Abstract: In the production of automobile fuel ethanol from biomass by dilute acid hydrolysis/fermentation process, degradation products from the hydrolysis substantially inhibit the bioconversion of sugar to ethanol. Majority of these inhibitors have not been previously identified due to the complexity of biomass hydrolyzate. This paper presents an analytical procedure for identification of biomass degradation products, which entails flash evaporation, anion exchange, chloroform and ethyl acetate extraction, HPLC and GC-MS analyses. More than 35 potential inhibitors to S. cerevisiae fermentation in dilute nitric acid hydrolyzates of hybrid poplar were identified by correlating the fermentability of the anion exchange treated and untreated hydrolyzate samples with their chemical compositions, and by chemical analysis of the regeneration eluate from the ion exchange resin saturated by the hydrolyzate.

Abstract: The bioconversion of vineyard pruning and grape pomace by Pleurotus spp. using a solid state fermentation (SSF) was evaluated. Fruiting body production and chemical changes in the substrates after harvesting were measured. Biological efficiency and bioconversion ranged from 37.2 to 78.7% and from 16.7 to 38.8%, respectively. The best substrates for mycelial growth and mushroom yield were the mixtures with higher vineyard pruning content. Inclusion of pruning content had higher phenolic components and total sugars, better C/N ratio, and lower crude fat and total nitrogen than pomace. On the contrary, mycelium grew more slowly and scarcely in all treatments with 100% grape pomace. Moisture, protein, fat, and lignin contents were generally higher in mixtures with higher pomace proportion, whereas neutral detergent fiber, hemicellulose, and cellulose contents were higher with pruning content. Pleurotus strains may act depending on the availability of fiber fractions of substrate, and dynamic changes in digest...

Abstract: Biocatalysis driven by D-amino acid oxidase is a significant example of the commercial production of high value-added intermediates using enzyme-based technology. The results of the most recent research on this FAD-dependent catalyst are reported here. In particular, insight is given of how in the past few years the main industrial application of this enzyme, i.e. the stereospecific bioconversion of cephalosporin C to glutaryl-7-amino cephalosporanic acid in the two-step production of 7-amino cephalosporanic acid, has been implemented by improving its production and by engineering of the biocatalyst. The set-up and the optimization of different conditions for carrying out the process under different procedures have also been updated.

Abstract: Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state. The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures. High volumetric mannitol productivity (26.2 g l−1 h−1) and mannitol yield (97 mol%) were achieved. Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches. Applying response surface methodology, the temperature and pH were studied with respect to specific mannitol productivity and yield. Moreover, increasing the initial fructose concentration from 100 to 120 and 140 g l−1 resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase (MDH). Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters. Journal of Industrial Microbiology & Biotechnology (2002) 29, 44–49 doi:10.1038/sj.jim.7000262

Abstract: Cost reductions for pretreatment and bioconversion processes are key objectives necessary to the successful deployment of a bioethanol industry. These unit operations have long been recognized for their impact on the production cost of ethanol. One strategy to achieve this objective is to improve the pretreatment process to produce a pretreated substrate resulting in reduced bioconversion time, lower cellulase enzyme usage, and/or higher ethanol yields. Previous research produced a highly digestible pretreated yellow poplar substrate using a multistage, continuously flowing, very dilute sulfuric acid (0.07% (w/v)) pretreatment. This process reduced the time required for the bioconversion of pretreated yellow poplar sawdust to ethanol. This resulted in a substantially improved yield of ethanol from cellulose. However, the liquid volume requirements, steam demand, and complexity of the flow-through reactor configuration were determined to be serious barriers to commercialization of that process. A reconfigured process to achieve similar performance has been developed using a single-stage batch pretreatment followed by a separation of solids and liquids and washing of the solids at a temperatures between 130 and 150 degrees C. Separation and washing at the elevated temperature is believed to prevent a large fraction of the solubilized lignin and xylan from reprecipitating and/or reassociating with the pretreated solids. This washing of the solids at elevated temperature resulted in both higher recovered yields of soluble xylose sugars and a more digestible pretreated substrate for enzymatic hydrolysis. Key operating variables and process performance indicators included acid concentration, temperature, wash volume, wash temperature, soluble xylose recovery, and performance of the washed, pretreated solids in bioconversion via simultaneous saccharification and fermentation (SSF). Initial results indicated over a 50% increase in ethanol yield at 72 h for the hot washed material as compared to the control (no washing, no separation) and a 43% reduction of in the bioconversion time required for a high ethanol yield from cellulose

Abstract: The multi-step bioconversion of β-sitosterol by Mycobacterium sp. NRRL B-3805 for the formation of androstenedione (AD) as major product, a key intermediate in the production of therapeutic steroids, is a well established industrial application of a biocatalytic process. In the present work, this sterol side-chain cleavage was selected as model system for a study of whole-cell biocatalyst operational stability in prolonged bioconversions in two-phase aqueous-organic media. The aim was to evaluate the effect of phase composition in the process yields by using four high log Poctanol phthalates as organic phases and phosphate buffer and nutritional media as aqueous phases. The effect of a non-ionic surfactant added to the aqueous phase was also studied. Results show no relation between bioconversion yields and solvent log Poctanol values, suggesting that, in the used range, solvent molecular structures and the resulting specific interactions with the cell envelope are determining for the biocatalyst behaviour. Different responses were obtained from the use of different aqueous media, the complex nutritional media leading to the highest conversion yields, while the lowest were observed with the mineral nutritional media. The effect of surfactant addition in the bioconversion yields was apparently not significant. In the systems with bis-(2-ethylhexyl) and bis-(3,5,5-trimethylhexyl) phthalates as the organic phases, the fed-batch operational mode allowed the maintenance of stable biocatalytic activity levels up to at least 6 days of operation, with high androstenedione yields.

Abstract: We demonstrate a general approach for metabolic engineering of biocatalytic systems comprising the uses of a chemostat for strain improvement and radioisotopic tracers for the quantification of pathway fluxes. Flux determination allows the identification of target pathways for modification as validated by subsequent overexpression of the corresponding gene. We demonstrate this method in the indene bioconversion network of Rhodococcus modified for the overproduction of 1,2-indandiol, a key precursor for the AIDS drug Crixivan.

Abstract: D-Mannitol (here: mannitol) is a naturally occurring sugar alcohol with six carbon atoms. It is only half as sweet as sucrose. However, mannitol and other sugar alcohols exhibit reduced caloric values compared to the respective value of most sugars, which make them applicable as sweeteners in so-called “light” foods. Moreover, sugar alcohols are metabolized independently of insulin and are thus also applicable in diabetic food products. Besides applications in the food industry, mannitol is also used in the pharmaceutical industry. In medicine, mannitol is used to decrease cellular edema (excessive accumulation of fluid) and increases the urinary output. In this doctoral thesis, the development of a new bioprocess for the production of mannitol is described. For this purpose, aspects such as strain selection, choice of process method, optimization of process parameters, scale-up, and metabolic engineering were studied. At present, mannitol is produced commercially by catalytic hydrogenation of fructose-containing syrups. The existing chemical production methods are, however, characterized by several drawbacks. The uppermost being that when fructose is catalytically hydrogenated only about 50% of it is converted into mannitol, whereas the rest is converted into another sugar alcohol, sorbitol. In addition, ultra-pure (expensive) raw materials (fructose and hydrogen gas) are required for efficient conversion. When more cost-effective raw materials, such as glucose-fructose syrups are used as starting material for catalytic hydrogenation, the main product is sorbitol and mannitol is formed as a by-product. Hence, mannitol production becomes very dependent on the market demand of sorbitol. Furthermore, mannitol is relatively difficult to purify from sorbitol. In addition, ion exchange is required for removal of the metal catalyst from the production solution. This results in even higher production costs and decreased yields. The microbial mannitol production process described in this thesis is based on high cell density cultures of slowly growing heterofermentative lactic acid bacteria. The bioconversion of fructose to mannitol was performed in a slowly agitated membrane cell-recycle bioreactor equipped with pH and temperature control. Neither aeration nor nitrogen flushing of the bioconversion medium was required, which drastically lowers the investment costs of such a plant. An important detail in the new bioprocess was the re-use of cell biomass in successive bioconversions. In a semicontinuous production experiment, the initial cell biomass provided stable mannitol productivities and yields for at least 14 successive batches. Moreover, using a simple

Abstract: The production of xylitol from D-glucose occurs through a three-step process in which D-arabitol and D-xylulose are formed as the first and second intermediate product, respectively, and both are obtained via microbial bioconversion reactions. Catalytic hydrogenation of D-xylulose yields xylitol; however, it is contaminated with D-arabitol. The aim of this study was to increase the stereoselectivity of the D-xylulose reduction step by using enzymatic catalysis. Recombinant xylitol dehydrogenase from the yeast Galactocandida mastotermitis was employed to catalyze xylitol formation from D-xylulose in an NADH-dependent reaction, and coenzyme regeneration was achieved by means of formate dehydrogenase-catalyzed oxidation of formate into carbon dioxide. The xylitol yield from D-xylulose was close to 100%. Optimal productivity was found for initial coenzyme concentrations of between 0.5 and 0.75 mM. In the presence of 0.30 M (45 g/L) D-xylulose and 2000 U/L of both dehydrogenases, exhaustive substrate turnover was achieved typically in a 4-h reaction time. The enzymes were recovered after the reaction in yields of approx 90% by means of ultrafiltration and could be reused for up to six cycles of D-xylulose reduction. The advantages of incorporating the enzyme-catalyzed step in a process for producing xylitol from D-glucose are discussed, and strategies for downstream processing are proposed by which the observed coenzyme turnover number of approx 600 could be increased significantly.

Abstract: An organic-aqueous two-liquid phase system was developed in order to perform the reduction of 3-thiophenecarboxaldeyde (3-TC) to 3-thiophenemethanol (3-TM), two intermediate log P compounds, using Pseudomonas putida S12 cells. Although, high bioconversion yields were observed in a bioconversion medium composed by Tris–HCl pH 8.5 buffer and n -octanol, adverse phase toxicity effects were observed following incubation periods in excess of 24 h. Furthermore, a stable emulsion was formed in two-liquid phase system in the presence of free cells. This prevented effective separation of the biocatalyst and of the two-liquid phases. Whole cells were effectively entrapped in Ca-alginate beads, thus enhancing biocatalyst recovery. No shifts in the pH versus activity profile resulted from the immobilization. Successive batches were carried out with the immobilized biocatalyst, but a decrease in the specific activity was observed.

Abstract: Bioconversion of higher strength of domestic wastewater biosolids (sludge) (4% w/w of TSS) by mixed fungal culture of Aspergillus niger and Penicillium corylophilum was studied in a laboratory. The effect of potential mixed fungi on domestic wastewater sludge accelerated the liquid state bioconversion (LSB) process. The highest production of dry sludge cake (biosolids) was enriched with fungal biomass to about 85.66 g/kg containing 25.23 g/kg of protein after 8 days of treatment. The results presented in this study revealed that the reduction of chemical oxygen demand (COD), total suspended solid (TSS), and specific resistance to filtration (SRF) of treated sludge were highly influenced by the fungal culture as compared to control (uninnoculated). The maximum removal rates in treated sludge (biosolids) supernatant recorded were 92% of COD and 98.8% of TSS. Lower SRF (1.08 x 10(12) m/kg) was perceived in microbially treated sludge after 6 days of fermentation. The observed parameters were highly influenced after 8 days of treatment. The influence of pH was also studied and presented in the paper.

Abstract: The yeast strain CGMCC 0573 was identified as Citeromyces matriensis and shown to be capable of enantioselectively hydrolyzing ethyl ester of (R)-Ketoprofen (2-(3-benzoylphenyl)propionic acid). The strain was isolated for the first time from soil samples through a new and efficient screening procedure in which the probability of obtaining active strains was greatly increased by using ethanol and Tween-80 alternatively as additives during the enrichment culture. Studies of the culture conditions and catalytic performance of Citeromyces matriensis CGMCC 0573 showed that the enzyme occurs constitutively in the cells and its production is enhanced by feeding with Tween-80 during the early period of cultivation. Yeast extract was found to be beneficial both for growth and for esterase production. The optimal temperature and pH for the bioconversion were 40 °C and pH 8.0, respectively. Biotransformation using resting cells cultured in a flask with baffles and magnetic stirring and in the presence of 50 mM substrate resulted in the production of (R)-ketoprofen at 93% ee (enantiomeric excess) and at 42.6% conversion.