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Applied Microbiology and Biotechnology (v.51, #4)

Chemically defined media for commercial fermentations by J. Zhang; R. Greasham (pp. 407-421).

The use of chemically defined media is gaining popularity in some commercial fermentations, particularly for the preparation of biological products. Although these media are still not frequently developed for industrial processes, they do exhibit favorable characteristics at large scale that are not observed with traditional complex media. This review focuses on the application, development, and practical considerations, especially process economics, of fermentations in chemically defined media in an industrial environment.

High-cell-density cultivation of microorganisms by D. Riesenberg; R. Guthke (pp. 422-430).

High-cell-density cultivation (HCDC) is required to improve microbial biomass and product formation substantially. An overview of HCDC is given for microorganisms including bacteria, archae and eukarya (yeasts). Problems encountered by HCDC and their possible solutions are discussed. Improvements of strains, different types of bioreactors and cultivation strategies for successful HCDC are described. Stirred-tank reactors with and without cell retention, a dialysis-membrane reactor, a gas-lift reactor and a membrane cyclone reactor used for HCDC are outlined. Recently modified traditional feeding strategies and new ones are included, in particular those for unlimited growth to very dense cultures. Emphasis is placed on robust fermentation control because of the growing industrial interest in this field. Therefore, developments in the application of multivariate statistical control, artificial neural networks, fuzzy control and knowledge-based supervision (expert systems) are summarized. Recent advances using Escherichia coli– the pioneer organism for HCDC – are outlined.

Production of ketocarotenoids by microalgae by P. Z. Margalith (pp. 431-438).

Among the highly valued ketocarotenoids employed for food coloration, astaxanthin is probably the most important. This carotenoid may be produced biotechnologically by a number of microorganisms, and the most promising seems to be the freshwater flagellate Haematococcus pluvialis (Chlorophyceae), which accumulate astaxanthin in their aplanospores. Many physiological aspects of the transition of the flagellate into aplanospores have been described. Mixotrophic cultivation and suitable irradiance may result in fairly good yields (up to 40 mg/l; 43 mg/g cell dry weight) within a reasonable time, under laboratory conditions. In order to compete with synthetic astaxanthin, suitable scaling-up is required. However, large-scale production in open ponds has proved unsatisfactory because of severe contamination problems. A selective medium might overcome this difficulty. Further research for the development of suitable strains is thus warranted.

New approaches to control plant parasitic nematodes by C. Jung; U. Wyss (pp. 439-446).

Plant parasitic nematodes are a serious threat for crop production worldwide. This review summarizes our understanding of plant nematode interactions and presents new alternatives for nematode control in the field. Breeding for resistance has been a major goal for many important crop species like soybean, potato, tomato and sugar-beet. As a result numerous nematode-resistance genes have been identified, two of which have been cloned recently, Hs1 pro-1 from sugar-beet, giving resistance to the beet cyst nematode Heterodera schachtii, and Mi from tomato, giving resistance to the root-knot nematode Meloidogyne incognita. Also artificial resistance genes, coding for nematotoxic proteins or causing rapid death of feeding cells, have been elucidated. In the future, genetic engineering of nematode resistance will become more and more important for plant breeding. Transformation techniques will allow genes to be quickly introduced into susceptible breeding lines and then combined with each other to produce plant varieties with durable resistance.

Cultivation of Tetrahymena thermophila in a 1.5-m³ airlift bioreactor by D. Hellenbroich; U. Valley; T. Ryll; R. Wagner; N. Tekkanat; W. Kessler; A. Roß; W.-D. Deckwer (pp. 447-455).

A large-scale cultivation system for the mass cell production and extraction of the protozoon Tetrahymena thermophila has been developed on the basis of a low-cost complex nutrient medium. Cell growth and the production of extracellular proteases were investigated using a 15-l stirred-tank reactor and 13-l and 1500-l airlift reactors. Processes using defined and complex medium formulations were compared. After cell mass production by 1200 l cell suspension in the large airlift bioreactor, two different extraction methods, based on the use of an extraction decanter and a sedimentation procedure, were compared and followed by cell lyophilization. Cell sedimentation was shown to be the more efficient extraction method as it enabled cell retention/separation while preserving the cell structure. Maximum cell growth was achieved in the stirred-tank bioreactor, supporting the hypothesis that higher shear forces reduce the particle size of the medium, which is responsible for an optimized nutrient supply. The highest glucose uptake rates were found in defined medium lacking the nutrient particles that are present in complex medium formulations. The cell-specific proteolytic activity in culture supernatants of airlift bioreactors using complex medium conditions was higher than that of a culture broth with cells grown under defined medium formulations.

Towards a high-yield bioconversion of ferulic acid to vanillin by A. Muheim; K. Lerch (pp. 456-461).

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l⁻¹. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l⁻¹ could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l⁻¹ and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l⁻¹ were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes.

Mathematical evaluation of plantaricin formation supports an auto-induced production mechanism by P. Klostermaier; C. Heiko Scheyhing; M. Ehrmann; R. F. Vogel (pp. 462-469).

The production rate of a bacteriocin, produced by Lactobacillus plantarum TMW1.25 and previously named plantaricin1.25, was studied during pH-constant batch fermentations under various growth media conditions. The growth of L. plantarum and production of bacteriocin during the retardation phase were modelled, using 11 different empirical and mechanistic approaches. The optimal pH for bacteriocin production was 4.5. Among the different nitrogen sources tested, yeast extract was the most important, on the basis of the fact that the maximum growth rate decreased 16% without yeast extract, and only 7.2% or 8.1% without meat extract or peptone respectively. However, the change of nitrogen source did not have a significant effect on bacteriocin production. The progression of plantaricin1.25 production during the retardation phase and growth of L. plantarum TMW1.25 could be described by a structured model in which the bacteriocin concentration induces its own production. Among those models not implementing bacteriocin induction, only the one with an exponential increase of bacteriocin yield per unit biomass was suitable to describe bacteriocin production. Computer-aided evaluation of experimental data appears to be helpful in elucidating the relationship between the growth of lactic acid bacteria and bacteriocin production.

Enzymatic synthesis of silicon-containing dipeptides with 3-trimethylsilylalanine by H. Ishikawa; H. Yamanaka; T. Kawamoto; A. Tanaka (pp. 470-473).

In this study, the enzymatic synthesis of silicon-containing dipeptides with a silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), was attempted in ethyl acetate, and the effects of TMS-Ala on thermolysin-catalyzed dipeptide synthesis are also discussed. Benzyloxycarbonyl(Z)-TMS-Ala was recognized by thermolysin as a better substrate than Z-Leu, and various silicon-containing dipeptides, Z-(TMS-Ala)-Xaa-OMe (Xaa = Leu, Ile, Phe, etc.), could be obtained. The acceleration of the reaction rate in the synthesis of Z-(TMS-Ala)-Leu-OMe compared with Z-Leu-Leu-OMe synthesis was explained by the higher hydrophobicity of the side-chain of TMS-Ala containing a trimethylsilyl group. On the other hand, TMS-Ala-OMe was not accepted as the amino component because of the bulkiness of the trimethylsilyl group. The enantioselectivity of thermolysin was very high. Z-d-TMS-Ala was not a substrate, while Z-l-TMS-Ala served as a good substrate.

Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67 by M. Yasuda; M. Aoyama; M. Sakaguchi; K. Nakachi; N. Kobamoto (pp. 474-479).

Bacillus pumilus TYO-67 was isolated from tofu (soybean curd) as the best producer of a soybean-milk-coagulating enzyme, induced by the addition of soybean protein to the growth medium. The enzyme was purified approximately 30-fold with an 11% yield. The homogeneous preparation of the enzyme showed that it is a monomer with a molecular mass of about 30 kDa and has an isoelectric point at pH 9.75. The results of amino acid composition analyses showed that the enzyme is rich in alanine, aspartic acid, glycine, serine and valine. Although the amino-terminal amino acid (alanine) was identical with that of subtilisins, the amino-terminal sequence was different from those of subtilisins. The α-helix content of the enzyme was calculated to be 28.2%. The optimum pH and temperature were observed at 6.0–6.1 and 65 °C respectively. The enzyme was significantly activated by the addition of 1 mM Mn²⁺, Ca²⁺, Mg²⁺, and Sr²⁺ ions in the reaction mixture, and its thermal stability was significantly increased by Ca²⁺ ion.

Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid by R. Borgdorf; S. Warwel (pp. 480-485).

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the CC double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3–4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candidaantarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid.

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes by M. Kataoka; K. Yamamoto; H. Kawabata; M. Wada; K. Kita; H. Yanase; S. Shimizu (pp. 486-490).

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP⁺ to CHBE formed was 13 500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.

Characterisation of cellulose-binding proteins that are involved in the adhesion mechanism of Fibrobacter intestinalis DR7 by J. Miron; C. W. Forsberg (pp. 491-497).

Cellulose-binding proteins (CBP) isolated from cell envelopes of the cellulolytic bacterium Fibrobacter intestinalis strain DR7 were studied in order to investigate the adhesion mechanism. The proteins were examined for their reaction with antibodies that specifically block bacterial adhesion, response to glycosylation staining and monosaccharide composition. To this end, the effect of some monosaccharides (CBP components) on blocking of DR7 adhesion to cellulose was determined. Previous study had shown the occurrence of 16 CBP in the outer membrane and periplasm of DR7, of which 6 had endoglucanase activity (Miron and Forsberg 1998). Data from the present study show that most of the 16 CBP of DR7, except for the 38-, 90- and 180-kDa proteins, are glycosylated. Rabbit antibodies that specifically block DR7 adhesion were prepared by affinity preabsorption of antiserum against wild-type DR7 with bacterial cells of its adherence-defective mutant (DR7-M). The preabsorbed antibodies reacted positively in Western blotting with glycosylated CBP of 225, 200, 150, 70, 45 and <38 kDa from the DR7 outer membrane, and reacted weakly with CBP of DR7-M. Modification of glycosidic residues attached to the CBP of DR7 by periodate oxidation prevented any reaction with the preabsorbed antibodies. Monosaccharide analysis by HPLC of isolated CBP from the outer membrane and periplasm of DR7 cells, showed that galactosamine, glucosamine, galacturonic acid, and glucuronic acid were the predominant monosaccharide components of CBP that can block the adhesion of DR7 cells to cellulose. It is suggested that some glycosylated residues of CBP may have a predominant role in the adhesion of DR7 to cellulose.

Cometabolic biodegradation of methyl t-butyl ether by Pseudomonas aeruginosa grown on pentane by P. M. Garnier; R. Auria; C. Augur; S. Revah (pp. 498-503).

A bacterial strain identified as Pseudomonas aeruginosa was isolated from a soil consortium able to mineralize pentane. P. aeruginosa could metabolize methyl t-butyl ether (MTBE) in the presence of pentane as the sole carbon and energy source. The carbon balance for this strain, grown on pentane, was established in order to determine the fate of pentane and the growth yield (0.9 g biomass/g pentane). An inhibition model for P. aeruginosa grown on pentane was proposed. Pentane had an inhibitory effect on growth of P. aeruginosa, even at a concentration as low as 85 μg/l. This resulted in the calculation of the following kinetic parameters (μ_max = 0.19 h⁻¹, K _s = 2.9 μg/l, K _i = 3.5 mg/l). Finally a simple model of MTBE degradation was derived in order to predict the quantity of MTBE able to be degraded in batch culture in the presence of pentane. This model depends only on two parameters: the concentrations of pentane and MTBE.

Purification and properties of a novel raw starch degrading cyclomaltodextrin glucanotransferase from Bacillus firmus by B. N. Gawande; A. Goel; A. Y. Patkar; S. N. Nene (pp. 504-509).

A novel raw starch degrading cyclomaltodextrin glucanotransferase (CGTase; E.C. 2.4.1.19), produced by Bacillus firmus, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The molecular weight of the pure protein was estimated to be 78 000 and 82 000 Da, by SDS-PAGE and gel filtration, respectively. The pure enzyme had a pH optimum in the range 5.5–8.5. It was stable over the pH range 7–11 at 10 °C, and at pH 7.0 at 60 °C. The optimum temperature for enzyme activity was 65 °C. In the absence of substrate, the enzyme rapidly lost its activity above 30 °C. K _m and k _cat for the pure enzyme were 1.21 mg/ml and 145.17 μM/mg per minute respectively, with soluble starch as the substrate. For cyclodextrin production, tapioca starch was the best substrate used when gelatinized, while wheat starch was the best substrate used when raw. This CGTase could degrade raw wheat starch very efficiently; up to 50% conversion to cyclodextrins was obtained from 150 g/l starch without using any additives. The enzyme produced α-, β- and γ-cyclodextrins in the ratio of 0.2:9.2:0.6 and 0.2:8.6:1.2 from gelatinized tapioca starch and raw wheat starch with 150 g/l concentration respectively, after 18 h incubation.

Optimization of pyrene oxidation by Penicillium janthinellum using response-surface methodology by L. A. Launen; L. J. Pinto; M. M. Moore (pp. 510-515).

At present, there is little information on the optimization of the degradation of polycyclic aromatic hydrocarbons (PAH) by deuteromycete filamentous fungi, a reaction catalyzed by cytochrome P450 monooxygenases. We utilized response-surface methodology to determine the optimal growth conditions for the oxidation of the PAH pyrene by Penicillium janthinellum SFU403, with respect to the variables glucose concentration, nitrate concentration and bioconversion time. Models were derived for the relationship between the variables tested and the level of the pyrene oxidation products, 1-pyrenol (1-PY) and pyrenequinones (PQ). Production of 1-PY and PQ were optimized by the same glucose and nitrate concentrations: 2.5% glucose and 1.5% sodium nitrate. The optimized 1-PY and PQ bioconversion times were 71 h and 73 h respectively. These conditions improved the yield of 1-PY by fivefold and PQ were more than 100-fold higher than the baseline levels obtained in this study. The optimized PQ yield represented 95% of the initial pyrene, thus the total optimised pyrene bioconversion to 1-PY and PQ was approximately 100%. Concentrations of glucose exceeding 4.0% repressed pyrene hydroxylation. Pyrene hydroxylation occurred almost exclusively during the deceleration phase of culture growth.

Induction and functional role of cytochromes P450 in the filamentous fungi Mortierella alpina ATCC 8979 and Cunninghamella blakesleeana DSM 1906 during hydroxylation of cycloalkylbenzoxazoles by O. Asperger; H. Steinbrenner; A. Lehmann; M. Petsch; H. Griengl (pp. 516-522).

The occurrence and regulation of cytochrome P450 (P450) in Mortierella alpina and Cunninghamella blakesleeana have been studied to elucidate the enzymatic basis by which 2-cyclopentyl-1,3-benzoxazole is hydroxylated to 3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol by these organisms. The occurrence of P450 in M. alpina was first been shown after induction with n-hexane. An assay protocol was developed with n-hexane-induced cells and adapted to the handling of fungal mycelia. This allowed the direct spectral determination of P450 in non-fractionated whole-cell suspensions, and an investigation of its regulation. Small amounts of P450 have been detected in early-stationary-phase cells in the absence of exogenous inducers. Addition of 2-cyclopentyl-1,3-benzoxazole or n-hexane resulted in a significant induction of P450. Induction by n-hexane occurs in all phases of growth but decreases rapidly during the stationary phase. The rate of 2-cyclopentyl-1,3-benzoxazole hydroxylation correlated with the content of substrate-induced P450 but not with the level of n-hexane-induced P450. Hydroxylation rates were significantly diminished in the presence of typical P450 inhibitors, the interaction of which with P450 was shown with isolated microsomes of M. alpina. It is concluded that a P450 enzyme is responsible for the hydroxylation of 2-cyclopentyl-1,3-benzoxazole, but that multiple forms of P450 forms occur. Similarly, a dependence on P450 is shown by spectral as well as by inhibition studies for the hydroxylation of this substrate by C. blakesleeana.

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy by K. Hong; S. Sun; W. Tian; G. Q. Chen; W. Huang (pp. 523-526).

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm⁻¹, 1740 cm⁻¹ or 1732 cm⁻¹ respectively. Other accompanying bands near 1280 cm⁻¹ and 1165 cm⁻¹ helped identify the types of PHA. The intensity of the methylene band near 2925 cm⁻¹ provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm⁻¹, 1744 cm⁻¹ or 1739 cm⁻¹, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further confirmed by gas chromatography analysis.

Fungal laccase grafts acrylamide onto lignin in presence of peroxides by C. Mai; O. Milstein; A. Hüttermann (pp. 527-531).

Laccase (EC 1.10.3.2) from the white-rot basidomycete Trametes versicolor in the presence of organic peroxides, particularly dioxane peroxide, tetrahydrofuran peroxide and t-butylhydroperoxide, initiated free-radical copolymerization of acrylamide and lignin. Hydrogen peroxide showed no such effect. Both the type of peroxide and the catalytic efficiency of the enzyme were important to ensure a significant yield of copolymerisate and a high rate of acrylamide incorporation into a lignin backbone. The mechanism of the enzymatic grafting is discussed.

Aerobic degradation of a hydrocarbon mixture in natural uncontaminated potting soil by indigenous microorganisms at 20 °C and 6 °C by M. Eriksson; G. Dalhammar; A.-K. Borg-Karlson (pp. 532-535).

A hydrocarbon mixture containing p-xylene, naphthalene, Br-naphthalene and straight aliphatic hydrocarbons (C₁₄ to C₁₇) was aerobically degraded without lag phase by a natural uncontaminated potting soil at 20 °C and 6 °C. Starting concentrations were approximately 46 ppm for the aromatic and 13 ppm for the aliphatic compounds. All aliphatic hydrocarbons were degraded within 5 days at 20 °C, to levels below detection (ppb levels) but only down to 10% of initial concentration at 6 °C. Naphthalene was degraded within 12 days at 20 °C and unaffected at 6 °C. At 20 °C p-xylene was degraded within 20 days, but no degradation occurred at 6 °C. Br-naphthalene was only removed down to 30% of initial concentration at 20 °C, with no significant effect at 6 °C. The biodegradation was monitored with head space solid-phase microextraction and gas chromatography–mass spectrometry.

Effects of nitrogen limitation on biofilm formation in a hydrocarbon-degrading trickle-bed filter by P. Holubar; C. Andorfer; R. Braun (pp. 536-540).

The effect of nitrogen limitation on young and mature steady-state biofilm in a trickle-bed filter was studied. Toluene and n-heptane were the sole carbon source. Biomass concentration, respiration, substrate-induced respiration, metabolic quotient, and total hydrocarbon degradation efficiency were measured. The aim of the experiment was to control excess biomass production in the trickle-bed filter by limiting the mineral nutrients and to achieve increased mineralization of the carbon source. Biofilm growth responded strongly to the amount of available nitrogen, whereas hydrocarbon degradation efficiency reached a maximum of 60% and could not be increased even by further addition of nitrogen. The experiments showed that 95% of the adsorbed carbon was mineralized completely and only 5% was used for biofilm formation. This complete mineralization can also be concluded from the metabolic quotient. The value of the latter was about 6–10 mg CO₂-C g⁻¹C_mic h⁻¹, indicating an expanded energy demand due to stress effects in the presence of nutrient deficiency. It was postulated that determination of the metabolic quotient could be an simple instrument to measure the rate of mineralization of carbon sources and also the rate of biomass formation in trickle-bed filters or biofilters.

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Applied Microbiology and Biotechnology (v.51, #4)