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A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 ± 2.3% and 63.2 ± 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1–30 % v/v (10.0–238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034–0.087 g ethanol/L working with the on-line dilution integrated to the biosensor—FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

The engineering of microbial cell factories for production of fine or bulk chemicals is a multidisciplinary effort that involves genetic engineering (overexpression, deletion or introduction of genes), physiological engineering (cultivation and adaptation of the catalyst to the appropriate process conditions) and biochemical engineering (process configuration, protocols for down-stream processing etc.). Any application oriented project in the field of industrial biotechnology must involve an “omics” analysis. This does not necessarily mean the application of transcriptomics, proteomics, metabolomics, etc. but especially, and invariably, an economics analysis. As pointed out by Cameron and Lievense (Proceedings 25 Symposium p 805) any application oriented project should:

Bioabatement is a potential method to remove inhibitory compounds from lignocellulose hydrolysates that could be incorporated into a scheme for fermentation of ethanol from cellulose. NRRL30616, an Ascomycete that metabolizes furfural and 5-hydroxymethylfurfural, is a unique strain that may be useful for detoxifying biomass sugars. NRRL30616 and 23 related fungal strains were screened for the ability to metabolize furans and grow in dilute-acid hydrolysate of corn stover. NRRL30616 was the best strain for removal of inhibitors from hydrolysate, and abatement of hydrolysate by inoculation with the strain allowed subsequent yeast fermentation of cellulose to ethanol.

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few strains are used to develop an efficient process for mannitol bio-production; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from using genomic polymerase chain reaction. The clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing via pRSETa vector in BL21pLysS on isopropyl-β--thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered TF103 carrying the gene of indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress.

This study demonstrates, for the first time, that it is possible to couple sulfate-limited growth to continuous H photoproduction for more than 4000 h. A two-stage chemostat system physically separates photosynthetic growth from H production, and it incorporates two automated photobioreactors (PhBRs). In the first PhBR, the algal cultures are grown aerobically in chemostat mode under limited sulfate to obtain photosynthetically competent cells. Active cells are then continuously delivered to the second PhBR, where H production occurs under anaerobic conditions. The dependence of the H production rate on sulfate concentration in the medium, dilution rates in the PhBRs, and incident light intensity is reported.

is a polyol overproducing yeast that can have a potential use for upgrading lignocellulosic hydrolysates. Therefore, the establishment of its tolerance to metabolic inhibitors found in hydrolysates is of major interest. We studied the effects of selected aliphatic acids, phenolic compounds, and furfural. Acetic acid favored biomass production for concentrations <6.0 g/L. Formic acid was more toxic than acetic acid and induced xylitol accumulation (maximum yield of 0.21 g/g of xylose). All tested phenolics strongly decreased the specific growth rate. Increased toxicity was found for hydroquinone, syringaldehyde, and 4-methylcatechol and was correlated to the compound’s hydrophobicity. Increasing the amount of furfural led to longer lag phases and had a detrimental effect on specific growth rate and biomass productivity.

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mg) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mg) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation.

spp. synthesize a pure form of hydrophilic cellulose that has several industrial specialty applications. Literature reports have concentrated on intensive investigation of static and agitated culture in liquid media containing high nutrient concentrations optimized for maximal cellulose production rates. The behavior of these bacteria on semisolid and solid surfaces has not been specifically addressed. The species was examined for cellulose synthesis and colony morphology on a range of solid supports, including cotton linters, and on media thickened with agar, methyl cellulose, or gellan. The concentration and chemical structure of the thickening agent were found to be directly related to the formation of contiguous cellulose pellicules. Viability of the bacteria following freezer storage was improved when the bacteria were frozen in their cellulose pellicules.

Furfural and hydroxymethylfurfural (HMF) are representative inhibitors among many inhibitive compounds derived from biomass degradation and saccharification for bioethanol fermentation. Most yeasts, including industrial strains, are susceptible to these inhibitory compounds, especially when multiple inhibitors are present. Additional detoxification steps add cost and complexity to the process and generate additional waste products. To promote efficient bioethanol production, we studied the mechanisms of stress tolerance, particularly to fermentation inhibitors such as furfural and HMF. We recently reported a metabolite of 2,5-bis-hydroxymethylfuran as a conversion product of HMF and characterized a dose-dependent response of ethanologenic yeasts to inhibitors. In this study, we present newly adapted strains that demonstrated higher levels of tolerance to furfural and HMF. 307-12H60 and 307-12H120 and 30710H60 showed enhanced biotransformation ability to reduce HMF to 2,5-bis-hydroxymethylfuran at 30 and 60 mM, and 307-12-F40 converted furfural into furfuryl alcohol at significantly higher rates compared to the parental strains. Strains of converted 100% of HMF at 60 mM and 307-12-F40 converted 100% of furfural into furfuryl alcohol at 30 mM. The results of this study suggest a possible detoxification of the inhibitors by using more inhibitor-tolerant yeast strains for bioethanol fermentation. The development of such tolerant strains provided a basis and useful materials for further studies on the mechanisms of stress tolerance.

The bioprocessing session of the 26 Symposium attracted a large number of submissions, with over 70 oral and poster presentations. This magnitude of work reflected a tremendous variety in approaches and issues being addressed for the processing of biomass and related separations work.