Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

渤海沉积物产脂肪酶细菌的筛选及其多样性分析

【目的】从渤海沉积物中分离筛选产脂肪酶细菌,分析其物种多样性,增加人们对渤海生态系统中产脂肪酶菌多样性的认识,获取高效产脂肪酶菌株,为海洋产脂肪酶微生物的挖掘提供菌群资源。【方法】分别将8个渤海沉积物样品梯度稀释涂布至吐温-80筛选平板和三丁酸甘油酯筛选平板,选择性分离产脂肪酶细菌;分析基于16SrRNA基因序列的系统发育关系,揭示这些细菌的分类地位和遗传多样性;利用对硝基苯酚法测定胞外脂肪酶活性,筛选出高效产脂肪酶菌株。【结果】从8个渤海沉积物样品中分离获得51株产脂肪酶细菌,这些菌株隶属于Bacteroidetes、Proteobacteria和Firmicutes三个门的8个属,其中Pseudoalteromonas(35.2%)、Marinobacter(23.5%)和Sulfitobacter(17.6%)是优势菌群;脂肪酶酶活性实验表明所有测定菌株都能够分泌脂肪酶,菌株70623分泌的脂肪酶酶活最高,为42.4 U/m L。【结论】渤海沉积物中可培养产脂肪酶细菌类群较为丰富,Pseudoalteromonas、Marinobacter和Sulfitobacter菌株是优势菌群,测定菌株所产胞外脂肪酶能力不同,获得了一株高效产脂肪酶菌株Marinobacter sp.70623。     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Presence of galactose in precultures induces lacS and leads to short lag phase in lactose-grown Lactococcus lactis cultures

Lactose conversion by lactic acid bacteria is of high industrial relevance and consistent starter culture quality is of outmost importance. We observed that Lactococcus lactis using the high-affinity lactose-phosphotransferase system excreted galactose towards the end of the lactose consumption phase. The excreted galactose was re-consumed after lactose depletion. The lacS gene, known to encode a lactose permease with affinity for galactose, a putative galactose–lactose antiporter, was upregulated under the conditions studied. When transferring cells from anaerobic to respiration-permissive conditions, lactose-assimilating strains exhibited a long and non-reproducible lag phase. Through systematic preculture experiments, the presence of galactose in the precultures was correlated to short and reproducible lag phases in respiration-permissive main cultivations. For starter culture production, the presence of galactose during propagation of dairy strains can provide a physiological marker for short culture lag phase in lactose-grown cultures.

     

Production of a novel glycerol-inducible lipase from thermophilic <Emphasis Type="Italic">Geobacillus stearothermophilus</Emphasis> strain-5

In a screening program for isolation of thermophilic lipase-producing bacteria, a number of thermophilic bacteria were isolated from desert soil from Baltim, Egypt. Among 55 isolates, a potent bacterial candidate (starin-5) was characterized and identified by biochemical and PCR techniques, based on 16S rRNA sequencing. Phylogenetic analysis revealed its closeness to geobacilli especially the thermophilic Geobacillus stearothermophilus with optimal growth and lipolytic enzyme activity at 60°C and pH 7.0. An inducible nature of lipolytic enzyme synthesis using glycerol and glucose was demonstrated. Approximately, 94–100% of the original activity was retained due to thermal stability of the crude enzyme after heat treatment for 15 min at 30–60°C. The enzyme retained 84.84% of its original activity during incubation at 70°C (pH 8.0) for 15 min. Lipase enzyme from G. stearothermophilus strain-5 was immobilized on various carriers and the most suitable carrier was chitin that showed 73.03% of activity yield.     

Effect of cultivation conditions on spore production from Bacillus amyloliquefaciens B128 and its antagonism to Botrytis elliptica

Aims:  To maximize spore production by Bacillus amyloliquefaciens B128, and its antagonism to the fungal pathogen Botrytis elliptica B061. Methods and Results:  In the 5-l stirred-tank bioreactor (STR), with the 0·5 vvm aeration rate, an agitation rate of 200 rev min⁻¹ significantly enhanced the spore yield compared to the same in 300 rev min⁻¹ cultivations. In a 20-l airlift bioreactor (ALR) the maximal spore production was further increased with a controlled aeration rate of 2·5 vvm operated in a 24-mesh net-draft tube mode, and no pH control cultivation. This spore yield in the 20-l ALR was five- and eightfold higher; in addition the cultivation period was 19 h shorter, compared to that obtained from shaker flask and in the 5-l STR cultivations respectively. Conclusions:  Although culture conditions are still to be optimized, by using an ALR with net-draft tube, a scaling up from shaker flasks and STR to ALR of spore production by the strain B128 is technically feasible. Significance and Impact of the Study:  The spore yields obtained using bioreactors were much higher than those previously reported. The freshly produced spore preparations from the B128 strain significantly antagonized the grey mould pathogen B. elliptica.     

Stepwise reduction of the culture redox potential allows the analysis of microaerobic metabolism and photosynthetic membrane synthesis in Rhodospirillum rubrum

Bacterial growth under oxygen‐limited (microaerobic) conditions is often accompanied by phenomena of great interest for fundamental research and industrial application. The microaerobic lifestyle of anoxygenic photosynthetic bacteria like Rhodospirillum rubrum harbors such a phenomenon, as it allows the formation of photosynthetic membranes and related interesting products without light. However, due to the technical difficulties in process control of microaerobic cultivations and the limited sensitivity of available oxygen sensors, the analysis of microaerobic growth and physiology is still underrepresented in current research. The main focus of the present study was to establish an experimental set‐up for the systematic study of physiological processes, associated with the growth of R. rubrum under microaerobic conditions in the dark. For this purpose, we introduce a robust and reliable microaerobic process control strategy, which applies the culture redox potential (CRP) for assessing different degrees of oxygen limitation in bioreactor cultivations. To describe the microaerobic growth behavior of R. rubrum cultures for each of these defined CRP reduction steps, basic growth parameters were experimentally determined. Flux variability analysis provided an insight into the metabolic activity of the TCA cycle and implied its connection to the respiratory capacity of the cells. In this context, our results suggest that microaerobic growth of R. rubrum can be described as an oxygen‐activated cooperative mechanism. The present study thus contributes to the investigation of metabolic and regulatory events responsible for the redox‐sensitive formation of photosynthetic membranes in facultative photosynthetic bacteria. Furthermore, the introduced microaerobic cultivation setup should be generally applicable for any microbial system of interest which can be cultivated in common stirred‐tank bioreactors. Biotechnol. Bioeng. 2013; 110: 573–585. © 2012 Wiley Periodicals, Inc.     

Production of polyhydroxyalkanoic acids by Ralstonia eutropha and Pseudomonas oleovorans from an oil remaining from biotechnological rhamnose production

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy decanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done. Received: 15 June 1999 / Received revision: 10 August 1999 / Accepted: 13 August 1999     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

Growth of Chlorella vulgaris and associated bacteria in photobioreactors

The aim of this study was to test three flat plate photobioreactor configurations for growth of Chlorella vulgaris under non‐axenic conditions and to characterize and quantify associated bacterial communities. The photobioreactor cultivations were conducted using tap water‐based media to introduce background bacterial population. Growth of algae was monitored over time with three independent methods. Additionally, the quantity and quality of eukaryotes and bacteria were analysed using culture‐independent molecular tools based on denaturing gradient gel electrophoresis (PCR‐DGGE) and quantitative polymerase chain reaction (QPCR). Static mixers used in the flat plate photobioreactors did not generally enhance the growth at the low light intensities used. The maximum biomass concentration and maximum specific growth rate were 1.0 g l^?1 and 2.0 day^?1 respectively. Bacterial growth as determined by QPCR was associated with the growth of C. vulgaris. Based on PCR‐DGGE, bacteria in the cultures mainly originated from the tap water. Bacterial community profiles were diverse but reproducible in all flat plate cultures. Most prominent bacteria in the C. vulgaris cultures belonged to the class Alphaproteobacteria and especially to the genus Sphingomonas. Analysis of the diversity of non‐photosynthetic microorganisms in algal mass cultures can provide useful information on the public health aspects and unravel community interactions.     

Production of lignin peroxidase and laccase by Phlebia radiata

Summary A cultivation method using carrierbound mycelium was developed for the production of lignin-modifying enzymes by Phlebia radiata. Laccase and lignin peroxidase were produced in batch and semi-continuous cultivations. Laccase activity was clearly enhanced by veratryl alcohol. The presence of both veratryl alcohol and Tween 80 was required for lignin peroxidase production in submerged cultivations. During the course of the semi-continuous cultivations production of lignin peroxidase activity increased fourfold compared with static cultivations.     

Experience in using an ethanol sensor to control molasses feed-rates in baker's yeast production

An ethanol sensor has been tested for feed-rate control of baker's yeast prouction. The yeast was grown on molasses in an 8 dm³ fed-batch reactor up to a cell concentration of 60–70 kg/m³. Studies were made on three levels: reliability of the sensor system, characterisation of the control problem, and evaluation of ethanol-controlled cultivations in terms of yield and production rate. Arguments are given for the conceptual advantages of ethanol control compared to other methods of substrate control. It is also shown that ethanol control allows for a simple regulator structure. In fact, a PID regulator, with constant parameters, was used around an exponential dosage scheme. Tuning of the regulator parameters was performed by using simulation on a simplified model of the process. Several cultivations have been carried out. Results from four comparable cultivations are given in detail, and the experience from many others is summarized.     

Semicontinuous system for the production of recombinant mCherry protein in Chlamydomonas reinhardtii

Biotechnology advances have allowed bacteria, yeasts, plants, mammalian and insect cells to function as heterologous protein expression systems. Recently, microalgae have gained attention as an innovative platform for recombinant protein production, due to low culture media cost, compared to traditional systems, as well as the fact that microalgae such as Chlamydomonas reinhardtii are considered safe (GRAS) by the Food and Drug Administration (FDA). Previous studies showed that recombinant protein production in traditional platforms by semicontinuous process increased biomass and bio product productivity, when compared to batch process. As there is a lack of studies on semicontinuous process for recombinant protein production in microalgae, the production of recombinant mCherry fluorescent protein was evaluated by semicontinuous cultivation of Chlamydomonas reinhardtii in bubble column photobioreactor. This semicontinuous cultivation process was evaluated in the following conditions: 20%, 40%, and 60% culture portion withdrawal. The highest culture withdrawal percentage (60%) provided the best results, as an up to 161% increase in mCherry productivity (454.5 RFU h⁻¹ – Relative Fluorescence Unit h⁻¹), in comparison to batch cultivation (174.0 RFU h⁻¹) of the same strain. All cultivations were carried out for 13 days, at pH 7, temperature 25°C and, by semicontinuous process, two culture withdrawals were taken during the cultivations. Throughout the production cycles, it was possible to obtain biomass concentration up to 1.36 g L⁻¹.     

Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase

High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available.     

Modeling of lutein production by heterotrophic <Emphasis Type="Italic">Chlorella</Emphasis> in batch and fed-batch cultures

Chlorella is a promising alternative resource of lutein (xanthophyll) production as it can be cultivated heterotrophically in fermentors. In this paper, a kinetic model for lutein production by heterotrophic Chlorella pyrenoidosa was developed based on batch cultivations in 250-ml flasks and a 19-l fermentor. The model was validated by experimental data from two fed-batch cultivations performed in the same fermentor. The dynamic behavior of lutein production by C. pyrenoidosa with various concentrations of glucose and nitrogen was analyzed based on the kinetic model. Model-based analyses suggested that glucose concentrations between 5 and 24 g/l and nitrogen concentrations between 0.7 and 12 g/l during the cultivation were favorable for lutein production by heterotrophic C. pyrenoidosa. It also showed that fed-batch cultivations are more suitable for efficient production of lutein than batch ones. The results obtained in this study may contribute to commercial lutein production by heterotrophic Chlorella.     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.     

<Emphasis Type="Italic">Dermacentor marginatus</Emphasis> and <Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks versus L929 and Vero cell lines in <Emphasis Type="Italic">Rickettsia slovaca</Emphasis> life cycle evaluated by quantitative real time PCR

Ticks transmit many different pathogens to animals, humans and their pets. Rickettsia slovaca, as a member of the spotted-fever-group rickettsiae is an agent of the human disease Tick-borne lymphadenopathy (TIBOLA), also called Dermacentor-borne necrosis erythema and lymphadenopathy (DEBONEL), which occurs from the Mediterranean to central Europe, transmitted by Dermacentor reticulatus and Dermacentor marginatus (Acari: Ixodidae). In this study, quantitative real time PCR was used to characterize the growth of R. slovaca, strain B in static (mammalian L929 and Vero cells without replacement of growth medium) and dynamic (D. marginatus and Ixodes ricinus ticks) cultivation systems. Curves of bacterial growth in static cultivations were modeled with exponential, stationary and death phases, whereas in dynamic systems the stationary phase was absent. The highest point of multiplication of R. slovaca was recorded on the 4th day post infection in both cell lines and the rickettsial DNA copy number in L929 and Vero cells at this point was 21 and 27 times greater than rickettsial DNA copy number of inoculum, respectively. In the dynamic system, the highest point of multiplication was on the 21th and 12th day after feeding of ticks and rickettsial DNA copy numbers were 7,482 and 865 times greater than the inoculum in D. marginatus and I. ricinus, respectively. Life cycle of R. slovaca in mammalian cell lines was shorter; supposedly, bacteria destroyed these cells and ticks, especially D. marginatus, were considered a more appropriate environment.     

Diversity,dynamics and reproduction in a community of small mammals in Upper Guinea,with emphasis on pygmy mice ecology

As part of a large survey on reservoirs of Lassa fever in Guinea, three villages were investigated in high endemic zone, close to Sierra Leone border. Biodiversity of the small mammal community is presented in this study through a standardized trapping in houses, cultivations and forest. Identification of the small mammals was based on morphology and by molecular technique for sibling species. Of the 1123 specimens collected in 2003–2005, we identified seventeen species (thirteen Muridae, four Soricidae), leading to high diversity (Shannon index = 1.6–1.8) and high equitability (evenness index = 0.7–0.8) in cultivations and forest. In houses conversely, the rodent community was dominated by Mastomys natalensis (95–98%), leading to low diversity and equitability. Dynamics and reproduction were investigated in two species of pygmy mice, Mus mattheyi and Mus minutoides, two species of Praomys, P. daltoni and P. rostratus, and in Mastomys erythroleucus. The pygmy mice were abundant in cultivations in early rainy season, and reproduced from rainy to dry season. Praomys daltoni was also found more abundant in cultivations and seemed to reproduce between rainy and dry season, whereas P. rostratus preferred forest and cultivations in late rainy season, and reproduced throughout the year. Finally, M. erythroleucus was more abundant in forest in dry season, and seemed to reproduce from late rainy to dry season. This species had a low occurrence (6.5%) in the Faranah’s zone, and probably lived at its southern limit in Guinea. The presence of other Murinae, such as M. natalensis, Praomys spp as possible competitors in the same habitats, is discussed. For the first time, this study relates population biology of pygmy mice with molecular identification.     

Effect of carbon dioxide on the rheological behavior of submerged cultures of Chlorella minutissima in stirred tank reactors

The objective of this study was to quantify the effect of algal biomass concentration on the rheology of the algal culture broth. Batch cultivations of Chlorella minutissima were carried out with air and carbon dioxide in a stirred tank bioreactor with a working volume of 1.8 L. The apparent viscosity of the culture broth was significantly affected by the cell mass concentrations in the bioreactor. Culture broth containing 50 g/L cell mass from air fed was found to exhibit an apparent viscosity of 1.52 mPa.s. The apparent viscosity of the carbon‐dioxide‐fed cultivations was found to increase by 20% at a shear rate of 100 s^?1. The flow behavior of the system was adequately described by the Herschel–Bulkley model with a small yield stress.     

Erhaltung und Wiederansiedlung des Kleinen Rohrkolbens (Typha minima) – Vegetationsaufnahmen, Monitoring und genetische Herkunftsanalysen

Galeuchet D. J. and Holderegger R. 2005. Conservation and re-introduction of Dwarf Bulrush (Typha minima) – vegetation surveys, monitoring and genetic analysis of origin. Bot. Helv. 115: 15–32.Typha minima was formerly widespread along fast flowing alpine rivers but is now red-listed as critically endangered. To assess its conservation perspectives, we surveyed the few remaining natural populations along the alpine part of the River Rhine from 1997 to 2002 and determined their genetic diversity using isozyme electrophoresis. Six of the populations became extinct or extremely small, probably due to shading by taller plants and trampling, while six other populations remained stable or increased, partly due to habitat restoration measures. The largest populations, with areas of more than 10000 m², are found in secondary habitats which are regularly disturbed due to water regime management. Of the 19 investigated isozyme loci, only six were polymorphic. Allelic diversity (1.4–1.8) was low in all populations, and the number of multilocus genotpyes (1–18) was low for 11 of 13 investigated populations. Genetic diversity was also studied for ex-situ cultivations of T. minima in Swiss botanical gardens and reintroduced stands.These artificial populations (each with 1–3 multilocus genotypes) were genetically similar to natural populations (average genetic distance 0.094). For two ex-situ cultivations with unknown origin, the likely origin could genetically be defined. Hybridisation between two ex-situ cultivations of different origin (i.e. a potential risk of genetic introgression) was detected in one botanical garden. It is concluded that the long-term conservation of T. minima requires both the restoration of regularly disturbed, sparsely vegetated river margins and the re-introduction of plants from ex-situ cultivations with appropriate origin.Manuskript angenommen am 2. Februar 2005