Simple processes for optimized growth and harvest of Ettlia sp. by pH control using CO2 and light irradiation.

Microalgae cultures show wide range of pH depending on the availability of light and CO2 for their strain specific photosynthesis. Thus, the modulation of light irradiation and CO2 supply can be applied for the pH control of microalgae cultures. The optimal pH of Ettlia sp. YC001, for phototrophic growth and auto-flocculation was investigated by controlling light irradiation and 10% CO2 supply. Ettlia sp. YC001 showed the highest biomass productivity, 96.7 mg L(-1) d(-1) , at pH 8.5. The flocculating activity of Ettlia sp. YC001 showed a sigmoid pattern with pH increase and was above 70% at pH 10.5. Based on these differentiated optimal pH regimes for the growth and flocculation, an integrated process consisting of cultivation and settling vessels was proposed. The integrated process demonstrated that high flocculation activity of Ettlia sp. YC001 could be achieved in the settling vessel with various hydraulic retention times by only irradiation of light to maintain high pH while maintaining the optimal growth in cultivation vessel with the light irradiation and CO2 supply at pH 8.5. Thus, the proposed strategy for pH control would provide a simple, cost-effective, and flexible design and operation for microalgae cultivation-harvest systems.

Caulobacter profunda sp. nov., isolated from deep freshwater sediment.

The Gram-stain-negative, aerobic, non-spore-forming, motile, with a single polar flagellum, or non-motile (stalked) and rod-shaped bacteria, DS48-5-2(T) and DS48-6-3, were isolated from a sediment sample collected from a depth of 48 m taken from Daechung Reservoir, Republic of Korea. Comparative 16S rRNA gene sequence studies showed that the two isolates had clear affiliation with Alphaproteobacteria and the closest relatedness to Caulobacter mirabilis FWC 38(T), Caulobacter fusiformis ATCC 15257(T) and Caulobacter daechungensis H-E3-2(T) showing 98.5%, 97.3% and 97.3% 16S rRNA gene sequence similarity, respectively, and 96.1-96.7% similarity to all other species of the genus Caulobacter. The two isolates shared 100 % 16S rRNA gene sequence similarity. The predominant ubiquinone was Q-10. The major fatty acids were summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c), C16:0, C18:0ω7c 11-methyl and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c). The G+C contents of the genomic DNA of strains DS48-5-2(T) and DS48-6-3 were 66.7 mol% and 66.2 mol%, respectively. DNA-DNA hybridization values of strains DS48-5-2(T) and DS48-6-3 with C. mirabilis FWC 38(T), C. fusiformis ATCC 15257(T) and C. daechungensis H-E3-2(T) were 19.3 %-24.4 %. Thus, based on the evidence from polyphasic studies, it is proposed that strains DS48-5-2(T) and DS48-6-3 are representatives of a novel species in the genus Caulobacter, for which the name Caulobacter profunda sp. nov. is proposed. The type strain is DS48-5-2(T) ( = KCTC 32480(T) = JCM 19440(T)).

Ferruginibacter profundus sp. nov., a novel member of the family Chitinophagaceae, isolated from freshwater sediment of a reservoir.

A Gram-negative, aerobic, non-motile, and rod-shaped bacterium, designated strain DS48-5-3(T), was isolated from a 48 m sediment sample taken from Daechung Reservoir, Republic of Korea. Comparative 16S rRNA gene sequence studies showed a clear affiliation of this strain to the Bacteroidetes, notably most closely related to Ferruginibacter alkalilentus HU1-GD23(T), Ferruginibacter lapsinanis HU1-HG42(T) and Ferruginibacter yonginensis HME8442(T), showing 16S rRNA gene sequence similarities to the type strains of these species of 95.2-96.4 % similarity. The predominant ubiquinone was identified as MK-7. The major fatty acids were identified as iso-C15:0, iso-C17:0 3-OH, and iso-C15:1 G. The G+C content of the genomic DNA of strain DS48-5-3(T) was determined to be 37.2 %. On the basis of polyphasic evidence, it is proposed that strain DS48-5-3(T) should belong to a novel species, for which the name Ferruginibacter profundus sp. nov. (type strain DS48-5-3(T) = KCTC 32478(T) = JCM 19431(T)), is proposed.

Influence of plasmid pO157 on Escherichia coli O157:H7 Sakai biofilm formation.

The role of plasmid pO157 in biofilm formation was investigated using wild-type and pO157-cured Escherichia coli O157:H7 Sakai. Compared to the wild type, the biofilm formed by the pO157-cured mutant produced fewer extracellular carbohydrates, had lower viscosity, and did not give rise to colony morphology variants that hyperadhered to solid surfaces.

Evolution of diversity in spatially structured Escherichia coli populations.

The stochastic Ricker population model was used to investigate the generation and maintenance of genetic diversity in a bacterial population grown in a spatially structured environment. In particular, we showed that Escherichia coli undergoes dramatic genetic diversification when grown as a biofilm. Using a novel biofilm entrapment method, we retrieved 64 clones from each of six different depths of a mature biofilm, and after subculturing for approximately 30 generations, we measured their growth kinetics in three different media. We fit a stochastic Ricker population growth model to the recorded growth curves. The growth kinetics of clonal lineages descendant from cells sampled at different biofilm depths varied as a function of both the depth in the biofilm and the growth medium used. We concluded that differences in the growth dynamics of clones were heritable and arose during adaptive evolution under local conditions in a spatially heterogeneous environment. We postulate that under nutrient-limited conditions, selective sweeps would be protracted and would be insufficient to purge less-fit variants, a phenomenon that would allow the coexistence of genetically distinct clones. These findings contribute to the current understanding of biofilm ecology and complement current hypotheses for the maintenance and generation of microbial diversity in spatially structured environments.

Light intensity as major factor to maximize biomass and lipid productivity of Ettlia sp. in CO2-controlled photoautotrophic chemostat.

The optimal culture conditions are critical factors for high microalgal biomass and lipid productivity. To optimize the photoautotrophic culture conditions, combination of the pH (regulated by CO2 supply), dilution rate, and light intensity was systematically investigated for Ettlia sp. YC001 cultivation in a chemostat during 143days. The biomass productivity increased with the increase in dilution rate and light intensity, but decreased with increasing pH. The average lipid content was 19.8% and statistically non-variable among the tested conditions. The highest biomass and lipid productivities were 1.48gL-1d-1 and 291.4mgL-1d-1 with a pH of 6.5, dilution rate of 0.78d-1, and light intensity of 1500µmolphotonsm-2s-1. With a sufficient supply of CO2 and nutrients, the light intensity was the main determinant of the photosynthetic rate. Therefore, the surface-to-volume ratio of a photobioreactor should enable efficient light distribution to enhance microalgal growth.

Evaluation of various techniques for microalgal biomass quantification.

Biomass concentration is one of the most important parameters in the biotechnology processes. Its measurement relies on the physical, chemical or biological properties of the cells. Several techniques were applied in this work to measure the cell concentration of four microalgae: Botryococcussp., Botryococcusbraunii, Chlorella vulgaris, and Ettlia sp. The experiments were performed using samples taken from a chemostat for each strain to provide microalgal cell suspensions in a stable physiological state and concentration. The dry cell weight (DCW) was used as the reference method for the evaluation of other methods. The two commercial sensors used to determine optical density and dielectric permittivity showed a broad effective measurement range up to more than 20gl(-1). A Red-Green-Blue model analysis of microalgal digital images in combination with Fourier equation significantly extended the measurements range up to 6gl(-1). Cell count using a flow cytometer showed a broad range of linearity to DCW in washed samples, but other counting methods using hemocytometer and microscopic automated count were limited. Finally, the oxygen production rate, representing the photosynthetic activity, showed a linear regression with DCW at cell concentrations lower than 1gl(-1).

Aeration effects on metabolic events during sporulation of Bacillus thuringiensis.

The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO2 production rates during sporulation, although a maximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.

Effective screening of Scenedesmus sp. from environmental microalgae communities using optimal sonication conditions predicted by statistical parameters of fluorescence-activated cell sorting.

The effects of the sonication parameters, including the power and time, were investigated for the effective isolation of Scenedesmus sp. from environmental microalgae communities when using fluorescence-activated cell sorting (FACS). The selectivity, defined as the percentage of Scenedesmus sp. successfully isolated and grown in microplates, appeared as peaks in contour plots spanned by the sonication power and time. For fast screening of the optimal sonication conditions, correlations between the selectivity and the statistical parameters from the FACS analysis were investigated. A graphical comparison analysis of the contour plots showed a pattern similarity of over 82% between the coefficients of variation for the side scatter (SSC-CV) and the selectivity. This predictability of the optimal sonication conditions enabled a Scenedesmus sp. selectivity of ca. 2 times using only one-third of the sonication condition sets arbitrarily chosen around the peaks of the SSC-CV, thereby saving resources and time for subsequent processes.

Simple, rapid and cost-effective method for high quality nucleic acids extraction from different strains of Botryococcus braunii.

This study deals with an effective nucleic acids extraction method from various strains of Botryococcus braunii which possesses an extensive extracellular matrix. A method combining freeze/thaw and bead-beating with heterogeneous diameter of silica/zirconia beads was optimized to isolate DNA and RNA from microalgae, especially from B. braunii. Eukaryotic Microalgal Nucleic Acids Extraction (EMNE) method developed in this study showed at least 300 times higher DNA yield in all strains of B. braunii with high integrity and 50 times reduced working volume compared to commercially available DNA extraction kits. High quality RNA was also extracted using this method and more than two times the yield compared to existing methods. Real-time experiments confirmed the quality and quantity of the input DNA and RNA extracted using EMNE method. The method was also applied to other eukaryotic microalgae, such as diatoms, Chlamydomonas sp., Chlorella sp., and Scenedesmus sp. resulting in higher efficiencies. Cost-effectiveness analysis of DNA extraction by various methods revealed that EMNE method was superior to commercial kits and other reported methods by >15%. This method would immensely contribute to area of microalgal genomics.

Harvest of Scenedesmus sp. with bioflocculant and reuse of culture medium for subsequent high-density cultures.

The optimal flocculating conditions for harvesting high-density cultures of Scenedesmus sp. were investigated using inorganic coagulants and the bioflocculant produced by Paenibacillus polymyxa AM49. The flocculated medium as nutrients for subsequent algal cultivation was also tested. Consecutive treatment with 8.5 mM CaCl(2) and 0.2 mM FeCl(3) as coagulants and 1% bioflocculant from the culture broth of P. polymyxa AM49 showed the highest flocculating activity of up to 95% for high density algal cultures. The medium flocculated with the coagulants and bioflocculant showed less than 8% decrease in the growth yield in the subsequent algal cultivation. Furthermore, a 20% or 50% fresh BG11 medium supplement allowed the flocculated medium to maintain a high growth yield in subsequent algal cultivation. These results suggest that the flocculation method presented here is efficient and bio-friendly, and allows the reuse of the flocculated medium, thereby contributing to the economic cultivation and harvest of microalgae.

System for determining the relative fitness of multiple bacterial populations without using selective markers.

A device for simultaneously measuring the relative fitness of multiple bacterial populations was developed and evaluated. The new device eliminates the need to construct strains with selectively neutral markers so that strains can be readily distinguished, and it provides a means to perform multispecies competition experiments.

Enhancement of sulfate reduction activity using granular sludge in anaerobic treatment of acid mine drainage.

Three laboratory-scale, upflow anaerobic reactors were operated for about 250 d to determine the effect of activated granular sludge with high density of sulfate reducing bacteria in the treatment of artificial acid mine drainage. Sulfate reducing bacteria in the granular sludge taken from the upflow anaerobic sludge blanket reactor were 1-2 x 10(6) c.f.u. g(-1), which is at least 10 times higher than that of organic substrates such as cow manure and oak compost. The reactors with granular sludge effectively removed over 99% of heavy metals, such as Fe, Al, Cu, and Cd during the experiment. This result suggests a feasibility of the application of granular sludge as a source of sulfate reducing bacteria for the treatment of acid mine drainage.