Search

Lactic acid bacteria (LAB) have long been used to produce safe and high quality products as they are potential producers of a wide range of antimicrobial compounds that exert either narrow or wide spectrum antimicrobial activity towards spoilage or disease-causing organisms. The present investigation aimed to study the antimicrobial effect of three LAB strains, viz., Lactobacillus plantarum (86), Lactobacillus fermentum (AI2) and Weissella confusa (AI10), against two clinical pathogenic strains viz., Escherichia coli NG 502121 and Staphylococcus aureus AY 507047 in co-culture. Effects of change in inoculum size, and growth measurement at different time intervals were also studied. The pH and viable count were measured for initial as well as 24 h incubated samples. A significant (P

The possibility of improving the physical quality of extruded fish feed using transglutaminase (TGase) treatment at different stages of the production process was investigated. The addition of TGase to the raw material mix and processing under high and medium moisture conditions significantly increased (p > 0.05) the durability, hardness and elasticity of fish feed pellets. However, the water stability of feeds was only improved when the TGase was applied in the vacuum coater; when it was mixed with the dry raw materials, the water stability of the product decreased. By further optimization of the enzyme dose when added to the vacuum coater, an increase in pellet durability was observed at enzyme dosages between 2.5 and 5 g kg−1. Application of TGase in the coating step allowed an 87.5 % decrease in the dose of enzyme in feed produced under high moisture conditions. TGase treatment improves the physical quality of extruded fish feed, and the importance of optimization of enzyme dosage and processing conditions was demonstrated.

An acute meal study was performed to determine postprandial glucose and insulin responses after consumption of two fermented oat bran-based beverages (with and without exopolysaccharides) and yoghurt. This randomized, single-blind, within-subject study included 18 healthy, overweight participants. Four breakfast meals, including a reference meal, were tested; all meals contained 50 g of available carbohydrates, but differed in energy and macronutrient composition. All experimental meals reduced the postprandial glucose response compared with the reference meal. The oat drinks as well as the yoghurt elicited higher early (0-15 min) insulin responses, but the overall insulinaemia were similar to the reference meal. A new food product containing fermented liquid oat bran and milk reduced the postprandial blood glucose response as efficiently as yoghurt after a high-glycaemic index white wheat bread meal, but the presence of microbial exopolysaccharides did not affect the outcome.

Exopolysaccharide producing lactic acid bacterial (LAB) were tested in vitro to select a candidate probiotic strain by testing their tolerance to low pH and bile, bile salt hydrolase (BSH) activity, antibiotics susceptibility pattern and antimicrobial activity. Results indicated that LAB isolates were highly sensitive against oxbile (0.3%) but could grow in the presence of sodium taurocholate (0.3%). Seven out of 9 isolates were found to be BSH positive. Two of the isolates, Lactobacillus plantarum 86 and Weissella cibaria 92 showed considerable antimicrobial activity against Gram-positive and Gram-negative pathogens. The study reveals that traditional fermented products of India could be an alternative and readily available resource for LAB starter cultures with interesting functional characteristics.

Recovering nutrients from urine as solid minerals such as struvite can eliminate many hurdles related to the use of liquid urine as fertilizer in the current urine separation systems. The focus of this study has been to increase nitrogen and phosphorus removal efficiencies by developing a struvite formation and recycling process. Struvite crystallization was carried out in urine samples by addition of MgO where 92% of phosphorus as orthophosphate was recovered. In order to optimize the use of calcined struvite in the struvite recycling step, four calcination treatments were tested and calcination at 200 degrees C for 3 h was chosen for further experiments. To remove the remaining ammonia present in urine a struvite recycling step was used subsequently. Through sequential struvite formation and struvite recycling process up to 90% of ammonium nitrogen was successfully removed. (Less)

Pediococcus parvulus 2.6 (previously Pediococcus damnosus 2.6, here confirmed as P. parvulus by 16S DNA sequencing) displayed antibacterial activity toward several bacterial species, including isolates found as contaminants in oats, herein genetically identified as Bacillus cereus. No inhibition of Listeria monocytogenes was found under the conditions used. Antibacterial activity was retrieved after ammonium sulfate or acetone precipitation showed it to be peptide mediated. P. parvulus 2.6 has previously shown good technological properties in oat-based products. This, together with the currently found inhibition of food spoilage microorganisms like B. cereus, makes it suitable as a food protective culture. Survival trials of P. parvulus 2.6 at conditions mimicking the gastrointestinal tract were prompted by previously found cholesterol-lowering effects in humans after consumption of oat products cofermented by using P. parvulus 2.6 and Bifidobacterium spp. Viability was measured with in vitro, gutlike simulations at 37 degrees C. High survival was shown under two of three conditions (gastric juice, bile, and small intestine juice), defined as main obstacles of the gastrointestinal tract. The critical step was bile exposure. At a concentration of 20%, viability was low, but 0.3% bile (mean concentration in the intestine) did not have a major influence on growth. Viability of P. parvulus 2.6 was significantly decreased in gastric juice at pH 1.5 (with pepsin), but it was not significantly affected at pH 2.5, and was also improved at a lower pH in 20% oat milk. Viability was judged sufficient for colonization at gutlike conditions, qualifying the strain for further probiotic studies.

A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae alpha-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T(m)) was 78 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.

Escherichia coli is a microorganism routinely used in the production of heterologous proteins. The overexpression of a xylanase (Xyn10A Delta NC), which originated from the thermophile Rhodo-thermus marinus cloned under the control of the strong T7/lac promoter in a defined medium (mAT) using a substrate-limited feed strategy, was however shown to impose a significant metabolic burden on host cells. This resulted in a decreased cell growth rate and ultimately also a decreased target protein production. The investigation hence centers on the effect of some selected nutrient feed additives (amino acid [Cys] or TCA-intermediates [citrate, succinate, malate]) used to relieve the metabolic burden imposed during the feeding and postinduction phases of these glucose-limited fed-batch cultivations. The use of either succinic acid or malic acid as feed-additives resulted in an increase in production of approximately 40% of the heterologous thermostable xylanase. Furthermore, use of lactose as an alternative inducer of the T7/lac promoter was also proven to be a suitable strategy that significantly prolonged the heterologous protein production phase as compared with induction using isopropyl P-D-thiogalactopyranoside (IPTG). (Less)

The formation of exopolysaccharides (EPS) by Pediococcus damnosus strain 2.6 was investigated in two different high P-glucan suspensions of oat (OBC) and barley (BBC). Bacterial growth was confirmed by measuring pH, colony forming units (cfu), viscosity and ropiness during growth. Growth and an increase in viscosity of the beta-glucan suspension based on oat were observed whereas only a minor growth and no significant effect on viscosity was seen in the beta-glucans suspension based on barley. Higher bacterial growth and higher final viscosity and ropiness were observed in samples that were mixed with the OBC and an ordinary commercial oat-base (Adavena(R) G40). These results show the potential of using a combination of a high beta-glucan suspension of cereals to generate fermented products containing both high levels of native beta-glucans from oats or barley and also microbial beta-glucans from the starter culture.

Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.

A cultivation strategy combining the advantages of temperature limitedfed-batch and probing control is presented.This is a suitable way to produce recombinant proteins while minimizing therelease of endotoxins which complicates the downstream processing.The downside with the temperature limited fed-batch technique has been the difficulty to achieve a controlled excess of glucose inthe reactor without the accumulation of acetic acid when usingstandard sensors.A method using a probing feeding strategy withdown-pulses is here suggested. The technique is investigated insimulations and experiments.

Biotechnological leaching has been proposed as a suitable method for extraction of vanadium from spent catalysts and oil ash. In the biological leaching process, the vanadium(V) can be reduced to vanadium(IV), which is a less toxic and more soluble form of the vanadium. The present investigation showed that Acidithiobacillus ferrooxidans efficiently reduced vanadium(V) in the form of vanadium pentaoxide, to vanadyl(IV) ions, and tolerated high concentrations of vanadium(IV) and vanadium(V). A. ferrooxidans was compared with Acidithiobacillus thiooxidans, which has previously been utilized for vanadium leaching and reduction. Vanadium pentaoxide and sodium vanadate were used as model compounds. The results of this study indicate possibilities to develop an economical and technically feasible process for biotechnological vanadium recovery.

The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.

The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that...

The fermentation characteristics of two strains of lactic acid bacteria (LAB), Lactobacillus brevis G-77 and Pediococcus damnosus 2.6 were compared in an oat-based, nondairy milk medium (Adavena® G40). Viscosity and ropiness were the main growth parameters studied. Both strains are reported to produce an exopolysaccharide (EPS) with a -glucan structure; in addition, the L. brevis strain produces also an EPS with an -glucan structure. Both strains were able to ferment and produce EPS in the oat-based, nondairy medium to the extent that an obvious change was observed in terms of viscosity and ropiness during the fermentation period. These results show the potential of both LAB strains as possible starter cultures in new kinds of fermented, nondairy milk products. (Less)

Three fermented, ropy, non-dairy, oat-based products were evaluated for their effect on serum lipids, faecal cholesterol and faecal short chain fatty acids in germfree and conventional rats. Three different exopolysaccharide (EPS) producing lactic acid bacteria strains were used to ferment the non-dairy oat-base (Adavena ® G40) (Ceba Foods AB, Lund, Sweden). Two commercial non-dairy products based on oats (Mill Milk™) (Ceba Foods AB, Lund, Sweden) and rice (Rice Dream ® ) (Imagine Foods, London, UK) were used as non-ropy and unfermented controls. All the standardized feeds were sterilized before being fed to the animals. Adult, germfree and conventional AGUS rats

Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.

Four strains of lactic acid bacteria were used as pure or mixed cultures to investigate the texture-promoting capacity and exopolysaccharides (EPS) formation in three different oat-based, non-dairy milk products, Adavena G40, Adavena M40 (both with a dry matter content of 20%) and Mill Milk (with a dry matter content of 10%). Viscosity was measured at two different shear rates during 2 min of shear thinning. The highest viscosity was measured at a shear rate of 129 s–1 when a mixed culture consisting of Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 and Lactobacillus brevis DSM 1269 was grown at 30 °C in the medium containing mostly glucose as the carbohydrate source (Adavena G40). The mixed culture consisting of Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 and Streptococcus thermophilus DSM 20259 gave the highest viscosity when using the medium with a dry matter content of 20% and maltose as the main carbohydrate source (Adavena M40). The EPS formation in the Adavena G40 medium was confirmed by isolating the soluble polymer fraction after fermentation. A higher yield of polymer dry mass was obtained from the samples with higher viscosity. The study shows that the co-operative growth that occurs when using mixed cultures also influences the EPS formation and final viscosity in the different oat-based media. This knowledge is of importance when strains are selected for the development of new kinds of fermented, oat-based, non-dairy products.

In an attempt to develop a fermented, non-dairy product based on oats, a new kind of oat base, Adavena® M40, was fermented with two different yoghurt cultures. Adavena® M40 is a concentrated liquid (with a dry matter content of 16 or 18%) derived entirely from oats, with maltose as the main carbohydrate source and an intact β-glucan content. The oat base was heat treated for Smin at 85°C prior to inoculation. Additives in the form of stabiliser, fat and flavours were used. Texture, syneresis, colour and sensory parameters were evaluated. Yoghurt was used as a control. The final product had an acidity and viscosity similar to those of yoghurt. Addition of xanthan gum (0.03% w/v) improved the texture and overall appearance of the product. The product had the same texture as yoghurt but showed less syneresis. The mixture was less white than the control. The oat-based, yoghurt-like product showed high acceptability in terms of acidity, texture and overall appearance. The addition of flavours resulted in a higher acceptance of the final products by the panellists. The β-glucan content was still high after the fermentation process. The results indicated the potential for a new, fermentable, oat-based product with high acceptance and a high final β-glucan content. Adavena® M40 est un produit liquide derive de l'avoine. Il subit un traitement thermique, est fermente et est additionne de stabilisants, graisses et aromes. Les proprietes texturales, physicochimiques et sensorielles sont comparees a celles du yaourt. Le produit obtenu est satisfaisant quant a son gout et son apparence. L'ajout de bacteries lactiques permet d'obtenir un produit acidifie, et ayant developpe des flaveurs. La teneur en β-glucane du produit n'est pas modifiee par la fermentation et l'entreposage.

Cryo-ground tire rubber was treated with the sulfur oxidizing bacterium Thiobacillus ferrooxidans in order to devulcanize the surface of the ground particles. The devulcanized rubber (15% (w/w)) was mixed with carbon black filled natural rubber, and subsequently vulcanized. The physical properties of the resulting material were examined. This study shows that the stress relaxation properties are improved due to the microbial treatment. The stress at break and swelling properties are also positively affected by this process.

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 degrees C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 degrees C and unfolding temperatures up to 103 degrees C.

Nine strains of lactic acid bacteria were tested for fermentation characteristics in an oat- based, non-dairy milk substitute, Mill MilkTM (MM medium). Viable counts, aroma formation, lactic acid production and viscosity were the parameters investigated. In general, bacterial strains grown at 30°C yielded a better flavour and higher viable counts than those incubated at 37°C. Strains were selected for their capacity to produce exopolysaccharides in a semi-defined medium. Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 was chosen for further fermentation experiments, using yoghurt as a control. Production of exopolysaccharide and shear thinning properties were measured using a viscosimetric method. A combination of temperature stress and an increased carbon/nitrogen ratio was shown to affect the production of exopolysaccharide. Increased viscosity was found after incubation at a low temperature using glucose as a supplementary carbon source. The production of exopolysaccharides was also favoured by prolonged incubation times. The use of physical factors such as time and temperature in combination with chemical factors, i.e. media and carbon/nitrogen levels, were crucial in the course of improving exopolysaccharide production during fermentation in the MM medium.

α-Galactosidase activity in Rhodothermus marinus is dependent on the composition of the growth media. A maximum of 46 μU g−1 cell dry weight was obtained using minimal medium with galactooligo- or polysaccharides as single carbon source. An enzyme hydrolysing both high and low molecular weight galacto-saccharides was partly purified from the cell fractions. The molecular weight was 200 kDa (native) and 50 kDa (monomer). It was optimally active at 85 °C, with a half-life of 2 h at 75 °C, and had a broad pH range (4–8).

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichiacoli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

A cultivation strategy for the production of two truncated thermostable recombinant xylanases (Xyn1deltaN and Xyn1deltaNC) was developed. Fed-batch cultivations of Escherichia coli strain BL21(DE3) with a controlled exponential glucose feed led to high specific production of the recombinant proteins. Addition of complex nutrients (e.g. Tryptone Soya Broth (TSB)) to the media were shown to increase both the specific growth rate during the production phase and the production per cell. The final cell-mass concentration depended on the time of induction in relation to both the feed-start and the expected time at which the cultivation had to be terminated due to oxygen transfer limitations or cell lysis. The gene used for the genetic constructions (encoding Xyn1deltaN and Xyn1deltaNC) was originally isolated from Rhodothermus marinus. Recombinant protein expression was controlled by the T7 lac-promoter and induced in the fed-batch phase at low glucose concentrations by the single addition of either lactose or isopropyl-thio-beta-d-galactoside (IPTG). In lactose-induced cells, the production of recombinant xylanase was delayed for approximately 30 min in comparison with those induced with IPTG, but the specific product levels were comparable at 3 h after induction. At this time, approximately 35% of the intracellular protein content was constituted by recombinant xylanase. Under the cultivation conditions used, production of the shorter deletion derivative (Xyn1deltaNC) led to nonspecific leakage and cell lysis, starting 1.5 or 2 h after induction with IPTG or lactose, respectively. At 3 h after induction, 50% of the produced protein (Xyn1deltaNC) was found in the culture medium. This was not the case for the longer protein (Xyn1deltaN), where only 10% of the xylanase leaked into the medium.

The catalytic domain of a xylanase from Rhodothermus marinus was produced in Escherichia coli . The catalytic domain belongs to glycosyl hydrolase family 10. The produced protein has a 22-amino acid leader peptide followed by a 411-amino acid truncated xylanase. The molecular mass was 48 kDa and the recombinant xylanase had a pI of 4.9. The pH and temperature optima for activity were determined to be 7.5 and 80°C, respectively. At that temperature the enzyme had a half-life of 1 h 40 min. An addition of 1 mM calcium stabilized the activity of the enzyme at 80°C. The xylanase had its highest specific activity on oat spelt xylan but was active also on other xylans and to a limited extent on some other polysaccharides (soluble glucans). No exo- or endo-cellulase activity was observed. Hydrolysis of xylo-oligomers and oat spelt xylan was studied and the predominant products of hydrolysis were xylobiose and xylotriose. The enzyme was inactive on xylobiose, xylotriose and on the soluble fraction from oat spelt xylan. The R. marinus xylanase is shown to have a strong preference for internal linkages and is therefore classified as an endo-xylanase.

Thiobacillus ferrooxidans, T. thioparus, Acidianus brierleyi and TH2 Lund, an archaeal isolate, were tested for sulphur oxidising capacity on ground rubber from spent car tyres. T. ferrooxidans converted 8 % (w/w) of the sulphur in cryoground spent rubber to sulphate in 20 days. Desulphurization of fresh made natural rubber was studied with scanning electron microscope with energy-dispersive x-ray spectrometer and electron spectroscopy for chemical analysis. The average depletion of sulphur on the treated surface was 24 % compared to the bulk region. Oxidised sulphur species were detected on the surface after microbial treatment. © Rapid Science Ltd. 1998

Every year large amounts of spent rubber material, mainly from rubber tyres, are discarded. Of the annual total global production of rubber material, which amounts to 16–17 million tonnes, approximately 65% is used for the production of tyres. About 250 millions spent car tyres are generated yearly in USA only. This huge amount of waste rubber material is an environmental problem of great concern. Various ways to remediate the problem have been proposed. Among these are road fillings and combustion in kilns. Spent tyres, however, comprise valuable material that could be recycled if a proper technique can be developed. One way of recycling old tyres is to blend ground spent rubber with virgin material followed by vulcanization. The main obstacle to this recycling is bad adhesion between the crumb and matrix of virgin rubber material due to little formation of interfacial sulphur crosslinks. Micro-organisms able to break sulphur-sulphur and sulphur-carbon bonds can be used to devulcanize waste rubber in order to make polymer chains on the surface more flexible and facilitate increased binding upon vulcanization. Several species belonging to both Bacteria and Archaea have this ability. Mainly sulphur oxidizing species, such as different species of the genus Thiobacillus and thermoacidophiles of the order of Sulfolobales, have been studied in this context. The present paper will give a background to the problem and an overview of the biotechnological possibilities for solutions of waste rubber as an environmental problem, focusing on microbial desulphurization.

Thermophilic microorganisms have been of great scientific interest for several decades, principally in regard to their biotechnological potential and also of the thermostable enzymes they produce. Optimal cultivation techniques for these organisms are required, therefore, not only for basic study but also for evaluation of their thermostable microbial products. Operating a fermentor at elevated temperatures may be advantageous in terms of increased solubility of substrates, improved mass transfer due to decreased viscosity, and increased diffusion rates. However, the cultivation of thermophiles also has many associated problems. A high cultivation temperature can give unexpected problems affecting the choice of reactor design and construction materials, and with the heating and cooling of the fermentor. Other problems may be caused by the low solubility of gases and the instability of substrates and other reagents used. Furthermore, high productivity requires high cell densities to be achieved and in many cases thermophiles are characterised by low growth rates, low growth yields and susceptibility to substrate and product inhibition at low concentrations. Different ways to circumvent some of these problems, such as using gas-lift fermentors, dialysis fermentors or cultivation with cell recycling are discussed.

The growth behaviour of the thermophilic anaerobic bacterium Thermoanaerobium brockii for the production of its intracellular secondary alcohol dehydrogenase (sADH) has been studied in batch cultures as well as in continuous cultivation with complete cell recycling. In batch culture the maximum specific growth rate, μ max , was 0.5 h -1 , resulting in a cell density of 1.2 g l -1 and an sADH activity of 1.3 units ml -1 . Higher glucose concentrations resulted in a decrease in μ max enzyme productivity as well as biomass yield although an increase in total biomass was achieved. To improve cell density and productivity, continuous culture with complete cell recycling was used, resulting in an increase in cell density by 5 times and in productivity of the sADH by 3 times in comparison to those obtained in batch culture.

Growth of the thermophilic H-oxidizing bacterium Hydrogenobacter thermophilus (ITI 553) was achieved in continuous culture using a gas-lift bioreactor. Although the highest equilibrium cell density was obtained at a dilution rate of 0.05 h, the maximum productivity was obtained at 0.1h. At 0.05 h, the maintainable cell density was 7.10 cells/mL at 2-4% oxygen with a gas flow rate of 0.1vvm. The steady-state cell density increased further with increasing gas flow rates. Maximum specific activity of the peroxidase was obtained at 8% oxygen. The total productivity of the enzyme had its maximum at 6-8% oxygen. Increasing gas flow rates had hardly any effect upon peroxidase specific activity, however, productivity was increased due to the increased cell densities obtained.

A flow-injection system for determination of acetic acid in Escherichia coli cultivations with electrochemical detection based on immobilized acetate kinase (AK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) was developed to cope with the problems related to measurements under process conditions such as interferences from pyruvate, drift of electrode baseline and making the cultivation medium and reagents compatible. The results obtained by flow injection analysis were compared with those obtained with an enzymatic test kit.

Six strains isolated from fermented food were identified as Weissella species by 16S rDNA sequencing, clustering with the species pair W. confusa/W. cibaria. The strains were analysed for growth on glucose, xylose and xylooligosaccharides (XOS). All strains were xylose positive using the API CHL 50 test. Growth on XOS was observed for strains 85, 92, 145 and AV1, firstly by optical density measurements in microtitre plates and secondly in batch cultures also confirming concomitant decrease in pH. Analysis of XOS before and after growth established consumption in the DP2-DP5 range in the four XOS-fermenting strains. XOS were consumed simultaneously with glucose, while xylose was consumed after glucose depletion. Cell-associated β-xylosidase activity was detected in the XOS-fermenting strains. Analysis of genomic data suggests this activity to be linked with genes encoding glycoside hydrolases from family 3, 8 or 43. No endo-β-xylanase activity was detectable. Major end products were lactate and acetate. A higher ratio of acetic acid to lactic acid was obtained during growth on XOS compared with growth on glucose. This is the first report on utilization of XOS in Weissella, indicating an increased probiotic potential for XOS-utilizing strains from the species pair W. confusa/W. cibaria, but also showing that XOS utilization is strain dependent for these species.

Growing evidence indicates that the gut microbiota could have an important role in the development of diet- and lifestyle-induced diseases. It has been shown that modulation of the gut microbiota by means of probiotics and prebiotics could improve host health. An oat-based product fermented by the exopolysaccharide (EPS)-producing organism Pediococcus parvulus 2.6 has been reported to have a bifidogenic effect. To find out whether the effect could be attributed to the EPS or the bacterium, mice were fed a diet supplemented with 2% purified EPS or 10 8 CFU/g of live P. parvulus 2.6 for 6 weeks. Both supplementations altered the gut microbiota composition but in different directions. Purified EPS not only significantly lowered the microbial diversity ( P < 0.001) but decreased the bifidobacterial population ( P = 0.01). In contrast, the live EPS-producing bacterium P. parvulus 2.6 antagonized Enterobacteriaceae without disturbing the homeostasis of the cecal microbiota.

Amyloglucosidase (AMG) was produced by Aspergillus niger in solid-state fermentation (SSF), submerged fermentation (SmF) and an aqueous, two-phase system of polyethyleneglycol (PEG) and salt. In SSF, a fed-batch mode of operation gave a yield of 64 U/ml compared with 44 U/ml in batch mode. Similar trends were observed for SmF, where fed-batch cultivation gave a yield of 102 U/ml compared with 66 U/ml in batch. Shorter cultivation times (66 h) were required for SmF than for SSF (96 h). In the aqueous, two-phase cultivation, the productivity and yield of AMG were both twice those in the control fermentation.

The thermophilic hydrogen-oxidizing eubacterium, Hydrogenobacter thermophilus was grown with a semi-continuous gas feed in a 150 l fermentor, on different ratios of H 2 , O 2 (air) and CO 2 and on media of variable mineral salts composition. The best gas ratio during the exponential phase was approximately 0.5 :1.0 :0.03 atm of H 2 :air :CO 2 . Cell densities were increased by increasing the concentration of ammonium and phosphate in the medium. The maximum cell density obtained under the most favourable conditions was 2.62 g wet wt l -1 , corresponding to an absorption of 1.6 at 600 nm. The maximum specific growth rate (μ) was 0.44 h -1 .

Thiobacillus ferrooxidans andAcidianus brierleyi were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. First order reactions were assumed in the kinetic analysis performed. For oxidation of pure pyrite the rate constant was higher forA. brierleyi than forT. ferrooxidans. For sulfur removal from coal the values of the rate constants were comparable for the two microorganisms.

A high level expression system for production of an immunoglobulin-binding protein, in Escherichia coli was studied. The protein, called protein L I–IV , consists of four immunoglobulin-binding domains of the native protein L. A simple fed-batch cultivation strategy was used to investigate the influence of different induction times on cell growth, viability, acetic acid formation and product formation. Induction allowing product formation for several hours, i.e. , in this case in early exponential phase, was most favorable in terms of product yields. The highest specific yield obtained was 150 mg protein per gram cell dry weight (dw), corresponding to 360 mg per liter broth. The leakage of product into the media was less than 5%. Induction in early exponential phase lead to the highest amount of acetic acid, 1.47 g/g dw. Viability decreased significantly after induction.

Intake of dietary fibres may reduce the prevalence of physiological risk factors of the metabolic syndrome, such as high plasma lipid levels and low-grade inflammatory state. Dietary fibres are usually of plant origin however microbial exopolysaccharides (EPSs) have analogue structures that could potentially exert similar physiological effects. Pediococcus parvulus 2.6 (Pd 2.6) excretes a ropy EPS and has previously shown probiotic potential. The aim of this work was to evaluate physiological effects of Pd 2.6 and its EPS in vivo. The live Pd 2.6 (both the ropy and non-ropy isogenic variant) and its purified EPS were fed to hypercholesterolemic LDL-receptor deficient mice for 6 weeks to investigate their effects on cholesterol levels and the inflammatory tone of the animals. Both variants of Pd 2.6 survived passage through the mouse gut fulfilling an important criterion of probiotics. The ability to produce EPS was conferring an advantage to survival (faecal recovery of 3.7 (1.9-8.7) vs. 0.21 (0.14-0.34) *108 CFU, P

Several different microorganisms have been suggested for coal desulfurization. In the present investigation, the thermophilic archaea Acidianus brierleyi (DSM 1651), Sulfolobus acidocaldarius (DSM 639) and Sulfolobus solfataricus (DSM 1616) were compared with the mesophilic bacterium Thiobacillus ferrooxidans (DSM 583) concerning their capability of removing sulfur from coal. The desulfurization rate as well as the amount of sulfur removed by the microorganisms was studied. Two of the investigated microorganisms, Thiobacillus ferrooxidans and Acidianus brierleyi, were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. A kinetic analysis was performed assuming first order reactions. The rate constant for oxidation of pure pyrite by A. brierleyi was observed to be higher than for T. ferrooxidans, The values of the rate constants for sulfur removal from coal were comparable for the two microorganisms, but were higher than for oxidation of pure pyrite. (Less)

Process integration involving fermentation with cell recycling, affinity adsorption to remove the product, and on-line process monitoring of the product were investigated. α-Amylase was produced in continuous and batch fermentations of Bacillus amyloliquefaciens with complete cell recycling, which was achieved using a membrane unit coupled to the fermentor. An affinity column of crosslinked starch was employed in the recycling loop to adsorb the α-amylase before the cell-free broth was recycled to the fermentor. The concentration of α-amylase in the cell-free broth prior to and after the affinity separation step was continuously registered using a flow-injection, immunochemical monitoring system. The different unit operations were integrated pair-wise, and eventually as a complete system. The complete feasibility of monitoring a macromolecular product on-line during a cultivation/adsorption process was also demonstrated.

The extracellular β-xylosidase (EC 3.2.1.37) excreted by the thermophilic eubacteriumRhodothermus marinus when grown on xylan has been investigated. The enzyme has been partially purified by ultrafiltration and gel filtration, and some of its characteristics are presented.Rhodothermus marinus grew on xylan with μmax= 0.4 h− and the α-xylosidase activity was 50 nkat/mL after 24 h in a batch fermentation. The α-xylosidase activity had a half-life of more than 1 h at 90°C and of 14 h at 85 °C. At 80°C, 80% of the initial activity remained after 24 h. The initial activity increased with increasing temperature, showing maximal activity at 90°C. The β-xylosidase had a pH-optimum of 6 and was stable in the range between pH 5 and 9. At pH 10 and 11, 82 and 66%, respectively, of the initial activity remained after 24 h when incubated at 65°C. The molecular weight was estimated to be 169,000 dalton by gelfiltration.

The use of gas chromatography-mass spectrometry for early detection of bacterial contaminations in cultures of baker's yeast, Penicillium chrysogenum, and an animal cell line was evaluated; muramic acid and characteristic cellular fatty acids were used as analytes. By analyzing branched-chain and cyclopropane-substituted fatty acids as methyl esters, Staphylococcus epidermidis, Bacillus subtilis, Lactobacillus reuteri, Enterobacter cloacae, and Pseudomonas fluorescens were detected in a 500-fold excess (w/w) of baker's yeast; the amounts injected corresponded to 300 ng (dry mass) of the bacteria. Contamination with Bacillus was detected in cultures of Penicillium chrysogenum and animal cells by analyzing muramic acid, both as its alditol acetate derivative, using electron impact ionization, and its trifluoroacetyl methyl glycoside derivative, using negative ion-chemical ionization. The trifluoroacetylated derivative was detected in injected amounts corresponding to 1 x 10(3) bacterial cells in the contaminated animal cell line, whereas amounts corresponding to 1 x 10(5) bacteria were required for detection of the alditol acetate derivative; the amounts in the original samples were 5 x 10(5) and 5 x 10(6), respectively. However, the alditol acetate method exhibited lower chemical interferences than the trifluoroacetyl methyl glycoside procedure. The results show the potential of using gas chromatographic-mass spectrometric analysis of cellular constituents for the detection of bacterial contaminations in eucaryotic cultures as an alternative to conventional microbiological methods.