Optimisation of cultivation parameters in photobioreactors for microalgae cultivation using the A-stat technique

Light availability inside the reactor is often the bottleneck in microalgal cultivation and for this reason much attention is being given to light limited growth kinetics of microalgae, aiming at the increase of productivity in photobioreactors. Steady-state culture characteristics are commonly used for productivity optimisation and for cell physiology studies in continuous cultures, and are normally achieved using chemostat cultivations. In the present study, we investigated the applicability of a new and dynamic cultivation method called acceleration-stat (A-stat) to microalgae cultivations where light is the limiting substrate. In the A-stat, the dilution rate is increased at a constant rate. This acceleration rate should be a compromise between a short cultivation time, in order to make it a fast process, and the metabolic adaptation rate of the microorganism to changes in the environment. Simulations of the A-stat were done with different acceleration rates to have an indication of the best rate to use. An A-stat was performed in a pilot plant bubble column (65 l) with Dunaliella tertiolecta as a model organism, and results showed that a pseudo steady state was maintained throughout the experiment. From this work, it was concluded that the A-stat can be used as a fast and accurate tool to determine kinetic parameters and to optimise any specific type of photobioreactor.     

Isolation and characterization of acid- and Al-tolerant microorganisms

Acid- and aluminum (Al)-tolerant microorganisms were isolated from tea fields, from which six strains were selected and identified as Cryptococcus humicola, Rhodotorula glutinis, Aspergillus flavus Link, Penicillium sp., Penicillium janthinellum Biourge and Trichoderma asperellum. They were tolerant to Al up to 100-200 mM and could grow at low pH, 2.5-2.2. In a glucose medium (pH 3.5) the pH of the spent medium decreased to below 3.0. The toxic inorganic monomeric Al in the spent medium decreased with three strains (A. flavus F-6b, Penicillium sp. F-8b and P. janthinellum F-13), but the total Al remained constant for all strains. In a soil extract medium (pH 3.5), the pH of the spent medium of all strains increased to around 6.0-7. 2 and total Al in the spent medium was removed by precipitation due to pH increase. Thus, different tolerance mechanisms were suggested in glucose and soil extract media.     

Selection and characterization of active psychrotrophic microbial oil-degrading microorganisms

The ability of 96 microbial strains degrading oil and 32 strains degrading polycyclic aromatic hydrocarbons (PAHs) to consume diesel fuel and oil at 4–6 and 24°C and at elevated NaCl concentrations was studied. The temperature range, salt tolerance, ability to produce biosurfactants, range of substrates, and antibiotic resistance were determined. The eleven most active oil-degrading and PAH-degrading strains were genotyped by a polymerase chain reaction with BoxA1R primers and a restriction analysis of ribosomal DNA amplicons. For six strains, the degree of oil degradation at 4–6°C was higher than at 24°C. For the most active strains, the degree of oil degradation in liquid mineral medium ranged from 15 to 26% at 24°C and from 28 to 47% at 4–6°C. An association of six of the strains degraded the oil by 46% at 24°C.     

Spectroscopic characterization of microorganisms by Fourier transform infrared microspectroscopy

Spectroscopic fingerprints of bacteria were investigated by Fourier transform infrared (FTIR) microspectroscopy for the elucidation of chemical composition and structural information during growth. Good differentiation of six microorganisms was achieved down to the strain level. The inherent compositional and structural differences of cell envelopes and cytoplasm were investigated and utilized to obtain more detailed analysis of the spectroscopic features. Bands or regions of key functional groups were also identified in the original spectra. Microspectroscopic monitoring of bacterial growth demonstrated that FTIR spectroscopy cannot only provide molecular fingerprints of the cell envelope, but also compositional and metabolic information of the cytoplasm under different physiological conditions. This approach could be an effective alternative to traditional nutritional and biochemical methods to monitor and assess the effects of inhibitors and other environmental factors on microbial cell growth.     

The isolation and characterization of simultaneous saccharification and fermentation microorganisms for Laminaria japonica utilization

Brown seaweed contains various carbohydrates, such as alginate, laminaran, and mannitol, therefore ethanol fermentation was attempted with Nuruk and a mixed culture that included Laminaria japonica. Nuruk is used to make Korean traditional alcohol. In the research, four microorganisms that produced ethanol and had the ability to achieve alginate degradation were obtained on the L. japonica medium. Nuruk 4 was found to produce a better result than the other tested microorganisms, and the optimal substrate for ethanol production was found to be mannitol (2.59 g/L at 96 h). Nuruk 4 was more than three times better compared with Candida tropicalis in regards to ethanol production. When alginate lyase activity occurred, it appeared as a clear zone around Nuruk 3. The maximal ethanol production yield conditions were comprised of Nuruk 3 and 4 on the anaerobic culture. In this case, 2.0 g/L of ethanol were efficiently produced under the same conditions.     

Methods for the differentiation of microorganisms

Advances in analytical and diagnostic assays based on novel nucleic acid analyses techniques have revolutionized the application of molecular differentiation of microorganisms. Phenotypic typing schemes are now broadly supplemented by new genotyping methods which allow a more refined and detailed differentiation of closely related microorganisms, bacterial strains, isolates and pathogens on the DNA level. Bio-, sero- and phagetyping, antibiotic susceptibility tests, immunoblotting as well as multilocus enzyme- or polyacrylamide gel electrophoresis are now supported by the analysis of plasmid or chromosomal DNA restriction profiles, ribotyping, pulsed-field gel electrophoresis and polymerase- or ligase-chain reaction-based methods or direct sequencing technique to differentiate microorganisms. Some of these molecular techniques are also used in the field of virology to analyse and differentiate closely related sub- or genotypes. Few examples for the analysis and investigation of these usually small genomes will also be given.     

Ecology and characterization of polyhydroxyalkanoate-producing microorganisms on and in plants

Polyhydroxyalkanoates are energy reserve polymers produced by bacteria to survive periods of starvation in natural habitats. Little is known about the ecology of polyhydroxyalkanoate-producing bacteria. To analyse the occurrence of this specific group on/in seven different plant species, a combined strategy containing culture-dependent and -independent methods was applied. Using microbial fingerprint techniques (single-strand conformation polymorphism analysis with specific primers for phaC gene encoding the key enzyme of the polyhydroxyalkanoate synthesis), a high number of bands were especially found for the rhizosphere. Furthermore, cluster analysis revealed plant species-specific communities. Isolation of bacteria, recognition of brightly refractile cytoplasmatic inclusions, lipophilic stainings and a PCR strategy targeted on the phaC gene were used as a culture-dependent strategy for the detection of polyhydroxyalkanoate-producing bacteria. Results again represent a high degree of plant specificity: the rhizosphere of sugar beet contained the highest number of positive strains. This was confirmed by quantitative PCR: the relative copy number of phaC was statistically and significantly enhanced in all rhizospheres in comparison with bulk soil. New polyhydroxyalkanoate-producing bacterial species were detected: for example, Burkholderia terricola, Lysobacter gummosus, Pseudomonas extremaustralis, Pseudomonas brassicacearum and Pseudomonas orientalis . Our results confirm the hypothesis that the rhizosphere is an interesting hidden reservoir for polyhydroxyalkanoate producers.     

Modification and characterization of cellulosic cotton fibers for efficient immobilization of urease

Cotton fibers were first grafted by polyacrylonitril in the presence of KMnO(4) and oxalic acid as a combined redox initiator. Moreover, modification of the grafted cotton fibers was done by changing the nitrile group (-CN) into hydrazidine group through the reaction with hydrazine hydrate, then the fibers were activated by glutaraldehyde to introduce free aldehyde groups which were able to react with amino groups of urease to form Schiff's base, and result in cotton fibers immobilized urease. The efficiency of the immobilization was evaluated by examining the relative enzymatic activity of enzyme before and after the immobilization of urease. The results showed that the optimum temperature of immobilized urease was 35°C, which was higher than that of the free enzyme (30°C), and the immobilized urease exhibited a higher relative activity than that of free urease over 35°C. The optimal pH for immobilized urease was 6.5, which was lower than that of the free urease (pH 7.0), and the immobilization resulted in stabilization of enzyme over a wider pH range. The kinetic constant value (K(m)) of immobilized urease was higher than that of the free urease. However, the thermal and operational stabilities of immobilized urease have been improved greatly.     

Use of gas chromatography-ion trap tandem mass spectrometry for the detection and characterization of microorganisms in complex samples

Gas chromatography-mass spectrometry (GC-MS) can be applied to detect and characterize microorganisms in clinical and environmental samples, and microbial contaminants in biotechnological production cultures. With this approach, unique microbial monomeric compounds, known as chemical markers, are used as analytes. In the present article, two GC-MS-based techniques, viz. GC-ion trap tandem MS (GC-MS-MS) and conventional quadrupole GC-MS used in the selected ion monitoring mode, were compared regarding their ability to detect 3-hydroxy fatty acids, muramic acid, and ergosterol (markers for endotoxin, peptidoglycan, and fungal biomass, respectively) in complex matrices. When using GC-MS-MS, daughter ion spectra were obtained for all markers present in amounts close to the detection limit of the GC-MS. Ion-trap GC-MS-MS shows great promise as a chemical marker analysis technique for application in clinical diagnosis, occupational and public health care, and biotechnology.     

Isolation and characterization of novel atrazine-degrading microorganisms from an agricultural soil

Six previously undescribed microorganisms capable of atrazine degradation were isolated from an agricultural soil that received repeated exposures of the commonly used herbicides atrazine and acetochlor. These isolates are all Gram-positive and group with microorganisms in the genera Nocardioides and Arthrobacter, both of which contain previously described atrazine degraders. All six isolates were capable of utilizing atrazine as a sole nitrogen source when provided with glucose as a separate carbon source. Under the culture conditions used, none of the isolates could utilize atrazine as the sole carbon and nitrogen source. We used several polymerase-chain-reaction-based assays to screen for the presence of a number of atrazine-degrading genes and verified their identity through sequencing. All six isolates contain trzN and atzC, two well-characterized genes involved in the conversion of atrazine to cyanuric acid. An additional atrazine-degrading gene, atzB, was detected in one of the isolates as well, yet none appeared to contain atzA, a commonly encountered gene in atrazine impacted soils and atrazine-degrading isolates. Interestingly, the deoxyribonucleic acid sequences of trzN and atzC were all identical, implying that their presence may be the result of horizontal gene transfer among these isolates.     

Isolation and characterization of selenite- and selenate-tolerant microorganisms from selenium-contaminated sites

Eight bacterial strains designated ARI 1-8 were isolated from soil and water samples collected from selenium-contaminated sites in India using the enrichment culture technique. They exhibited very high MIC values ranging from 300 to 750 mM for different forms of selenium (selenite and selenate). On the basis of various biochemical tests, fatty acid methyl ester profile and 16S rRNA gene sequencing, these isolates were identified belonging to the classes β-Proteobacteria and Bacilli. These microorganisms could be further used for bioremediation of contaminated sites.     

Isolation and characterization of aromatics-degrading microorganisms from the gut of the lower termite Coptotermes formosanus

We isolated aromatics-degrading bacteria from the gut of a lower termite, Coptotermes formosanus, using a mineral salt medium containing various aromatic compounds as the sole carbon source. Two species, Burkholderia sp. strain VE22 and Citrobacter sp. strain VA53, were isolated by aerobic enrichment culture with veratraldehyde and vanillin, respectively. Strain VA53 could also grow and metabolize vanillin anaerobically.     

DNA probes for the identification of microorganisms

Summary The detection and identification of microorganisms is being carried out increasingly using DNA. Each organism has a unique DNA sequence which can be used to distinguish closely related organisms. Using PCR amplification and sequencing of ribosomal RNA genes we have developed DNA probes for a number of pathogenic bacteria and fungi. The development of DNA assays based on PCR has resulted in new questions which must be addressed including process carry-over contamination and inhibition of the PCR amplification reaction once the problems associated with the implementation of DNA assays are ironed out.     

提高微生物可培养性的方法和措施

目前自然界中只有极少部分微生物能够得到培养,严重阻碍了对微生物生命活动规律的研究和微生物资源的开发。改进传统培养方法,采用新型培养技术,提高微生物可培养性,大量培养自然界中存在的微生物,从而更全面、准确地了解微生物细胞的生命规律、获悉微生物群落中各种微生物之间的动态相互作用和相互协调的规律,对环境微生物工艺进行准确地设计、精细地调控和高效地利用。简要介绍了微生物不可培养的原因,系统总结了有关提高微生物可培养性方法的最新研究进展,提出研究中存在的问题,并阐述了模拟自然环境条件、强调微生物相互关系是提高微生物可培养性的关键。     

Effect of microorganisms on the phytotoxicity of herbicides. VI. Modification of venzar activity by bacterial cultures

Interactions between two bacterial strains and venzar were compared. It was found that the mechanism of interactions is various and causes the modification of herbicide phytotoxicity. Metabolites of Bacillus subtilis 72 interfered with herbicide by affecting physiological processes in plant tissues and enhancing its inhibitory influence. Arthrobacter sp. 18 strain decreased the phytoinhibitory effect of herbicide due to conjugation with the carrier from venzar.     

Screening and characterization of microorganisms with glutaryl-7 ADCA acylase activity

A screening of microorganisms producing glutaryl-7 ADCA acylase, an enzyme able to hydrolyse glutaric acid selectively from glutaryl-3-deacetoxy-7-aminocephalosporanic acid (glutaryl-7 ADCA), has been carried out in soil samples. Five microorganisms expressing acylase activity were isolated and classified as Bacillus cereus, Achromobacter xylosooxidans, Bacillus sp., Pseudomonas sp. and Pseudomonas paucimobilis. The screening was carried out by preparing enrichment cultures containing glutaryl-7-ADCA or cephalosporin C as the selective carbon source. Four model compounds (adipoyl-, glutamyl- and glutaryl-p-nitroanilide and glutarylcoumarin), mimicking the glutaryl-7 ADCA -lactam moiety, were synthesized as substrates suitable for the rapid screening of the microorganisms (2500) isolated from the enrichment cultures. A total of 300 strains were active on the model substrates and only 5 displayed acylase activity on glutaryl-7 ADCA. The fermentation parameters, such as pH and inducer concentration, for the optimal acylase expression and acylase specificity towards the model substrates were different for each strain.