Journal of Applied Bacteriology Symposium Supplement 1987, 71s-83s

Utilization of lignocellulosic wastes

J .M. LYNCH GCRI, Littlehampton, West Sussex BN176LP, U K

1. Introduction, 71s 2. Substrates, 72s 3. Metabolism, 73s 4. Lignocellulolysis in the environment, 74s 5. Waste treatment processes, 77s 6 . Controlled non-axenic fermentations, 78s 7. Industrial fermentations, 80s 8. Future prospects, 81s 9. Acknowledgements, 81s

10. References, 82s

1. Introduction

Lignocelluloses represent the most abundant natural material on earth, being about 50% of all bio- mass with an annual production of 50 x lo9 tons (Goldstein 1981). Some of the earliest studies of applied microbiology were in the field of lignocellulolysis because the characterization of natural processes was of primary concern in the early development of the discipline. Indeed, one of the most distinguished former members of the Society for Applied Bacteriology, H. J. Bunker, was co-author of a volume (Thaysen & Bunker 1927) recognizing the potential value of the natural process where it was stated that ‘the dead vegetation of the world might, of course, be converted into carbon dioxide and water by burning it, but its accumulated energy can be turned into better account and is, in fact, being far more economically utilized in the natural processes of decay’. This statement went virtually unchallenged and applied microbiologists made significant contributions to the understanding of lignocellulolysis in a diverse range of natural environments such as soil, rivers, sewage digestors, silage and the rumen. The emphasis of study was generally the microbiology of the process rather than its biochemistry, although some particularly useful biochemical studies were done in relation to the rumen.

With the escalation of oil prices in 1973 a new impetus for study was created. The abundance of cheap lignocellulosic materials was recognized as a potentially useful substrate for the direct pro- duction of fuels or to lessen our dependence on fossil fuels by acting as a substrate to yield products which would normally have been produced from fossil fuels. For example, this stimulated much support for research in the US by the Solar Energy Research Institute of the US Department of Energy, with an emphasis on alcohol production by fermentation, and in Europe by the European Economic Community under their Energy and Secondary Raw Materials Programme, which has a rather broader remit than the US programme and includes anaerobic digestion. During this second phase of lignocellulolysis research, many exciting developments have taken place, increasing our understanding of the biochemistry of the processes and creating new opportunities by genetic modifi- cations of the processes.

Straw is a major lignocellulosic waste and because microbial fermentation by soil micro-organisms can give rise to phytotoxic metabolites, and other straw-colonizing bacteria can invade roots (Lynch & Elliott 19841, farmers in Britain have tended to burn straw in the field, often generating environmental concern. The potential manipulation of straw breakdown in soil has been a further stimulus for lignocellulolysis research. On-farm and off-farm uses for straw have also been considered. One potential use of straw would be to upgrade it microbiologically or enzymically for use as an animal feed. All these options need careful economic as well as scientific evaluation, however, and they must also compete favourably with chemical processes such as the alkali treatment of straw for animal feed.

72s J . M . Lynch The year 1986 could herald the start of a new phase of study. Falling oil prices are removing some

research incentives, but with agricultural surpluses, a consequence of the European Common Agricul- tural Policy, there are new opportunities to utilize what are effectively higher quality lignocellulosic wastes. This would bring a new research perspective because, whereas the useful substrate for micro- bial processes could be more readily abailable material such as starch, lignocellulolysis might be a prerequisite to making all that substrate available.

2. Substrates

The lipnocelluloses which have received the greatest attention from microbiologists are wood and straw. Their availability as waste materials varies greatly between countries and between regions Rithin a country. A major problem with straw is that it has a low bulk density, which makes trans- port costly, limiting the scope for utilizing the material economically off-farm. Wood cannot generally be regarded as a waste with the exception of certain fractions, such as bark and forest thinnings.

I ignocelltilose is strictly made up of holocellulose bound to lignin. Holocellulose is cellulose (a polymer of glucose) and hemicellulose (a heterogeneous polymer of the hexoses glucose, mannose and galactose. and the pentoses xylose and arabinose). The major plant hemicellulose is D-xylan which has a backbone of poly-/l- 1. 4-xylan linked laterally to arabinose, glucuronic and arabinoglucuronic acid, mannans and galactans. Lignin is a polymer based on three phenolic acids (p-coumaryl alcohol, coniferyl alcohol and sinapyl dlcohol).

Both wood and straw are nearly 90‘% w/w lignocelluloses, wood being more lignified than straw (Fig. 1). There are many other plant materials which contain other major components in addition to the lignocellulose. The ‘quality’ of the lignocellulosic material as a biodegradation substrate is some- times related to its protein content and therefore its nitrogen (N) content, avoiding N-deficiency during biodegradation (Table 1). The lignocellulose content of bran is compared with straw and wood in Fig. 1 ; bran is a milling fraction for which there is less use today than there has been in the past and might therefore be regarded as a lignocellulosic waste. The other major components of bran are protein (17.1% wiw). starch (16% wjw), sugars (4.5% w/w) and lipids (94.5% w/w). Lignocellu- lose is concentrated in the bran component of whole grain and therefore grain has an even greater percentage of other components, especially starch which accounts for about 8@-85O/O w/w. With developing ‘grain mountains’, there is an argument that this could also become a Iignocellulosic waste. Inetitably, materials such as bran and grain have more potential as more complete and readily utilizable substrates for microbial fermentation. Wood, because of its very high lignin content, is one of the most recalcitrant natural substrates for micro-organisms. However, lignocelluloses obtained directly from nature are not the only substrates that have been considered for microbial degradation.

Table 1. Nitrogen content of some materials containing Iigno- cellulose

Straw type N’h w w Material Nu/> W ~ W

Barley 0.59 Barley feed 1.97

Oat 0.66 Rice 0.62 Rice, brown seed 1.46

Soybean 0.64 Soybean seed 6.06 Sunflower seed 2.69

Wheat bran 2.70 Grass 0.72 Grass hay 1.54

Cowpea 1 49 Cowpea seed 3.74

Rice, bran 2.00

Wheat 0.62 Wheat grain 2.1 1

Source US. -Canadian Tables of Feed Composition (1969) Publication 1684, Committee on Animal Nutrition and National Committee on Animal Nutrition, Canada, National Academy of Sciences - National Research Council, Washing- ton, DC.

Utilization of lignocellulosic wastes Wheat bran 17

-Wheat straw

Y l P i n e wood

-1 Bi rch wood

73s

0 10 2 0 30 40 50 Percentage iw/w)

Fig. 1. Chemical composition of some lignocellulosic materials. 0, lignin; m, cellulose; m, hemicellulose

Domestic refuse has received considerable attention for its potential to be composted. In practice, refuse is an extremely complex mixture of natural and processed lignocelluloses, ranging from paper (nearly all cellulose) to wood.

3. Metabolism

In recent years the cellulose enzyme complex has been studied extensively (Coughlan 1985) and the action of the three major components is as shown in Fig. 2.

Less attention has been paid to the xylanase complex but more is known about the endo-xylanases (p- 1, 4-~-xylan xylanohydrolase, EC 3.2.1.8) than the exo-xylanases. p-xylosidases (p-D-xylosidase xylohydrolase, EC 3.2.1.37) hydrolyse the p(1-4) links a t the non-reducing ends of xylo- oligosaccharides and p( l-4)-aryl xylopyranosides to produce xylose.

At least a part (probably about 50% w/w) of the cellulosic fraction of lignocellulosic is not tightly bound to lignin and is relatively available for microbial decomposition. To achieve maximum sub- strate utilization it is generally considered necessary to free the cellulosics from the lignin and this needs lignin catabolism. Some potential applications of lignocellulolysis might only become economic

74s

c, cornponent ). ciydrolyses amorphous and

Ticrocrystai line cel Iulose

ond Cx component).

Hydrolyses soluble cellulose derivatives

if this is achicved. The theories that have been put forward for lignin catabolism are (1) degradation by enzymes such as ligninases (Tien & Kirk 1984), and (2) oxidation by a non-specific ‘active-oxygen’ species such as singlet oxygen (Hall 1980; Janshekar & Fiechter 1983). The former option has also been investigated by Palmer er al. (1987) because this opened the opportunity of cloning genes with elevated enzyme activity (Paterson e f al. 1984).

The various enzyme components of the cellulase complex can act synergistically to enhance the overall cellulolysis (Wood & McCrae 1979). Nevertheless it can still be expected that some end- product accumulation will repress the enzyme system or inactivate it. It therefore might be expected that cellulolysis could be optimized by using mixed culture fermentations with a component member having an elevated //-glucosidase activity to match organisms such as Trichoderrna spp. with high b-1, 4-glucanase activity. We have been unsuccessful in our attempts to enhance cellulolysis by mixing Trichoderrna reesei with the yeast Hansenula californica isolated for its high b-glucosidase activity (D. M. Gaunt, A. P. J. Trinci & J. M. Lynch, unpublished). It could also be expected that mixing organisms with high cellulolytic and ligninolytic activity might result in greater lignocellulolysis but again we have been unsuccessful in accelerating substrate breakdown with T . reesei in co-culture with the ligninolytic actinomycete Therrnanospora mesophila. Such co-operations occur in nature and reconstructing dual- or multi-membered series should be feasible. Mixed culture fermentations for industrial applications might be expensive and technically difficult to control. Considering the cheap- ness of the lignocellulosic substrates, it might prove more satisfactory to accept a less technologically difficult process with a relatively low utilization of the substrate and in this respect ligninolytic activ- ity might be less important.

4. Lignocellulolysis in the environment

With the ubiquity of lignocellulose in the environment, there is a vast range of ecological niches where lignocellulolysis occurs. Examples can be found in soils, sediments, the rumen, silage, com- ponents and the sewage digestor. Lignocellulolytic pathways in the rumen are well understood (Latham 1979) and whereas there is considerable interest worldwide in the use of straw as animal feed, lignocelluloses with starch and pectin are the normal feed materials. Some of these systems, such as soil. proceed without any deliberate attempt to control the process whereas others (e.g. the sewage digestor) are highly managed with sophisticated technology. It is quite beyond the scope of this review to consider all of these in detail but an analysis will be given of our recent attempts to manage

Utilization of lignocellulosic wastes 7 5 s lignocellulolysis in soil, enhancing the positive value of plant residue decomposition at the expense of the negative value.

When straw is degraded in wet soils, anaerobic fermentation occurs, resulting in the formation of the fatty acids, acetic, propionic and butyric, with a concomitant decrease in pH (Lynch 1977). The redox potential for acids to accumulate is around zero (Lynch & Gunn 1978) and the process can occur in a range of ecological niches. In the sewage digestor it can cause problems in installations where methane produced is utilized as a fuel for the plant because it is indicative of a ‘sticking’ fermentation where the redox potential does not fall adequately to allow methanogenesis to take place. Whereas the process has been recognized for a long time (Marshall 1917), relatively little is known about the microbial species which are responsible. It is possible that cellulolytic/acid-forming bacteria will occur, although these have not been recognized, but it is more probable that cellulolytic fungi will generate simple sugars and utilize oxygen to create conditions for fermentative metabolism by bacteria. The problem for the plant is that the organic acids are phytotoxic in the millimolar concentration range and they appear to be one of the factors responsible for crop losses which can be between 13 and 29% in heavy clay soils when seed is direct drilled in the presence of surface straw (Graham et al. 1986).

Organic acids only accumulate around straw in the early stages of decomposition (Harper & Lynch 1981) and it therefore seemed possible that if straw breakdown could be accelerated by inoc- ulation or other means, then the ‘danger’ period of seeding for the farmer would be reduced. Defined mixed culture studies with T . harzianum (cellulase-positive, nitrogenase-negative) and Clostridium butyricum (cellulase-negative, nitrogenase-positive) on cellulose as the growth substrate indicated that the sugars generated by the fungus could be used by the bacterium and the N, fixed by the bacterium could be utilized by the fungus (demonstrated with a 15N label), although small additions of N (0.1 mg/ml as (NH,),SO,) promoted the process (Veal & Lynch 1984). The mixed culture degraded cellu- lose more rapidly than any of the pure cultures. Using non-axenic cultures with straw as substrate contained in glass columns and inoculated with C1. butyricum and Penicillium corylophilum as the cellulolytic fungus, accelerated rates of substrate decomposition and N-gain were observed as a con- sequence of the dual inoculation (Lynch & Harper 1983). Indeed with the T . harzianumlC1. butyricum system decomposition rates as great as 12 mg N/g straw have been observed (Lynch & Harper 1985). To assist this, a second bacterium, Enterobacter cloacae, which had frequently been isolated from straw-degrading communities, was added because it was felt that this would aid oxygen depletion and also provide respiratory protection to the anaerobe from the secretion of extracellular polysaccharide material. The challenge was to translate these laboratory observations to the field. Certainly it is possible to observe this three-membered series degrading straw in uitro (Fig. 3) and all three organisms can be recovered after 3 months from straw at levels similar to those inoculated in the field (P. Hand, N. Magan & J. M. Lynch, unpublished). Accelerated rates of straw decomposition have not been observed, however, in part caused by high natural populations of Fusarium spp. which, with Cl. butyricum and E. cloacae, degrades the straw naturally and these have similar cellulase activity to Trichoderma spp. This suggests that high levels of natural cellulolytic/N,-fixing activity may occur and this gains some support from field observations in Australia recording high rates of nitrogenase (acetylene reduction) in the presence of straw (Roper 1983). In nature it is unlikely that the fungal/ bacterial system will be the only one to contribute to such activity and Halsall et al. (1985) have indicated that the microaerophilic Azospirillum spp. which have nitrogenase activity also have xyla- nase activity, but little cellulase. To confirm the significance of such inoculated associations in the field it would be necessary to use I5N as a tracer, probably in closed systems such as lysimeters, to achieve an N budget as any N fixed can be easily lost by denitrification or mineralization and leaching.

A beneficial consequence of inoculation was that T . harzianum greatly reduced colonization of the straw by Fusarium culmorum, which is a plant pathogen. Such biocontrol potential was reported elsewhere (Papavizas 1985; Lynch 1987). It should also be recognized that E. cloacae is a powerful bio-control agent (Hadar et a/. 1983) and this could in part be responsible for the suppression of the Fusariurn. It thus seems that disease biocontrol could be a useful feature of the field inoculation of straw. Besides further evaluation of the field effects of inoculation, the inoculum production system needs investigation. Whereas fungi such as T . harzianum can be produced satisfactorily in submerged

76s J . M . Lynch

Fig. 3. Degradarion of H heat straw hy bacteria (Clo.srridium hutyricrtni and Eriterohacrer cloacae) and the hyphae (h) and spores (a) of the fungus Trichodrrmn hurziuriitm Bar markers 5 pm.

fermentation using soluble sugar substrates, the titre of conidia can be quite low. Some investigators have found that the homogenized mixtures of the fermenter medium are suitable as biocontrol inocula (Lewis & Papavizas 1985). but there are some situations where spore inocula might be more useful. Certainly conidia have long shelf-lives (P. Hand. unpublished). Furthermore, spores of strains with elevated levels of extracellular cell wall-degrading enzymes (Ridout ct al. 1986) can be obtained by growing T r i c h o d m n a spp. on solid substrates (fungal cell walls).

In regions where soil is poorly structured and plant growth is reduced as a consequence, straw can be retained as part of conservation tillage (Lynch & Elliott 1984). Straw-degrading micro-organisms generate the stabilization of soil aggregates by cementing and binding particles to form aggregates which are resistant to disruption by shaking in water, the laboratory simulation of rainfall (Lynch & Bragg 1985). Microbial exopolysaccharides are major factors in generating this stabilizing effect and whereas the chemical composition of some of these polymers has been determined, the effectiveness of the polysaccharides in aggregation seems to be more related to molecular weight (Chapman & Lynch 1985a). inoculation of straw with cellulolytic fungi and capsular bacteria can elevate the effect (Chapman & Lynch 1985b). Four co-cultures. Sordaria alcina with two strains of E. cloacae and a Pseudomonas sp. and T . /iar,-iunum with E . cloacae, were significantly more effective as inocula of sterile straw than a combined natural inoculum from straw. This suggests that inoculation of straw can produce a ‘compost’ to stabilize poorly structured soils. It also points to the potential damage of removing straw and other plant residues from soils which are inherently unstable.

One of the most obvious ‘products’ from straw is carbon dioxide. This is a relatively expensive commodity to enrich glasshouse atmospheres to obtain maximum photosynthesis capacity. Whereas

Utilization of lignocellulosic wastes 7 7 s

Fig. 4. Use of CO, generation from straw to enhance the CO, concentration in a glasshouse.

the gas can be generated by placing bales of dampened straw in a glasshouse, this is rather inconve- nient and it is diflicult to retain moisture. One enterprising West Sussex grower has built stacks of polyethylene-sheeted straw outside glasshouses and blown the gas into the house with a small fan, making a considerable cost-saving over the use of industrial carbon dioxide (Fig. 4). If the straw were inoculated with micro-organisms to carry out the functions described above, it is possible that the residue would provide a growing medium for plants which would act as a peat substitute or supple- ment that was resistant to disease. Preliminary experiments have demonstrated that a mixture of degraded straw with peat/grit can be as satisfactory as peat/grit alone for producing cucumbers (J. M. Lynch & E. K. Crawford, unpublished).

5. Waste treatment processes

Organic wastes containing lignocelluloses can cause a range of environmental problems through their degradation. For example, wastes from the pulp and paper industry can cause eutrophication of rivers and lakes and domestic municipal wastes can generate odour problems. An interesting tech- nique for deodorization of town wastes where sewage sludge storage produced a strong odour, has been to mix and cover the wastes with straw in stacks (J. Lopez-Real, personal communication) (Fig. 5). The static piles were aerated and composted according to the procedures developed by Finstein et al. (1983) at Rutgers University. The deodorized material so generated has provided a satisfactory growing medium for plants.

Another novel approach to utilization of straw would be to place bales on streams subject to eutrophication. Straw can cause mineral phosphorous (P) to be readily immobilized by the microbial biomass on the straw in slowly flowing feeder streams and prevent the P becoming available to algae and weeds in the streamjake, thus reducing algal growth (Wingfield et al. 1985).

There is a range of other agricultural wastes where enhanced microbial degradation might lead to useful products, for example sugar beet processing waste has been studied as a source of sugars using the cellulolytic complexes of Talaromyces emersoni and T . reesei (Coughlan et al. 1984).

78s J. M . Lynch

Fig. 5. Cornposting waste dewatered sewage sludge mixed with straw.

There have been extensive studies in Ohio on the utilization of composted municipal wastes and hardwood and softwood barks as horticultural growing media (Hoitink & Fahy 1987). Whereas the municipal wastes can give rise to satisfactory growing media, the composition is inevitably much more variable than the bark composts. Furthermore there is a risk of uptake of heavy metals by the plants and these could potentially be passed through food chains. There is now considerable Euro- pean interest in municipal waste composting, both as a means for waste disposal and to generate horticultural growing media (Gasser 1985). Relatively little is known about the ‘process’ microbiology of these composts and most microbiological investigations have been concerned with the retention of human or animal pathogens (mainly bacteria and viruses) in the composts.

The process microbiology of the bark composts has been studied in more detail and it has been observed that populations of Trichoderrna spp. are high in the mature composts and that these are important components of the population responsible for disease suppression (Hoitink & Fahy 1986). These ‘natural’ populations are somewhat variable and there is probably considerable scope for inoc- ulation of the composts with specific strains of Trichoderrna or other biocontrol fungi with a view to improving the consistency and effectiveness of the product.

Anaerobic waste treatment is frequently practised and sometimes fermentation products such as methane are recovered to fuel the treatment plant. The anaerobic treatment of liquid waste has been the subject of extensive research in Europe (Ferrero et al. 1984) but less scientific study has been made of ‘dry’ anaerobic digestion, particularly landfill technology (Grainger et al. 1984). Here there appears to be considerable commercial scope and inoculants are available to control the fermenta- tion.

6. Controlled non-axenic fermentations

Mushroom production can be considered as a controlled non-axenic fermentation and it is the only economically viable product from lignocellulosic residues produced on a world scale (Wood 1985). It is also one of the very few proven examples of agricultural biotechnology in commercial practice

Utilization of lignocellulosic wastes 79s (Tautorus & Townsley 1984). The most widely cultivated fungus on a world scale is Agaricus bisporus (about 75% of total production) which is grown on composted straw (Fig. 6). In Britain it is the major protected horticultural cash crop (ca €103 million per annum), being considerably higher in value than tomato (ca €53 million per annum) and with a greater consumption in many other parts of the world, it is of even greater importance.

In brief the production procedure is: (1) Growth of spawn on sterilized cereal grains. (2) Composting of straw with manure and fertilizers at 60"-70"C for 2 weeks. (3) 'Peak heating' of compost at 50"-5S"C for 5-7 d. (4) Spawn inoculation and 'run' at 25°C for 2-3 weeks. (5) Application of casing layer (peat and chalk). (6) Fructification in about four flushes for 4-6 weeks. After A. bisporus, Lentinus edodes or 'Shii-take', which is grown predominantly in Japan on whole

oak pieces (Chang & Hayes 1978), is the next most important mushroom with Voluariella uoluaceue and Pleurotus ostreutus following. In general the mushroom industry has grown constantly in volume output terms in the last decade although, with relatively decreased prices, profitability has not increased at the same rate. There must be scope, however, for further development of the industry, especially as mushrooms have sometimes been linked with the 'health food boom'. Also, with the increased cosmopolitan nature of British tastes, it is possible that there will be further scope for the introduction of 'exotic' mushrooms to the British market; one of the drawbacks at present is that a premium of about 100% needs to be charged for economic viability.

Wood (1985) has compared the economics of mushroom cultivation technology with feed or single cell protein (SCP) production and outlines some of the reasons why the former, unlike the latter, is viable commercially. The major problems with SCP production are that the feedstock frequently needs hydrolysing, 'high technology' fermenters are required for sterile culture and the product prices are non-competitive with feed such as soybean. Furthermore, the products are feed and not foods for humans so prices which can be charged are relatively low. Some options for using basidiomycetes to provide feeds have been suggested (e.g. Zadrazil 1980) but have never been fully evaluated. This could produce an exciting new perspective from lignocellulolysis research. Besides using the mushrooms as protein, it is likely that the enzymic treatment of straw would increase its digestibility, unlike the

Fig. 6. Production of mushroom compost

80s J . M . Lynch alkali hydrolysis of straw which has been somewhat dubious economically. Whereas the rumen itself provides a convenient. but somewhat inefficient, ‘mobile fermentation system’, it is relatively cheap and requires little technological input (Latham 1979). Grant er al. (1978) described a pilot-scale semi- solid fermentation of straw using the yeast Critidida utilis to increase the protein content, crude fat content and in r i m rumen digestibility. However this, in common with several other processes which have been described. has not become a commercial success.

7. Industrial fermentations

The prospects of industrial products from lignocellulose fermentations have been discussed by Evel- eigh 11983) and Kirk (1983). Wood (1985) has analysed lignocellulose bioconversion products in relation to other biotechnology products and bases this on a double logarithmic plot of the relation- ship between price and tonnage from the data of Dunnill (1983). The following points arise:

(1 ) Bioconversinn is used where chemical routes are too difficult or costly (e.g. penicillin, interferon).

( 2 ) High value products are used in health care and produced in low tonnages. (3) Low value products are bulk materials such as feed, beverages and related materials. Lignocellulose fermentations could only satisfactorily be linked to the latter category and, with the

exception of mushroom production, there is no economic process developed as yet. Even in stirred tank submerged fermentations. lignocellulose utilization must be regarded as solid substrate fermen- tation (Moo-Young ef a/ . 1983). an area of fermentation research which has been unfashionable but in which there is a growing interest.

The technology IS available for several potential bioconversion processes from wood and straw but there are constraints on the profitability. For example. biopulp is of dubious quality and high cost. Bulk chemicals such as ethanol, glucose and butanediol can be produced from other feedstocks more competitively. and speciality chemicals such as polymers and phenolics, and enzymes such as cellu- lases and hemicellulases suffer from high product cost compared with liquid fermentation or chemical processes. The major question which arises is whether the economics could be made competitive by modifying the organisms genetically or whether the fermentation process itself could be significantly modified. Using conventional mutagenic agents (u.v.-light and nitrosoguanidine) mutants of the fungus Petiicillium pinophilurn hyperproducing cellulase, P-glucosidase and xylanase have been selected (Wood er a/. 1984).

One of the major product targets from cellulose and hemicellulose has been glucose and other sugars but, with the availability of sugar from plant sources, the better economic prospect might be to use the sugars as intermediates for other as yet unidentified products. Ethanol has been the major true product target from celluloses (Eveleigh 1983) and CI. thermocellum has been a major focus of study even though i t produces ethanol and acetate in about equal quantities. Cloning of two different genes from this bacterium, both coding for an enzyme capable of hydrolysing carboxymethylcellulose (a soluble substrate for cellulase assays) has been reported (Cornet et al. 1983). Whereas many inter- esting scientific and technological developments have taken place in this direction in recent years, and there is now the potential for recombinant DNA technology to replace the regulatory pathway with a new one that can give higher yields, the sudden fall in world oil prices has reduced the incentives for further developments and the existing knowledge may need to be stored until a more favourable economic climate develops.

Coombs ( 1987) has analysed the total world market for various fermentation products (Table 2) and concludes that the only significant market is ethanol for use as an octane enhancer or petroleum additive or as a chemical feedstock. In Brazil, where cane juice is used as the fermentation feedstock, ethanol does not compete favourably with naptha as a feedstock for polyethylene production. Cane juice IS of course a high ‘quality’ fermentation substrate compared with most lignocelluloses and therefore the prospects from crop residues as feedstocks are not good. However, socio-economic and political factors must be considered and, for example, a useful guide to the economics of on-farm production and use of ethanol has been produced in the US (Anon. 1980).

The potential of the enzymic degradation of lignin to supply chemicals has been reviewed by

Utilization of lignocellulosic wastes Table 2. Some potential products of

lignocellulose biodegradation

81s

Cellulose/ hemicellulose Lienin

Acetic acid Acetic acid Acetone Acetovanillone Citric acid Cresol Ethanol Dimethylsulphide Fumaric acid Syringaldehyde Glucose Vanillic acid Lactic acid Vanillin Xylose

Harvey et at. (1985). The pulp and paper industry is probably the principal source of waste lignins. The potential advantages for enzyme over chemical processing are (1) greater substrate and reaction specificity; ( 2 ) lower energy requirements; (3) lower pollution generation; (4) higher yields of desired products and ( 5 ) a greater number of transformations are possible. Although many natural polymers are biodegraded hydrolytically, the intermonomeric units of lignin are non-hydrolysable. The lignin biodegradative processes would mainly provide sources of aromatic chemicals as outlined in Table 2 but as yet there have been no economic feasibility studies of such processes. Whereas enzymic degra- dation may offer more potential than chemical processes, the latter are not used industrially at present and therefore the major competition comes from chemical processes using other substrates. Whereas more detailed enzymological and genetical studies will undoubtedly lead to enhanced lignin biodegradation, even with the present state of knowledge it would be appropriate to set goals of what would be needed of a biodegradative process. To improve conversion efficiencies by one order of magnitude might be possible but if two orders of magnitude are needed economically, then the demands on the enzymes might prove too great. It is, of course, also possible that new chemicals might be found from biodegradative processes but that must be speculation at present.

8. Future prospects

This essay was started with a question of the relative value of natural us controlled processes for lignocellulose biodegradative processes. Clearly there are useful environmental processes of lignocel- lulolysis, including the rumen, and there is some prospect of enhancing these processes in nature by inoculation. The only proven controlled or industrial process of lignocellulolysis is that of mushroom production and there still seems considerable scope for the development of this industry worldwide. Microbiologists can make major contributions by selecting or engineering new strains with enhanced biodegradative and fruiting capacities.

At present there are no economically proven examples of utilizing industrially what would nor- mally be regarded as lignocellulosic wastes (straw, wood, municipal refuse, etc.). Composting as a means of waste disposal is not competing well with landfill but there is the possibility of microbiolo- gists making inputs to the technology of the latter with a view to controlling methanogenesis. However, with increasing grain surpluses, better ‘quality’ lignocelluloses are becoming available as wastes and with their additional more readily available utilizable energy sources such as starch, they may provide new opportunities for the utilization of lignocellulosic materials. There is also the opportunity for the utilization of fractionated products, such as bran. A new era of lignocellulolysis research could emerge.

9. Acknowledgements

My studies in this area have been supported financially by the EEC under Contract UW-033-UK, and the Agricultural Genetics Company in association with the Department of Industry. Collabo- iators who hbve produced much valued support are Mr S.H.T. Harper, Dr S.J. Chapman, Dr D.A. Veal, Dr D.A. Wood, Dr N. Magan, Dr P. Hand and Dr D.M. Gaunt.

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