Hydrolysis of Cellulose: Mechanisms of ... - American Chemical Society

14 Selective Screening Methods for the Isolation of High Yielding Cellulase Mutants of

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Trichoderma reesei B L A N D S. M O N T E N E C O U R T and D O U G L A S E . E V E L E I G H Department of Biochemistry and Microbiology, Cook College, Rutgers—The State University of New Jersey, New Brunswick, NJ 08903

Screening methods for the selection of high-yielding cellulase mutants of Trichoderma reesei have been devised that specifically detect hyperproducing, catabolite repression -resistant, and end-product inhibition-resistant strains. Indi vidual selection techniques employing specific substrates and end-product inhibitors allow for visual screening of a single enzyme in the cellulase complex: endo-β 1-4 glucanase and β-glucosidase (cellobiase) as well as the synergistic action of all the enzymes of the cellulase complex. By use of these methods, mutant strains have been isolated that hyperproduce cellulase and are resistant to catabolite repression by glucose or glycerol.

Tndustrial scale enzymatic hydrolysis of cellulose, the world's most abundant renewable carbon source, has been envisioned as a route toward the conversion of cellulose to fuels, food, and chemicals. En zymatic hydrolysis offers certain advantages in such a process, as enzymes are reusable, energy sparing, nonpolluting, and promote high conversion efficiencies without undesirable side products. To date, the overall con version of cellulose to glucose employing cellulase enzymes has been hampered by the low yields and associated high cost of the microbial enzymes used. Our specific emphasis has been to increase enzyme pro duction by Trichoderma reesei, since this organism is considered the best source of all of the enzymes in the cellulase complex (I). There are three primary enzymes thought to act synergistically to degrade crystal line cellulose in Trichoderma: l,4-/?-D-glucan 4-glucanohydrolase ( E C 0-8412-0460-8/79/33-181-289$05.00/0 © 1979 American Chemical Society

In Hydrolysis of Cellulose: Mechanisms of Enzymatic and Acid Catalysis; Brown, R., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1979.


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3.2.1.4) (endoglucanase), l,4-/?-D-glucan cellobiohydrolase ( E C 3.2.1.91) (cellobiohydrolase), and β-glucosidase ( E C 3.2.1.21) (cellobiase). The current concept of the interrelationship of these enzymes envisions an initial attack by the endoglucanases to yield oligosaccharides followed by the release of cellobiose from their nonreducing termini by the action of the cellobiohydrolase. Glucose is subsequently produced from cello biose through the action of β-glucosidases (2). Multiple forms of each of these enzymes have been isolated which show distinctly different biochemical properties and in some cases exhibit different modes of action {3,4,5,6). Mechanisms Controlling Enzyme Synthesis and Activity The mechanisms controlling the synthesis and activity of each of the cellulase complex of enzymes are summarized in Table I.

Table I.

Biochemical Mechanisms Controlling Cellulase Synthesis and Activity in Trichoderma reesei

Enzyme

End-Product Inhibitor

Catabolite Repressor

Cellobiohydrolase Endoglucanase

cellobiose cellobiose

Cellobiase

glucose

glucose or metabolites of glucose

Inducer cellulose sophorose cellobiose

Catabolite Repression The early observations on glucose and catabolite repression in micro organisms were reviewed as long ago as 1943 (7). The specific term "catabolite repression" was coined by Magasanik (8) and is defined as the mechanism by which organisms decrease the rate of synthesis of catabolic enzymes when the catabolites accumulate to an excess in the cell. The biochemistry and genetics of catabolite repression in prokaryotes are now largely understood and are described in several excellent reviews (9,10,11). It is generally accepted that catabolite repression in E. colt is mediated by the cyclic AMP system (11). However, far less is known about the biochemistry and genetics of glucose and catabolite repression of enzymes involved in carbon metabolism in eukaryotes. Cellobiohydro lase, endoglucanase, and cellobiase are all subject to catabolite repression in Trichoderma (12,13,14). A number of other enzymes, for example, invertase (15), maltase (16), and alcohol dehydrogenase (17) have also been shown to be sensitive to catabolite repression in fungi.


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Cellulase Mutant Isolation

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End Product Inhibition


In addition to catabolite repression, the cellulase enzymes themselves are subject to end-product inhibition. For example, as glucose accumu lates during saccharification, it interacts noncompetively with cellobiase to inhibit further activity of this enzyme (6). Similar inhibition of endoglucanases occurs when cellobiose accumulates in a saccharification reactor (18,19,20). Cellulase Induction All naturally occurring fungal strains of Trichoderma require an inducer for cellulase synthesis. In the absence of an inducer such as cellulose, cellobiose (21,22), or sophorose (12,13,14,23), Trichoderma does not make any detectable "cellulase complex" enzymes. The true physiological inducer of cellulase is currently unknown. Insoluble cellu lose is presumably not such an inducer since there is no way for the internal cell machinery to sense the presence of this insoluble material. However, a small transglycosylation product such as sophorose, 2-Ο-βglucopyranosyl-D-glucose, may well be the natural inducer. The simultaneous action of these physiological and enzymatic regula tory mechanisms during industrial enzyme production and large scale saccharification represents one of the primary bottlenecks in the eco nomical production of glucose syrups from cellulosic materials. Our objectives have been to devise selective screening techniques which allow for the isolation of mutant strains that can specifically overcome these catabolite repression and end-product inhibition regulatory mechanisms and at the same time hyperproduce one or all of the individual enzymes in the cellulase complex. Such mutants will be useful in reducing the cost of enzyme production as well as contributing knowledge towards a basic understanding of the mechanisms controlling enzyme synthesis, secretion and mode of action at the cellular level. Previous efforts to increase cellulase production by Trichoderma (24,25) have met with limited success primarily due to the lack of selective screening methods. Problem areas have included an inability to isolate the progeny of a single spore because of the fast-spreading nature of the fungus and lack of a visual screening method that would allow selection on agar medium. All survivors after mutation have previously been tested individually in shake flasks for evaluation of enzyme produc tion. Addition of colony-growth restrictors such as oxgall (26), rose bengal (27), Phosfon D (28), and nonionic detergents such as deoxycholate (29) and saponin (30,31), as well as other paramorphologic agents (32,33), have largely overcome thefirstproblem.



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Incorporation of such colony restrictors into agar medium allows development of small discrete colonies from individual spores; as many as 50-100 colonies can be screened per petri plate, depending on the colony-restricting substance used. Visual detection of cellulase activity on agar plates in a reasonably short period of time has proved to be more difficult. Plate assays based on clear zone diameters have been reported utilizing cellulose swollen with phosphoric acid as a substrate (34,35). However, these agar diffusion assays have not been coupled with a colony-growth inhibitor to allow for screening of more than one organism at a time and require 7-14 days for visual detection of clearing. Recently, tests have been described (36,37) which employ celluloseazure in an agar overlay for detecting fungal cellulase production. Again the test is limited to one organism per tube, which rules out its use in large-scale selective screening of mutants. In screening for mutants that hyperproduce all of the enzymes in the cellulase complex, we have effectively utilized Walseth acid swollen cellulose as a substrate in con junction with 1.5% oxgall and 500 /xg/mL Phosfon D as colony growth inhibitors (38). Rapid evaluation of the mutants after colony formation can be achieved after overnight incubation of the plates at 50°C. The oxgall and the Phosfon D act together to retard hyphal spreading and in some way enhance visualization of cellulase activity. Although 50 °C is a temperature which is not permissive for growth of Trichoderma, it is the optimum temperature for the activity of the cellulase enzymes. Incubation of the screening plates at this temperature allows rapid expression of the enzymatic activities, resulting in distinct clear zones surrounding the potential mutant colonies when acid swollen cellulose is incorporated as a substrate in the agar plates.

Table II. Screening Methodologies for Isolation of High-Yielding Enzyme

Colony Inhibitor

Substrate

Total cellulase complex

1.5% oxgall + Phosfon D

acid swollen cellulose

Endoglucanase

1.5% oxgall + Phosfon D

carboxymethylcellulose

β-Glucosidase

(1) 1.5% oxgall + Phosfon D

esculin


1% cellobiose


any of above


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293

Selection of Hyperproducing Mutants for Each of the Enzymes in the Cellulase Complex


The screening methodologies used in the selection of high yielding cellulase mutants are summarized in Table II. Mutants with enhanced production of all the enzymes in the cellulase complex can be isolated utilizing oxgall, Phosfon D, and acid swollen cellulose as described above. Sequential increased production can be detected by increasing the cellu lose concentration, the agar thickness, and ultimately selection for colonies with enhanced ratios of clear zone diameter to colony diameter. Selection of hyperproducing endoglucanase mutants can be achieved by substituting a modified cellulose such as carboxymethylcellulose ( C M C ) for the acid swollen cellulose and retaining the oxgall and Phosfon D as colony growth inhibitors. C M C is not attacked by cellobiohydrolase or cellobiase but is subject to random splitting by the endoglucanase to yield oligomers of glucose. After growth and colony formation, the colonies are replicated and then plates flooded with cetylpyridinium chloride (other quaternary ammonium compounds may be substituted (39)). These quaternary ammonium compounds react with the long chain polysaccharides to form a dense white precipitate. Where the organism has secreted large quantities of endoglucanases, the C M C will have been digested and clear zones will be formed. Again sequential enzyme increases may be visualized by raising the C M C concentration and selecting colonies with increased ratios of clear zone diameter to colony diameter. Cellobiase or β-glucosidase mutants can be detected by a number of plate assay techniques: (1) esculin-ferric ammonium citrate, (2) cello-

and Catabolite Repression-Resistant Cellulase Mutants Catabolite Repressor 5% glycerol

Visualization clear zones after incubation at 50°C

5% glycerol or 5% glucose clear zones after flooding with quaternary ammonium compounds 5% glucose

black rings in the presence of ferric ammonium citrate

0.2% 2-deoxyglucose

large versus pinpoint colonies

5% glycerol

overlay with Glucostat + cellobiose, 50 °C, 30 min, red spots



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biose-2-deoxyglucose, or (3) cellobiose-glucose oxidase. In each case oxgall and Phosfon D are incorporated as colony inhibitors. The first method, esculin-ferric ammonium citrate, is a modification of a method previously described by Eberhart (40) and employs 0.1% esculin (6,7dihydrocoumarin-6-glucoside) as a substrate. In the presence of arylglucosidase, esculin is split into glucose and esculetin. The esculetin reacts with the ferric ammonium citrate (0.05% ), which is present in the agar, to form a black precipitate. In the second method, cellobiose (1% ) and 2-deoxyglucose (0.2-0.5%) are incorporated in the agar. Those organisms which are unable to make cellobiase are forced to utilize the 2-deoxyglucose and after minimal growth die because of the toxicity of the antimetabolite. Those mutants capable of synthesizing cellobiase are able to "ignore" the 2-deoxyglucose and grow on the cellobiose. The size of the colony should be directly proportional to the amount of cellobiase and thus mutants are distinguished from wild type on the basis of colony size. The third method for detecting cellobiase activity on plates uses a modification of a method previously described for the selection of high-yielding yeast-invertase mutants (15). The surviving colonies are overlayed with a piece of filter paper, which is sprayed with a mixture of cellobiose and Glucostat reagent (Worthington Biochemicals, Freehold, NJ). After incubation at 50°C for 30 min, those colonies with cellobiase activity will yield red spots on the filter paper resulting from the reaction of the glucose, derived from cellobiose, with the Glucostat reagent. It should be noted in the above procedures that two independent enzymes have been considered, cellobiase with optimal activity towards cellobiose and aryl-/?-glucosidase with optimal activity towards aryl glucosides. As the substrate specificity of each enzyme is not absolute, the above screening methods do not distinguish between each type of enzyme. Differentiation can only be accomplished through enzyme purification and characterization. Isolation of Constitutive, Catabolite Repression, and End-Product Inhibition-Resistant Mutants Constitutive mutants may be isolated by plating the survivors from mutagen treatment on agar-salts medium containing only colony growth inhibiting substances and a carbon source (e.g., glucose or sucrose) that is not an inducing substrate. Following growth, the plates are overlayed with the specific selective-screening medium described in Table II that will allow visual detection of the desired enzymatic activity. The plates are generally incubated at 50°C overnight. This temperature is not permissive for growth and presumably will not allow de novo protein synthesis. An inhibitor of protein synthesis (e.g., actidione) can be



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incorporated into the overlay if preferred. The plates are subsequently screened for mutant colonies which have made the desired enzyme constitutively. Mutants that no longer require insoluble cellulosic material as an inducer during cellulase production should allow for less sophisticated fermentor design and could be used for continuous enzyme production, as opposed to the present batch production system. Catabolite repression-resistant mutants can be isolated by adding high concentrations (5% or more) of a catabolite repressor such as glucose or glycerol in combination with the specific cellulosic substrate. All those organisms that remain sensitive to carbon catabolite repression will preferentially grow on the repressor and not synthesize any of the cellulase complex enzymes. Those colonies that are resistant to catabolite repression will secrete the particular cellulase-complex enzyme in spite of the repressor and the activity of the enzyme can be visualized in the appropriate screening agar. The antimetabolite 2-deoxyglucose is particularly useful in selecting catabolite repression-resistant mutants since at concentrations above 0.5% it can be used as a catabolite repressor as well as an antimetabolite. By incorporating 2-deoxyglucose into any of the previously described selective-screening methodologies, it is possible to enrich for the desired phenotype. Catabolite repression-resistant mutants will be useful in lowering the cost of cellulase by allowing substitution of inexpensive crude medium components during enzyme production stage. It may be possible, by utilizing such mutants, to digest cellulosic materials directly to glucose syrups, eliminating the need for two-stage enzyme production and saccharification. Mutants resistant to end-product inhibition may be obtained by adding high concentrations of the end product to the selective-screening system. For example, high concentrations of glucose will noncompetitively inhibit cellobiase activity (6). Mutants screened on any of the three selective media for cellobiase detection that retain this activity in the presence of high concentrations of glucose should show increased resistance to end-product inhibition of the enzymatic activity. Enzymatic saccharifications to date have not achieved 100% conversion of cellulose to soluble sugars (41) primarily because of end-product inhibition of the enzymes. Enzymes which are resistant to such inhibition, perhaps used in an immobilized mode, could vastly change the economics of enzymatic hydrolysis of cellulose. Illustrative Mutants of T . reesei Isolated Utilizing These Selective Screening Techniques Utilizing these screening techniques roughly 800,000 colonies have been screened and approximately 100 mutants have been isolated. Complete characterization of these mutants is a slow and tedious process.



296

HYDROLYSIS

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CELLULOSE

Rut-NG14 and Rut-C30 have been most fully investigated to date. The genealogy of these mutants is shown in Figure 1. Rut-NG14 was isolated employing the acid swollen cellulose, oxgall, Phosfon D screening method in the presence of 5% glycerol as a catabo lite repressor (42). This mutant under fermentor conditions synthesizes approximately 20 times the filter paper activity (a measure of the synergistic activity of all the cellulase complex enzymes ) when compared with the wild type QM6a in shake flask. Endoglucanase and cellobiase activities are both increased in this mutant (42). Although Rut-NG14 was isolated in the presence of a catabolite repressor (5% glycerol) it is only partially resistant to catabolite repression. Significant amounts of endoglucanase and filter paper degrading activity can be detected, but the yield is not comparable to that under nonrepressed conditions. We insert here a note of caution. Rut-NG14 is representative of a group of mutants whose physiological characteristics in submerged culture do not parallel those on agar plates. In spite of the lack of complete catabolite resistance, Rut-NG14 is an extremely useful mutant. Figure 2 shows the production of cellulase under controlled fermentor conditions by Rut-

Trichoderma reesei QM 6a (wild type)(0.5-D*

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HOURS Figure 2. Cellulase synthesis by Rut-NG14 in a 6% cellulose fermenta tion. (Data from M. Mandels, U.S. Army Laboratories, Natick, Mass.) (O—O) Residual dry weight, (A—A) FP units/mL (42), (·—·) soluble protein, ( Δ — Δ ) mycelial protein. Substrate: 6% two-roll milled cotton; Mandels' (42) basic salts medium without urea; inoculum 20% υ/υ of a 3-day-old shakeflaskmycelium; pH > 3, continuous addition of NH OH. 4

NG14. This mutant is capable of elaborating 15 FP units/mL with a productivity of 45 units/L/hr. The final concentration of soluble extra cellular protein is 21.2 mg/mL. Samples of this enzyme preparation were sent to G. Pettersson at the University of Uppsala, Sweden, for quantifi cation of each of the enzymes in the cellulase complex using purified antibodies to the individual enzymes. The quantitative antigen-antibody reaction showed that 600 mg/g of this enzyme preparation was one enzyme, cellobiohydrolase. This represents a yield of 13 g/L of cellobio hydrolase, which is a 100-fold increase over the amount of cellobiohydro lase obtained with strain Q M 9414 (130 mg/L) the best previously existing cellulase mutant (G. Pettersson, personal communication). Since Rut-NG14 was only partially resistant to catabolite repression but was high yielding for cellobiohydrolase, we used Rut-NG14 as an initial mutagenized strain and after subsequent mutagenesis and selection on cellobiose-2-deoxyglucose medium, isolated Rut-C30. Figure 3 shows cellulase production in shake flasks by Rut-C30 in the presence and absence of a catabolite repressor (5% glycerol). For comparative pur-



298


poses, T. reesei Q M 9414, an organism which is sensitive to catabolite repression, has been run in parallel. Rut-C30 makes almost as much filter paper degrading activity in the presence of 5% glycerol as it does during growth on cellulose alone. If the carbon to nitrogen ratios in such an experiment are adjusted to 10:1 by the addition of 0.6% peptone to the growth medium to match the 6% carbohydrate (5% glycerol and 1% cellulose), no differences in cellulase yield from Rut-C30 can be detected under repressed and nonrepressed conditions. Moreover, the synthesis of cellobiase (Figure 4) and endoglucanase (data not presented) by RutC30 are also resistant to catabolite repression by either 5% glycerol or 5% glucose. Rut-C30 has been tested for enzyme production under controlled fermentor conditions and yields similar productivity to RutNG14 (15 FP units/mL). Thus both mutants show a 15-20-fold increase in cellulase activity over the wild type strain QM6a (0.5-1.0 FP units/ mL) in shake flasks. Quantitation of the specific amounts of the indi vidual enzymes of Rut-C30 with specific antibodies has not been carried out to date. Our objective in isolating catabolite-repression-resistant

Figure 3. Cellulase production in shake flasks by Rut-C30 and QM 9414 under repressed and nonrepressed conditions. Antibiotic disk as substrate (43). (·—·,) Rut-C30,l% cellulose, (O—O) Rut-C30,1% cellulose, 5% glycerol, (M—M) CM 9414, 1% cellulose, (Π—Π) QM 9414, 1% cellu lose, 5% glycerol. Vogel's medium (44), 0.1% peptone, 0.2% Tween 80, 1% SW40Solkafloc.


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1.0

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0.4

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Figure 4. Cellobiase production. Rut-C30 and QM 9414 under repressed and nonrepressed conditions. Growth conditions same as Figure 3. Cello biase activity was measured using cellobiose as a substrate and quantitating free glucose with Glucostat reagent (Worthington Biochemicah, Free hold, N.J.). ( · — · ; Rut-C30, 1% cellulose, (O—O) Rut-C30, 1% cellu lose, 5% glycerol, (M—M) QM 9414, 1% cellulose, (Π—*Π) QM 9414, 1% cellulose, 5% glycerol strains of T. reesei was to lower the cost of enzyme production through the use of inexpensive medium components. Rut-C30 has been tested for cellulase production in shake flasks after growth on cellulose and corn steep liquor (an inexpensive nitrogen source). There is no detectable loss in enzyme yield when compared with cellulase production on more costly chemically-defined medium. Summary A number of selective-screening methodologies have been devised that have allowed isolation of a series of hyperproducing and catabolite repression-resistant mutants of T. reesei. Yields of cellulase of 15 units/ mL under controlled fermentor conditions have been achieved with both Rut-NG14 and Rut-C30. Quantitative reaction of Rut-NG14 enzyme preparation with purified antibodies to cellobiohydrolase shows that in this mutant, the cellobiohydrolase is specifically hyperproduced relative to the rest of the enzymes in the cellulase complex. Rut-C30, which was derived from Rut-NG14, shows resistance to catabolite repression for


300


filter paper degrading enzymes, endoglucanase, and cellobiase and is capable of producing these enzymes during growth on cellulose and corn steep liquor alone. Future goals are to isolate constitutive mutants and end-product resistant strains as well as continually increase overall cellulase yields. Mutants Rut-NG14 and Rut-C30 are capable of elaborat ing 2% extracellular protein, the majority of which is cellulase. A yield of 10% extracellular protein as cellulase would greatly improve the economics of cellulose saccharification through enzymatic digestion.


Acknowledgments We are grateful to Mary Mandels and Raymond Andreotti of the U.S. Army Natick Laboratories for testing the mutants under controlled fermentor conditions (Figure 2); and to Goran Pettersson of the Univer sity of Uppsala, Sweden, for the antibody determinations. This work was supported by the Department of Energy Contract No. EG-78-S-02-4591 A000 and the New Jersey Agricultural Experiment Station. Literature Cited 1. Mandels, M.; Weber, J. In "Cellulases and Their Applications," Adv. Chem. Ser. 1969, 95, 391. 2. Brown, Halliwell, and Wood, unpublished data. 3. Shoemaker, S.; Brown, R. Biochim. Biophys. Acta 1978, 523, 133. 4. Ibid., p. 147. 5. Wood, T. M.; McCrae, S. I. "Bioconversion of Cellulosic Substances into Energy Chemicals and Microbial Protein"; Aroon Purieat Thomson: Faridabad, Haryana, India, 1978; pp. 111-141. 6. Gong, C.; Ladisch, M.; Tsao, G.Biotechnol.Bioeng. 1977, 19, 959. 7. Gale, E.Bacteriol.Rev. 1943, 7, 139. 8. Magasanik, B. Cold Spring Harbor Symp. Quant.Biol.1961, 26, 249. 9. Demain, A. Adv. Biochem. Eng. 1971, 1, 113. 10. Magasanik, B. "The Lac Operon"; Cold Spring Harbor, NY, 1970; pp. 189-219. 11. Jose, J. P.; Rickenberg, H. V. Biochem. Rev. 1971, 40, 741. 12. Mandels, M.; Parrish, F. W.; Reese, Ε. T. J. Bacteriol. 1962, 83, 400. 13. Nisizawa, T.; Suzuki, H . ; Nakayama, M.; Nisizawa, K. J. Biochem. 1971, 70, 375. 14. Nisizawa, T.; Suzuki, H.; Nisizawa, K. J. Biochem. 1971, 70, 387. 15. Montenecourt, B. S.; Kuo, S. C.; Lampen, J. O. J. Bacteriol. 1973, 114, 233. 16. Schamhart, D. H . ; tenBerge, A. M.; van der Poll, K. W. J. Bacteriol. 1975, 121, 747. 17. Ciriacy, M. Mol. Gen. Genet. 1976, 145, 327. 18. Halliwell, G. In "Symposium on Enzymatic Hydrolysis of Cellulose"; Bailey, M., Enari, T.-M., Linko, M., Eds.; The Finnish National Fund for Research and Development (SITRA): Helsinki, 1975; pp. 319-336. 19. Howell, J. Α.; Stuck, J. D.Biotechnol.Bioeng. 1975, 17, 873. 20. Wood, T. H . ; McCrae, S. I. In "Symposium on Enzymatic Hydrolysis of Cellulose"; Bailey, M., Enari, T.-M., Linko, M., Eds.; The Finnish National Fund for Research and Development (SITRA): Helsinki, 1975; pp. 231-254.


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21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45.

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Mandels, M.; Reese, Ε. T.J.Bacteriol.1960, 79, 816. Eberhart, Β. M.; Beck, R. S.; Goolsby, Κ. M. J. Bacteriol. 1977, 130, 181. Lowenberg, J. R.; Chapman, C. M. Arch.Microbiol.1977, 113, 61. Mandels, M.; Weber, J.; Parizek, R. Appl. Microbiol. 1971, 21, 152. Morozova, E. In "Symposium on Enzymatic Hydrolysis of Cellulose"; Bailey, M., Enari, T.-M., Linko, M., Eds.; The Finnish National Fund for Research and Development (SITRA): Helsinki, 1975; pp. 193-211. Papovizas, G. C. Phytopathology 1967, 57, 848. Martin, J. P. SoilSci.1950, 69, 215. Curl, E. A. Can. J. Microbiol. 1968, 14, 182. Steiner, G. W.; Watson, R. D. Phytopathology 1965, 55, 1009. Ander, P.; Eriksson, K.-E. Sven. Papperstidn. 1975, 78, 643. Ander, P.; Eriksson, K.-E. Arch.Microbiol.1976, 109, 1. Mackintosh, M. E., Prichard, R. H. Genet. Res. 1963, 4, 320. Tatum, E. L.; Barratt, R. W.; Cutter, V. M. Science 1949, 109, 509. Rautela, G. S.; Cowling, Ε. B. Appl. Microbiol. 1966, 14, 892. Tansey, M. R. Arch.Mikrobiol.1971, 77, 1. Poincelot, R. P.; Day, P. R. Appl. Microbiol. 1972, 23, 875. Smith, R. E. Appl. Environ.Microbiol.1977, 33, 980. Montenecourt, B. S.; Eveleigh, D. E. Appl. Environ.Microbiol.1977, 33, 178. Hankin, L.; Anagnostakis, S. L. J. Gen. Microbiol. 1977, 98, 109. Eberhart, B.; Cross, D. F.; Chase, L. R. J. Bacteriol. 1964, 87, 761. Mandels, M.; Dorval, S.; Medeiros, J., presented at the Second Annual Symposium on Fuels from Biomass, Troy, NY, June, 1978. Montenecourt, B. S.; Eveleigh, D. E. Appl. Environ.Microbiol.1977, 34, 777. Mandels, M.; Andreotti, R.; Roche, C. Biotechnol. Bioeng. Symp., 6th 1976, 21. Montenecourt, B. S.; Eveleigh, R. E.; Elmund, G. K.; Parcells, J. Bio technol. Bioeng. 1978, 20, 297. Vogel, H. J. Am. Nat. 1964, 98, 435.

RECEIVED October 25,

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