Improvements in lipase production and recovery from Acinetobacter radioresistens in presence of polypropylene powders filled with carbon sources

Polypropylene powders as the adsorbent for organic solution containing n-hexadecane and olive oil were employed as the carbon source for producing an alkaline lipase from Acinetobacter radioresistens. The best volumetric ratio of n-hexadecane to olive oil around 5 for lipase production was determined from shake-flask and fermentation cultivations. The existence of a maximum time course lipase activity of the aqueous phase was attributed to the compensation effects of olive oil on cell growth and lipase production, repression of lipase synthesis by oleic acid, and lipase adsorption on the supports. A linear relationship between the average cell growth rate in the exponential phase and the ratio of surface areas of the supports was found. The benefits of using the present fermentation process include less foaming and emulsion of the broth, less organic phase used, higher lipase production, and easy recovery of the lipase in the centrifugation step.     

Beiträge zur Chemie der Pyrrolpigmente, 29. Mitt.: Zur anaeroben Photochemie von Gallenpigmenten: Zum Mechanismus der photochemischen Isomerisierung an exocyclischen Doppelbindungen

Studies of heavy atom effects as well as sensitization and quenching experiments with several model compounds suggested that in pyrromethenones and biladienes-ac the photoisomerization at exocyclic double bonds proceeds predominantly via the singlet path on direct irradiation. The energy of the triplet state of pyrromethenones and arylmethylenepyrrolinones was estimated by sensitization experiments to be in the region from 140 to 160 kJ/mol. On sensitization the reaction follows the triplet path. With bilatrienes-abc the situation is complicated by the fact that in homogenous phase there is only unidirectional isomerization but there is evidence that the direct process proceeds also via the singlet path. These experiments are supported by calculations using theFormosinho theory of radiationless deexcitation.

28. Mitt.:H. Falk undK. Grubmayr, Mh. Chem.110, 1237 (1979).     

Production of ceramide with Saccharomyces cerevisiae

The possibility of producing the biologically active material of the skin, ceramide, was studied using yeasts. The yeast strain that produced the most ceramide, Saccharomyces cerevisiae (KCCM 50515), was selected, and the optimal conditions for ceramide production were determined using shakeflask culture and batch fermentation. By measuring the production rate of ceramide at various pH values and temperatures, the optimal conditions for ceramide production were found to be pH 6.0 and 30°C. When heat shock was applied to the cells for 1 h by increasing the culture temperature from 30 to 40°C after cell growth, the amount of ceramide produced was increased 5.9-fold. A cell growth and ceramide production model was developed with Monod kinetics and the Leudecking-Piret model. It showed that ceramide production was increased when the cells were in the stationary phase.     

Beiträge zur Chemie der Pyrrolpigmente, 27. Mitt.

Pyrromethenones are isomerized into their diastereomers at the exocyclic double bond by light, whereas dipyrromethenes and methylenepyrrolylmethyllenepyrrolinones as well as integral bile pigments containing these two partial structures (biliviolines, biliverdines) are not. The reason for this dichotomy was shown by investigation of model compounds to be neither the thermodynamically and kinetically instable product nor the availability of low states of lone pair character. Instead the very efficient radiationless decay of the excited singlet proceeds via a desactivation process being a tunneling between excited and ground state potential surfaces. Calculations according to this theory ofFormosinho affords quantitative results which are in excellent agreement with experimental data. Another desactivation mode comes from the crossing of the potential surfaces of the ground state of one tautomeric form with those of the excited state of another, leading to a proton transfer process. The molecular basis of these processes is shown to be inter—as well as intramolecular hydrogen bonding.

H. Falk undF. Neufingerl, Mh. Chem.110, 987 (1979).     

Environmental Effects on Transglutaminase Production and Cell Sporulation in Submerged Cultivation of <Emphasis Type="Italic">Bacillus circulans</Emphasis>

In this research, the effects of pH, temperature, and oxygen on growth kinetics of a newly isolated strain of Bacillus circulans from the Amazon and their correlations with transglutaminase (TGase) production and cell sporulation were investigated. Statistical experimental methods were used to optimize these parameters, while induction of sporulation was achieved by oxygen culture control. Full factorial composite experimental design and response surface methodology were experimentally tested. The model showed that temperature has a positive and significant effect on TGase production (P < 0.05) while pH and temperature, associated with anoxic conditions, have a marked effect on cell sporulation which is consistently linked with TGase production. The contour plot of results showed that the best culture conditions for TGase production of B. circulans were 30°C, initial pH 8.5, and the highest production was obtained in late-stationary culture phase with maximal specific enzyme activity of 655 U g⁻¹ of cells (0.37 U/mL). A correlation between enzyme production and cell sporulation, as mediated by oxygen culture conditions, was also demonstrated and, although demonstrated only for B. subtilis, it corroborates the molecular mechanisms involved in this process. It can be suggested that B. circulans BL32 is a strong biological system for the industrial production of TGases.     

Inoculum studies in production of penicillin g acylase by Bacillus megaterium ATCC 14945

This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate for the production of semisynthetic β-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly, B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 × 10⁷ spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.     

Hydrogen production by the thermophilic bacterium Thermotoga neapolitana

Virtually all members of the order Thermotogales have demonstrated the ability to produce hydrogen; however, some members of this order produce considerably greater quantities than others. With one representative of this order, Thermotoga neapolitana, we have consistently obtained accumulation of 25–30% hydrogen with 12–15% carbon dioxide as the only other prominent product in the batch reaction. In contradistinction to information widely disseminated in the literature, we have also found that most members of this order tolerate and appear to utilize the moderate amounts of oxygen present in the gaseous phase of batch reactors (6–12%), with no apparent decrease in hydrogen production. Hydrogen accumulation has been widely reported to inhibit growth of Thermotogales. While this may be true at very high hydrogen tensions, we have observed log phase bacterial morphology (rods) even in the presence of 25–35% hydrogen concentrations. To maximize hydrogen production and minimize production of hydrogen sulfide, inorganic sulfur donors are avoided and the cysteine concentration in the medium is increased. We and others have demonstrated that different members of the order Thermotogales utilize a wide variety of feedstocks, including complex carbohydrates and proteins. Thus, it appears that organisms within this order have the potential to utilize a variety of organic wastes and to cost-effectively generate hydrogen.     

Oxygen-controlled Biosurfactant Production in a Bench Scale Bioreactor

Rhamnolipids have been pointed out as promising biosurfactants. The most studied microorganisms for the aerobic production of these molecules are the bacteria of the genus Pseudomonas. The aim of this work was to produce a rhamnolipid-type biosurfactant in a bench-scale bioreactor by one strain of Pseudomonas aeruginosa isolated from oil environments. To study the microorganism growth and production dependency on oxygen, a nondispersive oxygenation device was developed, and a programmable logic controller (PLC) was used to set the dissolved oxygen (DO) concentration. Using the data stored in a computer and the predetermined characteristics of the oxygenation device, it was possible to evaluate the oxygen uptake rate (OUR) and the specific OUR (SOUR) of this microorganism. These rates, obtained for some different DO concentrations, were then compared to the bacterial growth, to the carbon source consumption, and to the rhamnolipid and other virulence factors production. The SOUR presented an initial value of about 60.0 mgO₂/g_DW h. Then, when the exponential growth phase begins, there is a rise in this rate. After that, the SOUR reduces to about 20.0 mgO₂/g_DW h. The carbon source consumption is linear during the whole process.     

STUDIES ON THE PORE FORMATION MECHANISM OF β-CRYSTALLINE POLYPROPYLENE UNDER STRETCHING*

The pore formation mechanism of,β-crystalline polypropylene under stretching was investigated. The porosity of the samples increases rapidly with stretching, having a maximum at draw ratios around 2 and then decreases monotonically. An abrupt formation process of initial micropores at very low draw ratios was evidenced by in situ SAXS measurements. At the same time the phase transition from ,β-crystal to α-crystal proceeds slowly in the whole deformation process up to large draw ratios around 5. Comparative studies of α- and ,β-crystalline polypropylene samples before stretching indicate that in addition to difference in crystal forms the α- and β-crystalline polypropylene samples exhibit quite different morphological features. There are a lot of interfaces in ,β-crystalline polypropylene samples, which may have a lower density value and can be easily etched by argon ions and penetrated by small molecules. It was concluded from these experimental facts that the pore formation and crystal transition are two independent phenomena during the deformation of β-crystalline polypropylene samples, and phase transition from ,β-crystal to α-crystal could hardly be the origin of pore formation. A defect initiation mechanism was proposed to understand the pore formation behavior of β-crystalline polypropylenes.     

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis

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 Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK 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 mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-β-d-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 Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the names by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Early phase process scale-up challenges for fungal and filamentous bacterial cultures

Culture pelleting and morphology has a strong influence on process productivity and success for fungal and filamentous bacterial cultures. This impact is particularly evident with early phase secondary metabolite processes with limited process definition. A compilation of factors affecting filamentous or pelleting morphology described in the literature indicates potential leads for developing process-specific control methodologies. An evaluation of the factors mediating citric acid production is one example of an industrially important application of these techniques. For five model fungal and filamentous bacterial processes in an industrial fermentation pilot plant, process development strategies were developed and effectively implemented with the goal of achieving reasonable fermentation titers early in the process development cycle. Examples of approaches included the use of additives to minimize pelleting in inoculum shake flasks, the use of large-volume frozen bagged inoculum obtained from agitated seed fermentors, and variations in production medium composition and fermentor operating conditions. Results were evaluated with respect to productivity of desired secondary metabolites as well as process scalability. On-line measurements were utilized to indirectly evaluate the cultivation impact of changes in medium and process development. Key laboratory to pilot plant scale-up issues also were identified and often addressed in subsequent cultivations.     

Reduction of furfural to furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose

The ethanologenic bacteria Escherichia coli strains KO11 and LYO1, and Klebsiella oxytoca strain P2, were investigated for their ability to metabolize furfural. Using high performance liquid chromatography and ¹³C-nuclear magnetic resonance spectroscopy, furfural was found to be completely biotransformed into furfuryl alcohol by each of the three strains with tryptone and yeast extract as sole carbon sources. This reduction appears to be constitutive with NAD(P)H acting as electron donor. Glucose was shown to be an effective source of reducing power. Succinate inhibited furfural reduction, indicating that flavins are unlikely participants in this process. Furfural at concentrations >10 mM decreased the rate of ethanol formation but did not affect the final yield. Insight into the biochemical nature of this furfural reduction process may help efforts to mitigate furfural toxicity during ethanol production by ethanologenic bacteria.     

Inherent safety evaluation in process plants— a comparison of methodologies

A global population increase and an improved standard of living are generally expected. To meet these demands, an increased production of chemicals will be necessary while protecting human health and the environment. However, most current methods of chemical production are unsustainable. New designs must result in plants that assure process and operator safety, the sustained health of workers and the community, and the protection of the environment. Traditional safety precautions and process controls minimize risk but cannot guarantee the prevention of accidents followed by serious consequences. Therefore, the general approach to environmental and safety problems must be changed from reactive to proactive. One way is to further develop the concept of inherent safety. In this paper some methods for inherent safety evaluations are reviewed. The aim of the study is to analyze the different tools available for inherent safety evaluation and identify the most important criteria in determining the inherent safety of a process plant. A model is proposed to show the interactions of different factors on the inherent safety level of a process and the model is illustrated by a case study.     

Untersuchungen an Hydriden im Bereich der Phase Ti2CO1—x Fe x (x=0–0,5)

The hydrogen absorption of the phase Ti₆₄Co_32–x Fe _x (x=0...16) and its influence on the magnetic properties have been investigated. Measurements of the vapour-pressure, wide-line NMR and magnetic susceptibility have been performed. Substitution of Co by Fe does not change the amount of absorbed hydrogen. However the reaction rate of hydriding process, the activation energy of diffusion and the magnetic quantities change upon this substitution.

     

Influence of a new axial impeller on K L a and xylanase production by Penicillium canescens 10-10c

The effects of a new axial impeller (HTPG4) on oxygen volumetric transfer coefficient, K _L a, and xylanase production by Penicillium canescens 10-10c were studied and compared for dual-impeller systems, one with one DT4 impeller below and one HTPG4 above (DT4-HTPG4) and one with two DT4 (DT4-DT4) impellers, in a 5-L bioreactor. The volumetric coefficient of oxygen transfer was measured in culture medium using a gassing-out method at different gassing rates and agitation speeds. We observed that the DT4-HTPG4 combination provided better K _L a performance than the DT4-DT4 combination. The two combinations were also tested for their influence on xylanase production by a filamentous microorganism; P. canescens 10-10c. These experiments demonstrated that the DT4-HTPG4 combination impeller enhanced enzyme production up to 23% compared with the DT4-DT4 combination at an aeration rate of 1 vvm and an agitation speed of 600 rpm. The main cause for this difference is thought to be a higher shear stress generated by the DT4-DT4 combination, which damages the mycelium of P. canescens and decreases xylanase production.     

Beiträge zur Chemie der Pyrrolpigmente, 35. Mitt.: Eine regioselektive,reversible Addition an Bilatriene-abc

Using spectroscopic methods (UV-VIS,¹H-NMR) the reversible addition of donors (alcohols, amines, thiols, HCN, bisulfit and C-H acidic compounds) to bilatrienes-abc was investigated. This equilibrium is in close analogy to the addition of donors to a carbonyl group (as was deduced from thermodynamic and kinetic measurements) and is also the basis for understanding the photochemical isomerization of bilatrienes-abc in the adsorbed state. Based thereon a recipe for the isomerization of bilatrienes-abc in homogeneous phase giving high yields of the (E, Z, Z)-diastereomer is presented; in addition the (E, Z, E)-diastereomer of aetiobiliverdin-IV- was isolated and characterized for the first time. A model compound representing the intramolecular addition of a donor attached to a side chain (mimicking a peptide unit) was synthesized and studied with respect to its photochemical behaviour.

34. Mitt.:H. Falk undK. Thirring, Z. Naturforsch. im Druck.     

Evaluation of oily soil biodegradability by means of thermoanalytical methods

The oil content of an artificial polluted soil was estimated at different time period of a biodegradation process by means of thermogravimetry under non-isothermal conditions and in oxidative atmosphere. The model pollutant was hexadecane and for biodegradation a Pseudomonas aeruginosa culture was used. The intensity of the biodegradation process was correlated with the number of Colony Formation Units. In this study Pseudomonas aeruginosa was used, because these species are not pathogenic and easy to maintain in time and is resistant to hexadecane.     

Dynamics of cellulase production by glucose grown cultures of Trichoderma reesei Rut-C30 as a response to addition of cellulose

An economic process for the enzymatic hydrolysis of cellulose would allow utilization of cellulosic biomass for the production of easily fermentable low-cost sugars. New and more efficient fermentation processes are emerging to convert this biologic currency to a variety of commodity products with a special emphasis on fuel ethanol production. Since the cost of cellulase production currently accounts for a large fraction of the estimated total production costs of bioethanol, a significantly less expensive process for cellulase enzyme production is needed. It will most likely be desirable to obtain cellulase production on different carbon sources—including both polymeric carbohydrates and monosaccharides. The relation between enzyme production and growth profile of the microorganism is key for designing such processes. We conducted a careful characterization of growth and cellulase production by the soft-rot fungus Trichoderma reesei. Glucosegrown cultures of T. reesei Rut-C30 were subjected to pulse additions of Solka-floc (delignified pine pulp), and the response was monitored in terms of CO₂ evolution and increased enzyme activity. There was an immediate and unexpectedly strong CO₂ evolution at the point of Solka-floc addition. The time profiles of induction of cellulase activity, cellulose degradation, and CO₂ evolution are analyzed and discussed herein.     

Statistical optimization of conditions for protease production fromBacillus sp. and its scale-up in a bioreactor

A statistical approach, response surface methodology (RSM), was used to study the production of extracellular protease fromBacillus sp., which has properties of immense industrial importance. The most influential parameters for protease production obtained through the method of testing the parameters one at a time were starch, soybean meal, CaCl₂, agitation rate, and inoculum density. This method resulted in the production of 2543 U/mL of protease in 48 h fromBacillus sp. Based on these results, face-centered central composite design falling under RSM was employed to further enhance protease activity. The interactive effect of the most influential parameters resulted in a 1.50-fold increase in protease production, yielding 3746 U/mL in 48 h. Analysis of variance showed the adequacy of the model and verification experiments confirmed its validity. On subsequent scale-up in a 30-L bioreactor using conditions optimized through RSM, 3978 U/mL of protease was produced in 18 h. This clearly indicated that the model remained valid even on a large scale. RSM is a quick process for optimization of a large number of variables and provides profound insight into the interactive effect of various parameters involved in protease production.     

Influence of fadA G203R and ΔflbA mutations on morphology and physiology of submerged Aspergillus nidulans cultures

Morphologic and physiologic changes taking place in carbon-limited submerged cultures of Aspergillus nidulans ΔflbA and fadA ^G203R strains were studied. Loss-of-function mutation of the flbA gene resulted in an altered germination with unusually thick germination tubes, “fluffy” pellet morphology, as well as a reduced fragmentation rate of hyphae during autolysis. In the fadA ^G203R mutant strain, conidiophores formed in the stationary phase of growth, and the size of pellets shrank considerably. There were no significant differences in the generation of reactive oxygen species (ROS) and in the specific catalase and superoxide dismutase activities by the tested mutants and the appropriate parental strains. Therefore, the participation of ROS or antioxidative enzymes in FadA/FlbA signaling pathways seems to be unlikely in submerged cultures. On the other hand, earlier increases in the extracellular protease and ammonia production were recorded with the ΔflbA strain, whereas the protease and ammonia production of the fadA ^G203R mutant lagged behind those of the wild-type strains. Similar changes in the time courses of the induction of γ-glutamyltranspeptidase and the degradation of glutathione were observed. These results suggest that FadA/FlbA signaling may be involved in the mobilization of protein and peptide reserves as energy sources during carbon starvation.