03

0.4

a

A s i l l u s t r a t e d by the e x t r a c t l a b e l e d Β i n Figure 1.

b

A s i l l u s t r a t e d by the e x t r a c t l a b e l e d C i n Figure 1.

Curve 5

Ethanol (Methanol

2

I JUL_j Curve 4

100

120

140 160 Temperature, °C

180

600 ^400

t

-

Q "

18,800

m Curve 3

200 0»— Curve 2 Methanol Curve 1 40

Ethanol

A 60

80

A c e

A 100

120

,

t o n e

l

Ethyl ether

λ 140

160

Temperature, °C

Figure 2. Gas chromatographic curves for volatiles. Key: Curve 1, standard mix in water of less than 1% each of methanol, ethanol, diethyl ether, and acetone flushed with He, trapped and tested on Poropak Q as were the fermentation gases (FID); Curve 2, gas phase of eight-day fermentation trapped and tested as described in text (FID); Curve 3, collected and counted GC effluents (see text); Curve 4, standard mix of 0.1% each methanol, ethanol, acetone, and acetic acid; and Curve 5, water phase of fermentation.


19.

ABBOTT ET A L .


Table I I I .


277

Products

GC-MS I d e n t i f i e d TMS D e r i v a t i v e s o f Biodegraded and

O x i d i z e d Wheat Straw Q

Extract of E x t r a c t s o f Biodegradable Straw EtOH E x t r a c t

b

Extract

Nitrobenzene O x i d i z e d 0

Wheat Straw

P a l m i t i c acid'TMS

No monomeric

Phenol-TMS

L i n o l e i c acid^TMS

phenols

L a c t i c acid'TMS

Pentose·TMS Tetramer and A h i g h molecular pentamer weight (422) hydrocarbon, p o l y d i m e t h y l not TMS d e r i v a t i z e d siloxane which dominates the artifacts. GC curve i n s i z e . The same Probably a t r i t e r p e n e . 422 molecular weight compound as i n column 1.

G l y c o l i c acid^TMS p-Hydroxybenzaldehyde *TMS Vanillin-TMS Syringealdehyde *TMS 2 isomers o f

Nitrobenzene

a

K r a t o s MS-30 equipped w i t h a P e r k i n Elmer Sigma 3 gas chromatograph. An OV-1 surface-coated open t u b u l a r c a p i l l a r y column was h e l d a t 70° f o r 3 min, then programmed a t 4°/min t o 250° a f t e r sample i n j e c t i o n . ^ I s o l a t e d as i l l u s t r a t e d i n F i g u r e 1. I s o l a t e d as i l l u s t r a t e d by e x t r a c t C i n F i g u r e 1.



278


methylated f o r comparison to the unknown. Since methylated e x t r a c t A had not been separated i n t o v a r i o u s components before methylation, the gas chromâtograph curve shows many peaks (Figure 3, curve 1). The smaller peaks i n the area of methylated p o l y f u n c t i o n a l phenols are t h e r e f o r e d i f f i c u l t to i d e n t i f y from t h e i r mass spectrum alone. A technique c a l l e d mass chromatography was used wherein d i s t r i b u t i o n of s e v e r a l major masses that appear i n a standard spectrum i s p l o t t e d underneath the gas chromatograph curve as i n F i g u r e 3. In t h i s way, coincidence of s e v e r a l masses that are present i n a standard mass spectrum under a s i n g l e peak i n the gas chromatograph curve i d e n t i f i e s the l o c a t i o n of that standard. T h i s , i n conjunction with r e t e n t i o n time, confirms the presence of a compound i n the unknown, whereas the absence of such coincidence at a given r e t e n t i o n time confirms the absence of a compound to very low l e v e l s of- d e t e c t a b i l i t y i n the o r i g i n a l sample. Using t h i s technique, methylated v a n i l l i c a c i d , p-hydroxybenzoic a c i d , 2methoxy s u c c i n n i c a c i d and s y r i n g i c a c i d were found to be present. The p a l m i t i c a c i d , l i n o l e i c a c i d and t r i t e r p e n e s found e a r l i e r were the dominant m a t e r i a l s i n the water s o l u b l e s examined by t h i s technique, as can be seen i n Figure 3. The magnitude of these three m a t e r i a l s prevents p u t t i n g a l a r g e r sample on the GC-MS instrument to enhance the i n t e r p r e t a t i o n of small peaks. Larger samples p o s s i b l y could burn out f i l a m e n t s i n the mass d e t e c t i o n system. For t h i s reason, p r e - e x t r a c t i o n of the t r i t e r p e n e s and f a t t y a c i d s as much as p o s s i b l e was attempted with hexane. The f a t t y a c i d s and t r i t e r p e n e s c o n s t i t u t e the i n t e r f e r i n g substances r e f e r r e d to i n the i n t r o d u c t i o n . Since s e p a r a t i o n of these lower molecular weight compounds i s not e a s i l y achieved by ether e x t r a c t i o n , they are not q u a n t i t a t e d by the s e p a r a t i o n scheme of F i g u r e 1. Separation of higher molecular weight m a t e r i a l s f o r c h a r a c t e r i z a t i o n was a l s o necessary. Both of these goals were achieved by membrane f i l t r a t i o n , d i a l y s i s and GPC c h a r a c t e r i z a t i o n . Membrane f i l t r a t i o n r e s u l t s i n Table IV gave us considerable i n s i g h t i n t o the molecular weight d i s t r i b u t i o n of r e s i d u a l biodégradation products. We concluded from these r e s u l t s that few of the r e s i d u a l biodégradation products are present as lowmolecular-weight s p e c i e s . Compounds of l e s s than 1,000 nominal molecular weight c o n s t i t u t e 11% of the water-soluble biodégradation products, with monomeric aromatics only a p o r t i o n of t h i s . GPC curves of the v a r i o u s membrane-separated f r a c t i o n s confirm that the high-molecular-weight f r a c t i o n s c o n t a i n the m a j o r i t y of u l t r a v i o l e t absorbing species (Figures 4 and 5). F i g u r e 6 shows the C NMR spectrum of high-molecularweight biodégradation products from membrane f i l t r a t i o n and d i a l y s i s and the NMR spectrum of a l i g n i n - c a r b o h y d r a t e complex published by Himmelsbach and Barton (13). The spectrum of the biodégradation products shows predominantly carbohydrate polymers. The combination of weight and ^ C - l a b e l d i s t r i b u t i o n among s e q u e n t i a l membrane f i l t e r s , the UV absorbance of these species, 1 3



19.

ABBOTT ET A L .


279

Products

Mass 165

Curve 3

Mass 181 Curve 4

\jL„J_JijMJLJLJ^ Mass 196

Curve 5

70

150

100 Temperature, °C

Figure 3. GC-mass spectra curves. Key: Curve 1, total ionization vs. temperature curve for methylated water-soluble biodégradation products; Curve 2, a portion of Curve 1 for searching; Curve 3, plot of mass 165 ions received while Curve 2 was being collected; Curve 4, plot of mass 181 ions received while Curve 2 was being collected; and Curve 5, plot of mass 196 ions received while Curve 2 was being collected.



280

Table IV.

Membrane F i l t r a t i o n of Biodégradation Products

Percent of H 0 Solubles

Percent of H 0 Solubles

2

2

Retained b y

a


Membrane

14

Weight f o r the 40 g

f o r the

C Labeled L i g n i n

Fermentation

Wheat Straw Fermentation

Χ M 300

13.2

22.8

PM 10

10.9

23.9

UM 2

48.8

42.0

Through UM 2

21

11.3

Recovery

93.9

95.5

a

Nominal molecular weights r e t a i n e d by the XM300 membrane i s >300,000, by the PM10 membrane i s 10,000 to 300,000, and by the UM2 membrane i s 1,000 to 10,000.

A

J

.

v

V

Pu

Β

w

J 1

1

220

110

1 1 1 .

1 6 8 . 6 4.5

Calibrated Dextran Molecular Weight χ 1 0 - 3

I

ι

ι

ι

ι

0

5

10

15

20

.

1 25

Elution Volume, ml

Figure 4. Gel permeation chromatograms of the total water-soluble biodégradation product. Detection for Chromatogram A is by refractive index change, and Chromatogram Β is by UV absorbance at 254 nm.



19.

ABBOTT ET A L .


Products

Figure 5. Gel permeation chromatograms of the membrane filtration fractions using the same conditions as for the unseparated sample described in Figure 4.


281


282


I

I 160

180

I 140

I 120

I 100

I 80

I 60

I 40

I 20

L 0

PPM

13

Figure 6. C-NMR spectra of a lignin-carbohydrate complex A (13), and the high molecular weight water-soluble biodégradation products isolated by dialysis (B).


19.

ABBOTT ET A L .


Products

283

and the NMR spectrum i n F i g u r e 6 lead to the c o n c l u s i o n that the overwhelming m a j o r i t y of r e s i d u a l biodégradation products are l i g n i n - c a r b o h y d r a t e complexes.


Conclusions Although some low-molecular-weight o x i d i z e d aromatic species can be found i n biodégradation products of Cyathus stercoreus on wheat straw, the predominant r e s i d u a l biodégradation species are l i g n i n - c a r b o h y d r a t e complexes. The other major product of degraded l i g n i n i s carbon d i o x i d e . Lesser amounts of ethanol, methanol, acetone, a c e t i c a c i d , 2-methoxy s u c c i n n i c a c i d , v a n i l l i c a c i d , s y r i n g i c a c i d and p-hydroxy benzoic a c i d were a l s o found. These f i n d i n g s are c o n s i s t e n t with current t h i n k i n g on mode of a t t a c k of f u n g i on l i g n o c e l l u l o s i c s as described e a r l i e r . I f random o x i d a t i o n cleaves high molecular weight water-soluble f r a c t i o n s from l i g n o c e l l u l o s i c s , one would expect that they cannot be metabolized; however, as these a r e broken down to lowmolecular-weight m a t e r i a l s by continued oxygenation, these f r a c t i o n s a r e r a p i d l y metabolized to CO2. Thus, the r e s i d u a l fermentation b r o t h would c o n t a i n predominantly i n d i g e s t i b l e high-molecular-weight species and a few low-molecular-weight species and a s i g n i f i c a n t amount of CO2 would have been generated. We f i n d such a composition. We g r a t e f u l l y acknowledge Charles L. Swanson f o r h i s g e l permeation chromatography work and Gordon Adams f o r a s s i s t a n c e i n the fermentations. Literature Cited 1. 2. 3.

4. 5. 6. 7. 8. 9.

Wicklow, D. T.; Detroy, R. W.; Jessee, B. A. Appl. Environ. M i c r o b i o l . , 1980, 40, 169. Christmen, R. F.; Oglesby, R. T., i n L i g n i n s , Ch. 8, John Wiley and Sons, Inc., N.Y., N.Y., 1971. K i r k , T. K.; H i g u c h i , T.; Chang, Η., Ed. L i g n i n Biodegradation: M i c r o b i o l . , Chem., and P o t e n t i a l A p p l i c a t i o n s . V o l . I and V o l . I I , CRC Press, Boca Raton, F l o r i d a , 1980. K i r k , T. K.; S h u l t z , E.; Connors, W. J . ; Lorenz, L. F.; Zeikus, J. G. Arch. M i c r o b i o l . , 1978, 117, 277. Crawford, R. L.; Crawford, D. L.; D i z i k e s , G. J . Arch M i c r o b i o l . , 1981, 129, 204. Haider, K.; Trojanowski, J . Arch. M i c r o b i o l . , 1975, 105, 33. Bar-Lev, S. S.; K i r k , T. K. Biochem. Biophys. Res. Commun., 1981, 99, 373. E l i a s , H-G. Macromolecules. V o l . I I , Plenum Press, N.Y., N.Y., 1977, 548. Stone, J . Ε.; B l u n d e l l , M. J . ; Tanner, K. G. Can. J . Chem., 1951, 29, 734.


284 10. 11. 12.


13.

UNCONVENTIONAL SOURCES OF DIETARY FIBER Bagby, M. O.; Cunningham, R. L.; Maloney, R. L. Tappi, 1973, 56, 162. Roadhouse, F. E.; MacDougal, D. Biochem. J., 1956, 63, 33. Hedges, J . I.; Parker, P. L. Geochim. Cosmochim. Acta, 1976, 40, 1019. Himmelsbach, D. S.; Barton I I , F. E. A g r i c . Food Chem., 1980, 28, 1203.

RECEIVED October 29,

1982


Products of Wheat Straw Biodegradation by Cyathus

Recommend Documents