Summary• Air spaces in the leaf mesophyll generate deleterious optical effects that compromise confocal microscopy.• Leaves were mounted in the nontoxic, nonfluorescent perfluorocarbon, perfluorodecalin (PFD), and optical enhancement and physiological effect were assessed using confocal microscopy and chlorophyll fluorescence.• Mounting leaves of Arabidopsis thaliana in PFD significantly improved the optical qualities of the leaf, thereby enabling high-resolution laser scanning confocal imaging over twofold deeper into the mesophyll, compared with using water. Incubation in PFD had less physiological impact on the mounted specimen than water.• We conclude that the application of PFD as a mounting medium substantially increases confocal image resolution of living mesophyll and vascular bundle cells, with minimal physiological impact.
Botryococcus braunii can produce both long-chain hydrocarbons as well as carbohydrates in large quantities, and is therefore a promising industrial organism for the production of biopolymer building blocks. Many studies describe the use of different strains of Botryococcus braunii but differences in handling and cultivation conditions make the comparison between strains difficult. In this study, 16 B. braunii strains obtained from six culture collections were compared for their biomass productivity and hydrocarbon and carbohydrate content. Biomass productivity was highest for AC768 strain with 1.8gLday, while hydrocarbon production ranged from none to up to 42% per gram biomass dry weight, with Showa showing the highest hydrocarbon content followed by AC761. The total carbohydrate content varied from 20% to 76% per gram of the biomass dry weight, with CCALA777 as the highest producer. Glucose and galactose are the main monosaccharides in most strains and fucose content reached 463mgL in CCALA778.
Microalgae are widely viewed as a promising and sustainable source of renewable chemicals and biofuels. Botryococcus braunii synthesizes and secretes significant amounts of long‐chain (C30‐C40) hydrocarbons that can be subsequently converted into gasoline, diesel, and aviation fuel. B. braunii cultures are not axenic and the effects of co‐cultured microorganisms on B. braunii growth and hydrocarbon yield are important, but sometimes contradictory. To understand the composition of the B. braunii microbial consortium, we used high throughput Illumina sequencing of metagenomic DNA to profile the microbiota within a well established, stable B. braunii culture and characterized the demographic changes in the microcosm following modification to the culture conditions. DNA sequences attributed to B. braunii were present in equal quantities in all treatments, whereas sequences assigned to the associated microbial community were dramatically altered. Bacterial species least affected by treatments, and more robustly associated with the algal cells, included members of Rhizobiales, comprising Bradyrhizobium and Methylobacterium, and representatives of Dyadobacter, Achromobacter and Asticcacaulis. The presence of bacterial species identified by metagenomics was confirmed by additional 16S rDNA analysis of bacterial isolates. Our study demonstrates the advantages of high throughput sequencing and robust metagenomic analyses to define microcosms and further our understanding of microbial ecology.
Botryococcus braunii (Chlorophyta) is a green microalga known for producing hydrocarbons and exopolysaccharides (EPS). Improving the biomass productivity of B. braunii and hence, the productivity of the hydrocarbons and of the EPS, will make B. braunii more attractive for industries. Microalgae usually cohabit with bacteria which leads to the formation of species-specific communities with environmental and biological advantages. Bacteria have been found and identified with a few B. braunii strains, but little is known about the bacterial community across the different strains. A better knowledge of the bacterial community of B. braunii will help to optimize the biomass productivity, hydrocarbons, and EPS accumulation. To better understand the bacterial community diversity of B. braunii, we screened 12 strains from culture collections. Using 16S rRNA gene analysis by MiSeq we described the bacterial diversity across 12 B. braunii strains and identified possible shared communities. We found three bacterial families common to all strains: Rhizobiaceae, Bradyrhizobiaceae, and Comamonadaceae. Additionally, the results also suggest that each strain has its own specific bacteria that may be the result of long-term isolated culture.
Botryococcus braunii CCALA778 is a green microalga that can produce large amounts of extracellular carbohydrates and therefore is a potential host for industrial applications such as materials, food and pharmaceutical products. The downside of B. braunii is its slow growth and therefore, improvements on the biomass productivity or carbohydrate production will make this microalga more attractive for industrial exploitation. Microalgae grow naturally in the presence of bacteria and these can be beneficial or antagonistic. In outdoor cultivation systems, contamination by bacteria is common. The role or effects of bacteria present in B. braunii CCALA778 are not yet fully elucidated. We used UV-C treatment to reduce bacterial abundance in B. braunii CCALA778 cultures and 16S rRNA gene amplicon MiSeq sequencing for bacterial community analysis. The effect of the reduced amount of bacteria on biomass growth and production of extracellular carbohydrates was analysed. It is shown that UV-C treatment can reduce the bacterial population substantially without harming B. braunii. Bacteria removed by UV-C were antagonistic to B. braunii CCALA778 as in their absence production of biomass and extracellular carbohydrates was enhanced significantly. CCALA778 treated with UV-C accumulated 826 ± 61 mg L −1 of extracellular carbohydrates by day 15 compared with 422 ± 135 mg L −1 accumulated extracellular carbohydrates in the untreated culture.
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