A novel bacterium designated strain MV1 was isolated from a sludge enrichment taken from the wastewater treatment plant at a plastics manufacturing facility and shown to degrade 2,2-bis(4-hydroxyphenyl)propane (4,4'-isopropylidenediphenol or bisphenol A). Strain MV1 is a gram-negative, aerobic bacillus that grows on bisphenol A as a sole source of carbon and energy. Total carbon analysis for bisphenol A degradation demonstrated that 60%o of the carbon was mineralized to CO2, 20%o was associated with the bacterial cells, and 20%o was converted to soluble organic compounds. Metabolic intermediates detected in the culture medium during growth on bisphenol A were identified as 4-hydroxybenzoic acid, 4-hydroxyacetophenone, 2,2-bis(4hydroxyphenyl)-1-propanol, and 2,3-bis(4-hydroxyphenyl)-1,2-propanediol. Most of the bisphenol A degraded by strain MV1 is cleaved in some way to form 4-hydroxybenzoic acid and 4-hydroxyacetophenone, which are subsequently mineralized or assimilated into cell carbon. In addition, about 20%o of the bisphenol A is hydroxylated to form 2,2-bis(4-hydroxyphenyl)-1-propanol, which is slowly biotransformed to 2,3-bis(4hydroxyphenyl)-1,2-propanediol. Cells that were grown on bisphenol A degraded a variety of bisphenol alkanes, hydroxylated benzoic acids, and hydroxylated acetophenones during resting-cell assays. Transmission electron microscopy of cells grown on bisphenol A revealed lipid storage granules and intracytoplasmic membranes.

Clostridium thermocellum produces ethanol, acetate, H2, and CO2 as major fermentation products from cellulose and cellobiose. The performance of three strains of this microorganism was studied to assess the potential use in producing ethanol directly from cellulosic fiber. Depending on the bacterial strain, an ethanol/acetate product ratio from 1 to as high as 3 was observed in unstirred cultures. Vigorous stirring during growth resulted in a threefold decrease in the ethanol/acetate ratio. The H2 content in the unstirred culture broth was three times greater than that in the stirred one. Addition of exogenous H2 to the gas phase during growth increased the ethanol/acetate ratio much more in the stirred than in the unstirred fermentations. The addition of sufficient H2 to the gas phase almost relieved the effect of stirring, and the ethanol/acetate ratio approached that in the unstirred condition. Addition of tritium to the gas phase of the culture resulted in the formation of tritiated water (3H20), which indicates that C. thermocellum possesses hydrogenase(s) that catalyzes the reverse reaction. The rate of 3H2O formation was about three times higher in the stirred culture than in the unstirred culture. These results demonstrate that the H2 concentration in the broth plays an important role in the product formation. The H2 supersaturation present in the unstirred cultures is responsible for the observed effect of stirring. A hydrogen feedback control mechanism regulating the relative concentrations of reduced and oxidized electron carriers is proposed to account for the effect of hydrogen on the metabolite distribution.

Removal of Ethanol from the Thermophilic Ethanol Fermentation 4 4 Fiber Gradient Plate 11 Lignin and Cellulose Degradation by C. pruinosum Growing on a Damp Substrate. . ." 16 Lignin and Cellulose Degradation by Growing and Temperature-Shifted (Non-Growing) Cultures of C. pruinosum i 17 Survival Curve of Thermophilic Bacillus NW 36 Effect of Initial Ethanol Concentration on the Growth of Thermophilic Bacillus ZB-B2 38 9 Growth of ZB-B2 on 0.5% Xylose 40 Growth of ZB-B2 on 0.5% Glucose 41 Growth of ZB-B2 on 0.25% Xylose + 0.25% Glucose. . 42 HPLC Chromatograph of Glucose and Cellobiose .... 47 13 Standard Curves and High Pressure Liquid Chromatograph Analysis of Glucose and Cellobiose 48 Gas Chromatographic Analysis of Fermentation End-Products 49 Acetic Acid Standard Curve (from GLC analysis). . .51 Growth of Thermophilic Bacilli on 1% Glucose .... 54 Mixed Culture Fermentation of Amorphous Cellulose. 55 Mixed Culture Fermentation of Mycrocrystalline Cellulose 57 19 Fermentation Broth, Gas Liquid Chromatogram .... 59 Continuous Fermentation of Thermophilic Bacillus Growing on Glucose 62 iv LIST OF ILLUSTRATIONS (cont'd) Growth of Thermophilic Bacillus on Glucose in Continuous Fermentation .... 63 Continuous Fermentation of Thermophilic Bacillus (Glucose) Growth of C. thermocellum LQ8 on Microcrystalline Cellulose 69 Growth of C. thermocellum LQ8 on Microcrystalline Cellulose 71 Growth of C. thermocellum LQ8 on Microcrystalline Cellulose 73

Two approaches to ethanol production via thermophilic mixed culture fermentation of pretreated wood were investigated. The initial studies of wood biodelignification by Chrysosporium pruinosum and subsequent mixed culture fermentation to ethanol using a cellulolytic strain of sporocytophaga and a strain of Bacillus stearothermophilus proved to be premature for a development effort. Studies of the fermentation of S0 2 /steamtreated poplar by a mixed culture of C. thermocellum and C. thermosaccharolyticum were, however, technically and economically promising. Wood pretreatment to enhance microbial utilization, the microbiology and biochemistry of pure and mixed culture fermentation of cellulose by C. thermocellum and C. thermocellum and C. thermosaccharolyticum, and techniques for improving ethanol tolerance and yield were investigated. Considerable progress in overcoming the technical barriers to efficient ethanol production from wood have been demonstrated; however, additional studies and development work are required before technical feasibility can be established.

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