Byung-Hyuk Kim

Algae–bacteria interactions: Evolution, ecology and emerging applications

Algae and bacteria have coexisted ever since the early stages of evolution. This coevolution has revolutionized life on earth in many aspects. Algae and bacteria together influence ecosystems as varied as deep seas to lichens and represent all conceivable modes of interactions - from mutualism to parasitism. Several studies have shown that algae and bacteria synergistically affect each others physiology and metabolism, a classic case being algae-roseobacter interaction. These interactions are ubiquitous and define the primary productivity in most ecosystems. In recent years, algae have received much attention for industrial exploitation but their interaction with bacteria is often considered a contamination during commercialization. A few recent studies have shown that bacteria not only enhance algal growth but also help in flocculation, both essential processes in algal biotechnology. Hence, there is a need to understand these interactions from an evolutionary and ecological standpoint, and integrate this understanding for industrial use. Here we reflect on the diversity of such relationships and their associated mechanisms, as well as the habitats that they mutually influence. This review also outlines the role of these interactions in key evolutionary events such as endosymbiosis, besides their ecological role in biogeochemical cycles. Finally, we focus on extending such studies on algal-bacterial interactions to various environmental and bio-technological applications.

Two-stage cultivation of two Chlorella sp. strains by simultaneous treatment of brewery wastewater and maximizing lipid productivity.

A cultivation system in the two-stage photoautotrophic-photoheterotrophic/mixotrophic mode was adapted to maximize lipid productivity of two freshwater strains of Chlorella sp. grown in brewery wastewater (BWW). The endogenous Chlorella sp. isolated from BWW had a higher growth rate than wild-type Chlorella vulgaris (UTEX-265) while C. vulgaris (UTEX-265) had a higher maximal biomass and lipid contents than that of endogenous Chlorella sp., resulting in more than 90% of the inorganic nutrients in both total nitrogen (TN) and phosphorus (TP) was removed during the first stage in the two-stage photoautotrophic-photoheterotrophic mode in each Chlorella sp. The maximal biomass and lipid contents of C. vulgaris (UTEX-265) for single stage photoautotrophic cultivation were 1.5 g/L and 18%, respectively. Importantly, during two-stage photoautotrophic-photoheterotrophic cultivation for C. vulgaris (UTEX-265), the biomass was increased to 3.5 g/L, and the lipid productivity was increased from 31.1 to 108.0mg/L day.

Microalgae-associated bacteria play a key role in the flocculation of Chlorella vulgaris.

Flocculation is most preferred method for harvesting microalgae, however, the role of bacteria in microalgal flocculation process is still unknown. This study proves that bacteria play a profound role in flocculating by increasing the floc size resulting in sedimentation of microalgae. A flocculating activity of 94% was achieved with xenic Chlorella vulgaris culture as compared to 2% achieved with axenic culture. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene of xenic C. vulgaris culture revealed the presence of Flavobacterium sp., Terrimonas sp., Sphingobacterium sp., Rhizobium sp. and Hyphomonas sp. as microalgae-associated bacteria. However when Flavobacterium, Terrimonas, Sphingobacterium were eliminated by fluorescence activated cell sorter (FACS), flocculating activity reduced to 3%. Further studies with cell free extracts also suggest that bacterial extracellular substances might also have a role in enhancing flocculation. We conclude that the collective presence of certain bacteria is the determining factor in flocculation of C. vulgaris.

Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii

Phenotypic and genotypic changes in Chlamydomonas reinhardtii BafJ5, a starchless mutant, with respect to lipid metabolism was studied in different trophic states under nitrogen (N) sufficient and limited conditions. Interestingly, cellular lipid content increased linearly with input acetate concentration with highest lipid content (∼42%) under nitrogen limitation and mixotrophic state. RT‐qPCR studies indicate that key fatty acid biosynthesis genes are down‐regulated under N limitation but not under mixotrophic state, whereas, ACS2, encoding Acetyl‐CoA synthetase, and DGTT4, encoding Diacylglycerol O‐acyltransferase, are up‐regulated under all conditions. These results collectively indicate that acetate is the limiting factor and central molecule in lipid droplet synthesis. The study also provides further evidence of the presence of a chloroplast pathway for triacylglycerol synthesis in microalgae.

Enhancing microalgal biomass productivity by engineering a microalgal–bacterial community

This study demonstrates that ecologically engineered bacterial consortium could enhance microalgal biomass and lipid productivities through carbon exchange. Phycosphere bacterial diversity analysis in xenic Chlorella vulgaris (XCV) confirmed the presence of growth enhancing and inhibiting microorganisms. Co-cultivation of axenic C. vulgaris (ACV) with four different growth enhancing bacteria revealed a symbiotic relationship with each bacterium. An artificial microalgal-bacterial consortium (AMBC) constituting these four bacteria and ACV showed that the bacterial consortium exerted a statistically significant (P<0.05) growth enhancement on ACV. Moreover, AMBC had superior flocculation efficiency, lipid content and quality. Studies on carbon exchange revealed that bacteria in AMBC might utilize fixed organic carbon released by microalgae, and in return, supply inorganic and low molecular weight (LMW) organic carbon influencing algal growth and metabolism. Such exchanges, although species specific, have enormous significance in carbon cycle and can be exploitated by microalgal biotechnology industry.

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.

Novel approach for the development of axenic microalgal cultures from environmental samples.

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.

Organic carbon, influent microbial diversity and temperature strongly influence algal diversity and biomass in raceway ponds treating raw municipal wastewater.

Algae based wastewater treatment coupled to biofuel production has financial benefits and practical difficulties. This study evaluated the factors influencing diversity and growth of indigenous algal consortium cultivated on untreated municipal wastewater in a high rate algal pond (HRAP) for a period of 1 year using multivariate statistics. Diversity analyses revealed the presence of Chlorophyta, Cyanophyta and Bacillariophyta. Dominant microalgal genera by biovolume in various seasons were Scenedesmus sp., Microcystis sp., and Chlorella sp. Scenedesmus sp., persisted throughout the year but none of three strains co-dominated with the other. The most significant factors affecting genus dominance were temperature, inflow cyanophyta and organic carbon concentration. Cyanophyta concentration affected microalgal biomass and diversity, whereas temperature impacted biomass. Preferred diversity of microalgae is not sustained in wastewater systems but is obligatory for biofuel production. This study serves as a guideline to sustain desired microalgal consortium in wastewater treatment plants for biofuel production.

Microalgal diversity fosters stable biomass productivity in open ponds treating wastewater

It is established that biodiversity determines productivity of natural ecosystems globally. We have proved that abiotic factors influenced biomass productivity in engineered ecosystems i.e. high rate algal ponds (HRAPs), previously. This study demonstrates that biotic factors, particularly microalgal diversity, play an essential role in maintaining stable biomass productivity in HRAP treating municipal wastewater by mutualistic adaptation to environmental factors. The current study examined data from the second year of a two-year study on HRAP treating municipal wastewater. Microalgal diversity, wastewater characteristics, treatment efficiency and several environmental and meteorological factors were documented. Multivariate statistical analyses reveal that microalgae in uncontrolled HRAPs adapt to adverse environmental conditions by fostering diversity. Subsequently, five dominant microalgal strains by biovolume were isolated, enriched, and optimum conditions for high biomass productivity were ascertained. These laboratory experiments revealed that different microalgal strains dominate in different conditions and a consortium of these diverse taxa help in sustaining the algae community from environmental and predatory pressures. Diversity, niche or seasonal partitioning and mutualistic growth are pertinent in microalgal cultivation or wastewater treatment. Therefore, enrichment of selective species would deprive the collective adaptive ability of the consortium and encourage system vulnerability especially in wastewater treatment.

SIMPLE METHOD FOR RNA PREPARATION FROM CYANOBACTERIA

A simple and rapid method is presented for the preparation of RNA from various cyanobacteria. Unlike other methods that require a lysis solution, lysozymes, or proteinase K, the proposed method, called the bead–phenol–chloroform (BPC) method, uses silica/zirconia beads, phenol, and chloroform to break the cells and extract RNA more efficiently. Experiments confirm that the BPC method can successfully isolate total RNA from various cyanobacterial strains without DNA contamination, and the extracted RNA samples have a relatively high purity, concentration, and yield. Furthermore, the BPC method is more rapid, simple, and economical when compared with previously reported methods.