Gene. 115 (1992) 189-192 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00

GENE063~

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Actinomycetes as agents of biodegradation in the environment - a review*

(Composting; enzymology; lignocellulose degradation; microbial ecology; recombinant DNA technology)

Alan J. McCarthy and Stanley T. Williams

Department of Genetics and Microbiology, University of Liverpool, Liverpool L69 3BX (UK)

Received by T.W. Jeffries: 20 September 1991 Revised/Accepted: 5 November 1991 Received at publishers: 9 December 1991

SUMMARY

The diversity of form in the Actinomycetales is wcll-recognised, due to the sustained generation of environmental isolates for pharmaceutical screening. Actinomycetes isolated from soil and related substrates show primary biodegradative activ- ity, secreting a range of extracellular enzymes and exhibiting the capacity to metabolise recalcitrant molecules. Composting is one process which relies heavily on such prolific actinomycete activity. Amongst actinomycetes in soil, there are exam- ples of different strategies, from cycles ofrapid proliferation and sporulation to the maintenance of populations by prolonged slow growth and scavenging, and the evidence for this is examined. The mechanisms of lignocellulose degradation by actinomycetes are discussed in relation to functional conservation within the group, and correlations with those described in other bacteria and fungi.

INTRODUCTION

The application of recombinant DNA technology to the design of microorganisms which upon release might con- fer agricultural or environmental benefits has exposed the limitations of our underst~ding of microbial ecology. The intense debates on potential perturbation of microbial pop- ulations and processes quickly became focussed on the confidence limits for detection of gene survival and trans- fer in the environment. This led directly to the molecular approach to environmental microbiology where the goal was to develop highly sensitive techniques which would improve or even replace the traditional methods for recov-

Correspondence to: Dr. A.J. McCarthy, Genetics and Microbiology De- partment, University of Liverpool, Liverpool, L69 3BX (UK) Tel. (44-51)7944413; Fax (44-51)7086502. * Presented at the International Symposium on Biology of Actinomycetes, University of Wisconsin, Madison, WI (USA) 11-16 August 1991.

Abbreviations: cfu, colony-forming unit(s); P., Phanerochaete; T., Ther- momonospora.

ery and enumeration ofviable microorganisms as the means of monitoring populations. The ability to detect a popula- tion or even an individual by a method which does not rely on gene expression is seen as the ideal, and targeting ribo- somal RNA genes with amplification using the polymerase chain reaction can be regarded as the strategy with the widest appeal. Irrespective of views on the extent to wh~,.h this approach will change our perception of microbial ecol- ogy, it has already contributed by stimulating a new up- surge in research on environmental microbiology.

Three key elements in the analysis of natural populations can be identified - detection, diversity and activity. Most progress has been made in the former, where nucleic acid- based techniques have increased the sensitivity of detection for certain bacterial taxa, though not yet actinomycetes. The likelihood that diversity in natural microbial commu- nities has been underestimated by reliance on the identifi- cation of recovered isolates has been realised.

Direct analyses of 16S rRNA sequences present in a marine bacterioplankton community (Giovannoni et al., 1990) and a thermal spring habitat (Weller et al., 1991) have indeed revealed diversity far beyond that previously

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recognised in these environments. Similar insights can be predicted for a number of physiological groups such as autotrophs and o!igotrophs, but whether substantial diver- sity remains to be discovered in the biodegradative actino- mycetes is questionable. The development of molecular techniques for estimating microbial activity in the environ- ment, for example by quantifying differential hybridisation to ribosomal RNA indicative of gene expression, is the most demanding area and the one where least progress has been made to date. This is particularly relevant to actino- mycetes, since they are often likely to be present in the environment as dormant spores rather than active metab- olising hyphae. It is the role of biodegradative actino- mycetes in the environment, and particula,:!y their func- tional diversity which is discussed here.

GENERAL FEATURES AND ADAPTATIONS

Most biodegradative actinomycetes are saprophytes col- onising, or originating from, soil where they are usually present at 10 S- l0 s cfu/g. These organisms are aerobic and neutrophilic, comprising a range of forms belied by the predominance of streptomycetes in routine isolation sur- veys. They con utilise a wide range of carbon sources and it is this metabolic diversity together with the capacity for prolific sporulation which accounts for their ubiquity. In some of their physiological properties, actinomycetes do however show limited variation. For example, there appear to be no true 'extremophiles', thermophilic and acidophilic actinomycetes being largely classified as facultative (see Edwards, 1990).

Actinomycetes are adapted to growth on solid substrates. Their primary carbon sources in the soil are insoluble and polymeric, necessitating the secretion of a range of extra- cellular enzymes as hyphae penetrate and colonise the sub- strate. Nutrients are often present in low concentrations with sporadic temporary increases in availability. Williams (1985) discussed the distinction between autochthonous and zymogenous microbial populations in soil, and partic- ularly whether there was any evidence for the former. Bio- degradative actinomycetes appear to be good examples of zymogenous microorganisms as nutrient exhaustion leads to sporulation. The spores of most actinomycetes are borne on aerial hyphae and are hydrophobic, adapted for air dispersal. However, sporangia-forming actinomycetes and micromonosporas which do not form aerial hyphae, pro- duce hydrophilic spores dispersed by water movement through the soil. Actinomycete spores are functionally more akin to fungal conidia than to bacterial endospores, but they can survive prolonged desiccation prior to germinat- ing when nutrients again become available. There are some examples of autochthonous or autochthonous-fike behav-

iour, i.e. sustained growth at low nutrient concentrations, amongst actinomycetes. Micromonosporas are common in soil and all appear to be cellulolytic, but they are slow- growing with colonies requiring up to 21 days for develop- ment on isolation plates. It has been suggested (R. Cain, personal communication) that soil enrichment cultures for bacteria capable of aromatic ring cleavage yield pseudomonads at high substrate concentrations, but no- cardioform actinomycetes at low substrate concentrations. In contrast, Sorkhoh et al. (1990) report that the actino- mycete Rhodococcus rhodochrous greatly outnumbers other hydrocarbon-degrading bacteria in oil-saturated soil sites in Kuwait. Finally, Lemmer (1986) has observed both physiological strategies in a population of actinomycetes forming scum in a sewage treatment plant. At high sub- strate concentrations, growth rates and yields are commen- surate, while at low concentrations scavenging growth characterised by high nutrient uptake efficiency is adopted. Thus the population remains competitive across a range of nutrient concentrations. Although inconclusive, it is clear that natural actinomycete populations cannot be regarded simply as environmental housekeepers while the numbers of unicellular bacteria such as pseudomonads and bacilli dramatically rise and fall as easily metabolisable nutrients become available and are exhausted.

SAPROPHYTIC ACTIVITY

Plant biomass is the major carbon source in the terres- trial environment and that is composed largely of lignocel- lulose. This is a complex and highly interactive polymeric substrate whose degradation demands the cooperative ac- tivity of a range of hydrolytic and oxidative enzymes. Fungi are generally regarded as the most important primary iignocellulose-degraders, and although actinomycetes and other bacteria are recognised as being active, their relative ecological importance is often questioned. Actinomycete cellulases and hemicellulases exhibit a strong preference for neutral to alkaline pH conditions (McCarthy, 1987), while those of fungi show optimal activity under acid con- ditions. Large populations of lignocellulose-degrading acti- nomycetes develop in composts, particularly those pre- pared for the cultivation of mushrooms, where the alkaline conditions resulting from high ammonification is probably the most important parameter. There is no doubt that actinomycete-mediated degradation of lignoceUulose is crucial to the composting process.

The biochemistry of cellulose hydrolysis is the most intensely studied aspect of lignocellulose degradation. The complex physical structure of crystalline cellulose is responsible for its recalcitrance and several enzymatic mechanisms have evolved in microorganisms. Although

endoglucanase activity is reasonably common amongst actinomycetes, good activity against native cellulose is not. The thermophilic actinomycete Microbispora bispora ap- pears to be the most strongly cellulolytic (Yablonsky ctal., 1988; Ball and McCarthy, 1988); this has a similar strat- egy to that used by cellulolytic fungi, i.e., the secretion of multiple enzymes, including cellobiohydrolase, which act synergistically to effect degradation :rod permit the ramifi- cation of hyphae throughout the substrate. T.fusca en- doglucanases have been intensely studied at the molecular level, and here the multiple forms of this enzyme species show homologies to cellulases from a range of micro- organisms - fungi, actinomycetes and other bacteria (Lao et al., 1991). Finally, mycelium-bound cellulase reminiscent of the bacterial 'cellulosome' has been described in Streptomyces reticuli (Wachinger et al., 1989), thus dispens- ing with any simple conclusions about the functional evo- lution of cellulolytic activity in actinomycetes.

Hemicelluloses are chemically heterogeneous but rela- tively amenable to degradation such that activity against xylan, the predominant form of hemicellulose, is very com- mon in actinomycetes. The most important enzyme activ- ity is endoxylanase, and multiple fi~rms of this enzyme are secreted by microorganisms. Streptomycete endoxylanases can be differentiated from those of other bacteria by the relatively neutral pIs of their high-molecular-weight com- ponents (Wong et al., 1988) and this also applies to T. fusca endoxylanases (Bachmann and McCarthy, 1991). Like bacteria, but not fungi,//-xylosidase activity appears to be coil-associated and multimeric in actinomycetes (Bach- mann and McCarthy, 1989). Further evidence for func- tional conservation within actinomycete xylan-degrading systems is provided by the strong similarity between the only actinomycete arabinofuranosidases purified, from a streptomycete (Kaji et al., 1981) and T. fusca (Bachmann and McCarthy, 1991).

Degradation of the lignin component of plant biomass is the most contentious aspect, with no strong evidence that actinomycetes can depolymerise lignin on a scale compa- rable to that exhibited by wood-rotting fungi such as Phanerochaete chrysosporium. The ability to solubilise ligno- carbohydrate, recoverable from actinomycete culture su- pernatants by acid precipitation, was first described by Crawford et al. (1983) and now appears to be a common feature of biodegradative actinomycetes (Ball et al., 1990). This activity could make an important contribution to hu- mification in soil, since the material shows some of the properties of humic acid including a strong ability to bind active enzymes. Extracellular peroxidase activity is respon- sible for lignin depolymerisation in P. chrysosporium, and although it has been described in ligninolytic streptomycetes (Ramachandra et al., 1988) and other actinomycetes (Ball et al., 1990), there is a paucity of biochemical evidence for

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its role in plant biomass degradation by these organisms. In studies on the degradation and utilisation of lignin- related compounds, indicators of ligninolytic activity in P. chrysosporium were not found to be good criteria when applied to actinomycetes, nor are there any similarities in physiological regulation (Ball et al., 1989). However, the pathway for degradation of dimeric lignin model com- pounds appears to be similar in different actinomycetes (unpublished data) and can also be found in other micro- organisms.

CONCLUDING REMARKS

Nutrient recycling in the terrestrial environment requires the concerted action of a community of microorganisms in which actinomycetes are important as primary degraders. Lignoceilulose is the largest reservoir of carbon but its com- plex structure also provides a good model for studying interactions between biodegradative enzymes and micro- organisms. The common criticism that such studies con- centrate on purified substrates and axenic cultures is valid, but experiments with natural substrates and communities present many difficulties, not least with reproducibility. Simple laboratory experiments can, however, provide in- sights, a good example of which is the relationship between actinomycete-solubilised lignocarbohydrate and the humic complexes of soils and composts.

REFERENCES

Bachmann, S.L. and McCarthy, A.J.: Purification and characterization of a thermostable//-xylosidase from Thermomonospora fusca. J. Gen. Microbiol. 135 (1989) 293-299.

Bachmann, S.L. and McCarthy, A.J.: Puri.qcation and cooperative ac- tivity of enzymes constituting the xylan-degrading system of Thermomonospora fusca. Appl. Enviror, Microbiol. 57 ( ! 991) 2121 - 2130.

Ball, A.S. and McCarthy, AJ.: Saccharification of straw by actinomycete enzymes. J. Gen. Microbiol. 134 (1988) 2139-2147.

Ball, A.S., Betts, W.B. and McCarthy, A.J.: Degradation oflignin-related compounds by actinomycetes. Appl. Environ. Microbiol. 55 (1989) 1642-1644.

Ball, A.S., Godden, B., Helvenstein, P., Penninckx, M.J. and McCarthy, A.J.: Lignocarbohydrate solubilization from Araw by actinomycetes. Appl. Environ. Microbiol. 56 (1990)3017-3022.

Crawford, D.L., Pometto, A.L. and Crawford, R.L.: Lignin degradation by Streptomyces vin'dosporus: isolation and characterisation of a new polymeric iignin degradation intermediate. Appl. Environ. Microbiol. 45 (1983) 898-904.

Edwards, C. (Ed.).: Microbiology of Extreme Environments. McGraw- Hill, New York, 1990.

Giovannoni, S.J., Britschgi, T.B., Meyer, C.L. and Field, K.G.: Genetic diversity in Sargasso Sea bacterioplankton. Nature 345 (1990) 60-63.

Kaji, A., Sate, M. and Tsutui, Y.: An ~-L-arabinofuranosidase produced by wild-type Streptomyces sp. no. 17-1. Agri. Bio. Chem. 45 (1981) 925-931.

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Lao, G., Ghangas, G.S., Jung, E.D. and Wilson, D.B.: DNA sequence of three fl-l,4-endoglucanase genes from Thermomonospora fusca. J. Bacteriol. 173 (1991) 3397-3407.

Lemmer, H.: Actinomycete scum in sewage treatment plants. In: Szabo, G., Biro, S. and Goodfellow, M. (Eds.), Biological, Biochemical and Biomedical Aspects of Actinomycetes. Akademiai Kiado, Budapest, Hungary, 1986, pp. 731-733.

McCarthy, A.J.: Lignocellulose-degrading actinomycetes. FEMS Micro- biol. Rev. 46 (1987) 145-163.

Ramachandra, M., Crawford, D.L. and Hertel, G.: Description of an extracellular lignin peroxidase of the lignocellulolytic actinomycete actinomycete, Streptomyces viridosporus. Appl. Environ. Microbiol. 54 (1988) 3057-3063.

Sorkhoh, N,A., Ghannoum, M.A., Ibrahim, A.S., Stretton, R.J. and Rad- wan, S.S.: Sterols and diacylglycerophosphocholines in the lipids of the hydrocarbon-utilizing prokaryot¢ Rhodococcus rhodochrous. J. Appl. Bacterioi. 69 (1990) 856-863.

Wachingcr, G., Bronnemneier, K., Staudenbauer, W.L. and Schrempf,

H.: Identification of mycelium-associated cellulase from Streptomyces reticuli. Appl. Environ. Microbioi. 55 (1989) 2653-2657.

Weller, R., Weller, J.W. and Ward, D.M.: 16S rRNA sequences of un- cultivated hot spring cyanobacterial mat inhabitants retrieved as ran- domly primed cDNA. Appl. Environ. Microbiol. 57 (1991) 1146- 1151.

Williams, S.T.: Oligotrophy in soil: fact or fiction? In: Fletcher, M.M. and Floodgate, G.D. (Eds.), Bacteria in their Natural Environments. Ac- ademic Press, Orlando, FL, 1985, pp. 81-110.

Wong, K.K.Y., Tan, L.U.L. and Saddler, J.N.: Multiplicity of fl-l,4- xylanase in microorganisms: functions and applications. Microbiol. Rev. 52 (1988) 305-317.

Yablonsky, M.D., Bartley, T., Elliston, K.O., Kahrs, S.K., Shalita, Z.P. and Evcleigh, D.E.: Characterisation and cloning ofthe cellulase com- plex ofMicrobispora bispora. In: Aubert, J.-P., Beguin, P. and Millet, J. (Eds.), Biochemistry and Genetics of Cellulose Degradation. Ac- ademic Press, London, 1988, pp. 249-266.