Intron-containing tRNA genes ofSulfolobus solfataricus

Summary Nucleotide sequences of four tRNA genes from the archaebacteriumSulfolobus solfataricus have been determined. Based upon DNA sequence analysis, three of the four genes contain presumptive intervening sequences (introns) in their anticodon loops. The three introns can form similar, but not identical, secondary structures. The cleavage site at the 3 end of all three introns occurs in a three-base bulge loop. All four genes lack an encoded 3 CCA terminus and are flanked by A+T-rich DNA sequences. Two of the genes are located on antiparallel DNA strands, with their 3 termini separated by 414 bp of sequence. Including two previously published sequences, a total of five introns have now been detected among sixS. solfataricus tRNA genes. Occurrence of introns at corresponding locations in both archaebacterial and eukaryotic tRNA genes suggests that the intron/exon form of gene structure predates the evolutionary divergence of the archaebacteria and the eukaryotes.     

Adaptation ofSulfolobus solfataricus on minimal media

Summary An economic method to grow the thermoacidophilic archaebacteriumSulfolobus solfataricus is reported.     

Whey permeate as a growth medium for Pleurotus ostreatus and Lentinus edodes

Whey permeate, a dairy by-product, was studied as a growth medium for two white-rot Basidiomycetes, Lentinus edodes and Pleurotus ostreatus. Mycelial growth and phenoloxidase production appeared to be highly stimulated when the fungi were grown on the permeate instead of synthetic medium. No further increment in phenoloxidase production was observed when the permeate was supplemented with CuSO₄.     

Development of a low-cost fermentation medium for ethanol production from biomass

Nutrient cost is an important aspect in the fermentation of biomass to ethanol. With a goal of developing a cost-effective fermentation medium, several industrially available nutrient sources were evaluated for their effectiveness in the simultaneous saccharification and fermentation of pretreated poplar with Saccharomyces cerevisiae D₅A. These studies showed that a low-cost medium containing 0.3% corn steep liquor and 2.5 mM MgSO₄ · 7H₂O was similar in performance to a nutrient-rich medium. Besides its low cost, this alternative medium consists of components that are available on a commercial scale, thereby making it industrially relevant. Received: 14 August 1996 / Received revision: 7 January 1997 / Accepted: 24 January 1997     

Indirect method for quantification of cellular biomass in a solidscontaining medium used as pre-culture for cellulase production

A process that combines the advantages of solid state fermentation (SSF) and submerged fermentation (SmF) could increase the efficiency of cellulase production required in the cellulosic ethanol industry. Due to the difficulty of measuring cellular biomass in the presence of solids, we developed a novel methodology for indirect quantification of biomass during production of the preculture for a combined fermentation process. Cultivation of Aspergillus niger was initiated as SSF using sugar cane bagasse as a solid substrate. Experiments were conducted in the absence of bagasse to determine growth kinetic parameters. Changes in glucose and biomass concentrations were measured. and the data were used for simulation employing a simple unstructured model. Parameters were estimated by applying a combination of Simulated Annealing (SA) and Levenberg-Marquardt (LM) algorithms to search for minimization of the error between model estimates and experimental data. Growth kinetics followed the Contois model, with a maximum specific growth rate (μ_max) of 0.042/h, a yield coefficient for biomass formation (Y_x/s) of 0.30 g/g and a death constant (k_D) of 0.005/h.These parameters were used to simulate cellular growth in the solids-containing medium. The proposed model accurately described the experimental data and succeeded in simulating the cell concentration profile. The selected pre-culture conditions (24 h as SSF followed by 48 h as SmF) were applied for cellulase production using the combined fermentation process and resulted in an endoglucanase activity (1,052 ± 34 U/L) greater than that obtained using the conventional SmF procedure (824 ± 44 U/L). Besides the standardization of pre-culture conditions, this methodology could be very useful in systems where direct measurement of cell mass is not possible.     

Optimization of chemically defined medium for recombinant Pichia pastoris for biomass production

A chemically defined medium was optimized for the maximum biomass production of recombinant Pichia pastoris in the fermentor cultures using glycerol as the sole carbon source. Optimization was done using the statistical methods for getting the optimal level of salts, trace metals and vitamins for the growth of recombinant P. pastoris. The response surface methodology was effective in optimizing nutritional requirements using the limited number of experiments. The optimum medium composition was found to be 20 g/L glycerol, 7.5 g/L (NH4)2SO4, 1 g/L MgSO4.7H2O, 8.5 g/L KH2PO4, 1.5 mL/L vitamin solution and 20 mL/L trace metal solution. Using the optimized medium 11.25 g DCW/L biomass was produced giving a yield coefficient of 0.55 g biomass/g of glycerol in a batch culture. Chemostat cultivation of recombinant P. pastoris was done in the optimized medium at different dilution rates to determine the kinetic parameters for growth on glycerol. Maximum specific growth rate of 0.23 h(-1) and Monod saturation constant of 0.178 g/L were determined by applying Monod model on the steady state data. Products of fermentation pathway, ethanol and acetate, were not detected by HPLC even at higher dilution rates. This supports the notion that P. pastoris cells grow on glycerol by a respiratory route and are therefore an efficient biomass and protein producers.     

Rhizobium biomass production in batch and continuous culture with a malt-sprouts medium

Summary The growth of nine strains of four different species ofRhizobium (R. leguminosarum, R. meliloti, R. phaseoli andR. japonicum) was studied with media containing malt sprouts extract (MSE), in place of yeast extract (YE), as source of nitrogen and growth factors. The results obtained in batch cultures indicated that all the strains grew well in MSE medium. In the case of fast-growing strains the biomass increased with increase in the concentration of MSE in the medium, but with the slow-growing strains, likeR. japonicum, growth was inhibited completely by using a concentration of MSE (containing 3.75% w/v of solids) of 40% v/v. With all the strains the concentration of cells attained was greater than 5×10⁹ cells/ml, which clearly indicates that MSE is a suitable component of media, and one which can be used instead of YE. The results of continuous culture experiments showed that a high productivity of cells (15.3×10⁸ cells/ml/h) could be obtained using MSE medium.

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Producción de Rhizobium en cultivos batch y continuo sobre medio conteniendo extracto de raiz de malta

Resumen Se estudió el crecimiento de nueve cepas de cuatro especies diferentes deRhizobium (R. leguminosarum, R. meliloti, R. phaseoli y R. japonicum) en medios de cultivo conteniendo extracto de raíz de malta (ERM), en reemplazo de extracto de levaduras (EL), como fuente de nitrógeno y factores de crecimiento. Los resultados obtenidos en cultivos en batch indicaron que todas las cepas crecieron bien en los medios con ERM. En el caso de las cepas de crecimiento rápido la biomasa se incrementó con la concentración de ERM en el medio de cultivo, pero con las cepas de crecimiento lento(R. japonicum) el desarrollo celular se inhibió completamente para concentraciones de ERM (conteniendo 3,75% p/v de sólidos) de 40% v/v. Con todas las cepas empleadas la concentración celular alcanzada fue superior a 5×10⁹ cél/ml, lo que indica claramente que el ERM es un adecuado componente para los medios de cultivo que puede reemplazar al EL. Los resultados de los experimentos realizados en sistema continuo mostraron que se puede alcanzar una alta productividad (15,3×10⁸ cél/ml/h) utilizando un medio de cultivo suplementado con ERM.

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Production de Rhizobium en culture discontinue et continue sur milieu à base de germes de malt

Résumé La croissance de neuf souches appartenant à quatre éspèces différentes deRhizobium (R. leguminosarum, R. meliloti, R. phaseoli etR. japonicum) a été étudiée avec des milieux contenant, comme source d'azote et de facteurs de croissance, un extrait de germes de malt (MSE) au lieu d'extrait de levure (YE). Les résultats obtenus en culture discontinue (batch) montrent que toutes les souches poussent bien en milieu MSE. Dans la cas des souches à croissance rapide, la biomasse augmente proportionnellement à la concentration de MSE dans le milieu. Par contre, pour les souches à croissance lente, commeR. japonicum, la croissance est complètement inhibée lorsque la concentration en MSE (contenant 3.75% w/v de solides) atteint 40% (v/v). Avec toutes les souches, on obtient une concentration en cellules supérieure à 5×10⁹/ml, ce qui indique clairement que le MSE convient bien et peut être utilisé à la place de YE. Les expériences réalisées en culture continue montrent qu'on peut obtenir avec le milieu MSE une productivité en cellules très élevée (15.3×10⁸ cellules/ml/h.).

     

Pectin-rich biomass as feedstock for fuel ethanol production

The USA has proposed that 30 % of liquid transportation fuel be produced from renewable resources by 2030 (Perlack and Stokes 2011). It will be impossible to reach this goal using corn kernel-based ethanol alone. Pectin-rich biomass, an under-utilized waste product of the sugar and juice industry, can augment US ethanol supplies by capitalizing on this already established feedstock. Currently, pectin-rich biomass is sold (at low value) as animal feed. This review focuses on the three most studied types of pectin-rich biomass: sugar beet pulp, citrus waste and apple pomace. Fermentations of these materials have been conducted with a variety of ethanologens, including yeasts and bacteria. Escherichia coli can ferment a wide range of sugars including galacturonic acid, the primary component of pectin. However, the mixed acid metabolism of E. coli can produce unwanted side products. Saccharomyces cerevisiae cannot naturally ferment galacturonic acid nor pentose sugars but has a homoethanol pathway. Erwinia chrysanthemi is capable of degrading many of the cell wall components of pectin-rich materials, including pectin. Klebsiella oxytoca can metabolize a diverse array of sugars including cellobiose, one degradation product of cellulose. However, both E. chrysanthemi and K. oxytoca produce side products during fermentation, similar to E. coli. Using pectin-rich residues from industrial processes is beneficial because the material is already collected and partially pretreated to facilitate enzymatic deconstruction of the plant cell walls. Using biomass already produced for other purposes is an attractive practice because fewer greenhouse gases (GHG) will be anticipated from land-use changes.     

Alternative medium for production of Pleurotus ostreatus biomass and potential antitumor polysaccharides

Pleurotus species are recognized for producing beta-glucans with important medicinal properties as a constituent of the cellular wall of the fruiting body or of the mycelium. The aims of this work were to select a culture medium that maximized the production of biomass and polysaccharides produced by Pleurotus ostreatus DSM 1833 and to evaluate the selected medium in two values of initial oxygen transfer rate -K(L)a (10.2 and 19.3 h(-1)). A 2* *4 factorial design was constructed to evaluate the supplementation of wheat extract with corn steep liquor--CSL (10 or 20 g L(-1)), yeast extract--YE (2 or 5gL(-1)), ammonium sulfate--AS (0 or 5 g L(-1)) and glucose (20 or 40 g L(-1)). In terms of maximum productivity in biomass and global productivity in polysaccharides, the best values were obtained when 5 g L(-1) of YE and 40 g L(-1) of glucose were used. In terms of maximum concentration of biomass, the best results were obtained when 20 g L(-1) of CSL and 40 g L(-1) of glucose were used. The best results in terms of production of biomass and polysaccharides were achieved when lower initial K(L)a (10.2 h(-1)) was used.     

Use of waste Chinese cabbage as a substrate for yeast biomass production

The possibility of using waste Chinese cabbage (Brassica campestris) as a substrate for microbial biomass production was investigated. The juice from waste Chinese cabbage contains relatively high amounts of reducing sugars suitable for yeast culture. The cell mass and protein content of four species of yeast, Candida utilis, Pichia stipitis, Kluyveromyces marxianus, and Saccharomyces cerevisiae, were determined when cultured in juice extracted from cabbage waste. Compared to YM broth containing the same level of sugar, all the strains except C. utilis showed higher total protein production in cabbage juice medium (CJM).     

Spruce sugars and poultry hydrolysate as growth medium in repeated fed-batch fermentation processes for production of yeast biomass

Bioprocess and Biosystems Engineering - The production of microbial protein in the form of yeast grown on lignocellulosic sugars and nitrogen-rich industrial residues is an attractive approach for...     

Softwood forest thinnings as a biomass source for ethanol production: a feasibility study for California

A plan has been put forth to strategically thin northern California forests to reduce fire danger and improve forest health. The resulting biomass residue, instead of being open burned, can be converted into ethanol that can be used as a fuel oxygenate or an octane enhancer. Economic potential for a biomass-to-ethanol facility using this softwood biomass was evaluated for two cases: stand-alone and co-located. The co-located case refers to a specific site with an existing biomass power facility near Martell, California. A two-stage dilute acid hydrolysis process is used for the production of ethanol from softwoods, and the residual lignin is used to generate steam and electricity. For a plant processing 800 dry tonnes per day of feedstock, the co-located case is an economically attractive concept. Total estimated capital investment is approximately $70 million for the co-located plant, and the resulting internal rate of return (IRR) is about 24% using 25% equity financing. A sensitivity analysis showed that ethanol selling price and fixed capital investment have a substantial effect on the IRR. It can be concluded that such a biomass-to-ethanol plant seems to be an appealing proposition for California, if ethanol replaces methyl tert-butyl ether, which is slated for a phaseout.     

Improved biomass production of cyanobacteria by reutilization of the culture medium

The biomass production of a cyanobacterium (Nostoc sp.) in a photoreactor with a low illuminated surface area to volume ratio was improved by the reutilization of the culture medium. After six succesive utilizations the growth ofNostoc sp. amounted to 2.15 g/l with an average content in phycobiliproteins of 14.4% on dry weight basis. The procedure reported allowed an 80% increase in biomass. The cellular self-sedimentation proved to be effective for biomass separation between reutilization steps.     

Large-scale production of bacterial biomass from methanol for use as milk-replacer

Summary A mixed methanol-utilizing bacterial culture was utilized to produce bacterial biomass as milk replacer. This culture comprised three pure strains: KISRI-5 (NCIB 12135), KISRI-512 (NCIB 12137) and KISRI-5112 (NCIB 12138). Optimal concentrations of methanol (15 g 1^–1) and medium elements as well as optimal growth conditions, e.g., pH (6.8), temperature (38°C), dissolved oxygen and dilution rate, were established. The maximum biomass yield coefficient obtained under optimized conditions was 0.48 g g^–1.· Large-scale production was successfully carried out in a 1500 1 fermenter under chemostat conditions. A good product was obtained having high true protein content (59–62%) and low polysaccharides (5%) without microbial contamination.     

Evaluation of waste mushroom logs as a potential biomass resource for the production of bioethanol

In order to investigate the possibility of using waste mushroom logs as a biomass resource for alternative energy production, the chemical and physical characteristics of normal wood and waste mushroom logs were examined. Size reduction of normal wood (145 kW h/tone) required significantly higher energy consumption than waste mushroom logs (70 kW h/tone). The crystallinity value of waste mushroom logs was dramatically lower (33%) than normal wood (49%) after cultivation by Lentinus edodes as spawn. Lignin, an enzymatic hydrolysis inhibitor in sugar production, decreased from 21.07% to 18.78% after inoculation of L. edodes. Total sugar yields obtained by enzyme and acid hydrolysis were higher in waste mushroom logs than in normal wood. After 24h fermentation, 12 g/L ethanol was produced on waste mushroom logs, while normal wood produced 8 g/L ethanol. These results indicate that waste mushroom logs are economically suitable lignocellulosic material for the production of fermentable sugars related to bioethanol production.     

Whey proteins as a model system for chromatographic separation of proteins

Although chromatographic separation of whey proteins has been considered too expensive, whey may serve as an excellent model mixture to investigate and validate the use of simulation tools in the development and optimization of chromatographic separations and the outcome could easily be utilized since the model system has an intrinsic value. Besides, milk from transgenic animals could be an attractive source of pharmaceuticals which must be separated from the other proteins in the milk. Several whey proteins are of interest especially, alpha-lactalbumin, beta-lactoglobulins, immunoglobulins, lactoperoxidase, and lactoferrin. The scope of the project is to develop a consistent set of chromatographic data for whey proteins including isotherms, transport properties and scale-up studies and to develop the appropriate models for the anion exchangers Q-Sepharose XL, Source 30Q, Ceramic Q-HyperD F, and Merck Fractogel EMD TMAE 650 (S). In this work we have determined and correlated gradient and isocratic retention volumes in the linear range of the isotherm for alpha-lactalbumin, beta-lactoglobulin A and B, and bovine serum albumin at a pH from 6 to 9 at various NaCl concentrations.