Phycospheric Native Bacteria Pelagibaca bermudensis and Stappia sp. Ameliorate Biomass Productivity of Tetraselmis striata (KCTC1432BP) in Co-cultivation System through Mutualistic Interaction.
ABSTRACT: Effective sustainable algal cultivation techniques are essential for mass production of the marine microalga Tetraselmis for biofuel and array of co-products. The phycospheric communities affect the microalgal growth and metabolism through various allelochemical and nutrient interactions; hence, their potential to affect the quantity and quality of both biomass and bioproducts is significant. In the present study, we have screened the phycospheric communities of biofuel producing Tetraselmis striata (KCTC1432BP). A total of 26 bacterial strains were isolated and identified from the phycosphere of T. striata mass culture. Then, each bacterial strain was tested in co-cultivation conditions with T. striata for evaluating its growth promoting and inhibitory effects. Among these all strains, two promising strains (Pelagibaca bermudensis KCTC 13073BP and Stappia sp. KCTC 13072BP) were selected because of their maximum growth promoting effects and mutualistic interactions. The growth rate, biomass productivity, lipid contents, and fatty acids were analyzed during their combined growth in O3 media and compared with axenic growth of T. striata. Later, growth promoting mechanisms in the co-cultivation environment were investigated for these promising bacterial strains under replete and limited conditions of nutrients (nitrate, phosphate, and vitamin B12). The growth promoting potential of P. bermudensis was illustrated by the two fold enhancement in biomass productivity. These bacteria are promising for microalgal cultivation without any negative effects on the native seawater bacterial communities, as revealed by next generation sequencing analysis. This study represents, to date, the first report highlighting the role of phycospheric growth promoting bacteria of promising biofuel feedstock T. striata.
Project description:Amelioration of biofuel feedstock of microalgae using sustainable means through synthetic ecology is a promising strategy. The co-cultivation model (Tetraselmis striata and Pelagibaca bermudensis) was evaluated for the robust biofuel production under varying stressors as well as with the selected two-stage cultivation modes. In addition, the role of metabolic exudates including the quorum-sensing precursors was assessed.The co-cultivation model innovated in this study supported the biomass production of T. striata in a saline/marine medium at a broad range of pH, salinity, and temperature/light conditions, as well as nutrient limitation with a growth promotion of 1.2-3.6-fold. Hence, this developed model could contribute to abiotic stress mitigation of T. striata. The quorum-sensing precursor dynamics of the growth promoting bacteria P. bermudensis exhibited unique pattern under varying stressors as revealed through targeted metabolomics (using liquid chromatography-mass spectrometry, LC-MS). P. bermudensis and its metabolic exudates mutually promoted the growth of T. striata, which elevated the lipid productivity. Interestingly, hydroxy alkyl quinolones independently showed growth inhibition of T. striata on elevated concentration. Among two-stage cultivation modes (low pH, elevated salinity, and nitrate limitation), specifically, nitrate limitation induced a 1.5 times higher lipid content (30-31%) than control in both axenic and co-cultivated conditions.Pelagibaca bermudensis is established as a potential growth promoting native phycospheric bacteria for robust biomass generation of T. striata in varying environment, and two-stage cultivation using nitrate limitation strategically maximized the biofuel precursors for both axenic and co-cultivation conditions (T and T-PB, respectively). Optimum metabolic exudate of P. bermudensis which act as a growth substrate to T. striata surpasses the antagonistic effect of excessive hydroxy alkyl quinolones [HHQ, 4-hydroxy-2-alkylquinolines and PQS (pseudomonas quorum signal), 2-heptyl-3-hydroxy-4(1H)-quinolone].
Project description:Microalgae-based bioenergy production is a promising field with regard to the wide variety of algal species and metabolic potential. The use of liquid wastes as nutrient clearly improves the sustainability of microalgal biofuel production. Microalgae and bacteria have an ecological inter-kingdom relationship. This microenvironment called phycosphere has a major role in the ecosystem productivity and can be utilized both in bioremediation and biomass production. However, knowledge on the effects of indigenous bacteria on microalgal growth and the characteristics of bacterial communities associated with microalgae are limited. In this study municipal, industrial and agricultural liquid waste derivatives were used as cultivation media. Chlorella vulgaris green microalgae and its bacterial partners efficiently metabolized the carbon, nitrogen and phosphorous content available in these wastes. The read-based metagenomics approach revealed a diverse microbial composition at the start point of cultivations in the different types of liquid wastes. The relative abundance of the observed taxa significantly changed over the cultivation period. The genome-centric reconstruction of phycospheric bacteria further explained the observed correlations between the taxonomic composition and biomass yield of the various waste-based biodegradation systems. Functional profile investigation of the reconstructed microbes revealed a variety of relevant biological processes like organic acid oxidation and vitamin B synthesis. Thus, liquid wastes were shown to serve as valuable resources of nutrients as well as of growth promoting bacteria enabling increased microalgal biomass production.
Project description:Bioprospecting for novel microalgal strains is key to improving the feasibility of microalgae-derived biodiesel production. Tetraselmis sp. CTP4 (Chlorophyta, Chlorodendrophyceae) was isolated using fluorescence activated cell sorting (FACS) in order to screen novel lipid-rich microalgae. CTP4 is a robust, euryhaline strain able to grow in seawater growth medium as well as in non-sterile urban wastewater. Because of its large cell size (9-22 μm), CTP4 settles down after a six-hour sedimentation step. This leads to a medium removal efficiency of 80%, allowing a significant decrease of biomass dewatering costs. Using a two-stage system, a 3-fold increase in lipid content (up to 33% of DW) and a 2-fold enhancement in lipid productivity (up to 52.1 mg L-1 d-1) were observed upon exposure to nutrient depletion for 7 days. The biodiesel synthesized from the lipids of CTP4 contained high levels of oleic acid (25.67% of total fatty acids content) and minor amounts of polyunsaturated fatty acids with ≥4 double bonds (<1%). As a result, this biofuel complies with most of the European (EN14214) and American (ASTM D6751) specifications, which commonly used microalgal feedstocks are usually unable to meet. In conclusion, Tetraselmis sp. CTP4 displays promising features as feedstock with lower downstream processing costs for biomass dewatering and biodiesel refining.
Project description:Background:Microalgal starch is regarded as a promising alternative to crop-based starch for biorefinery such as the production of biofuels and bio-based chemicals. The single or separate use of inorganic carbon source, e.g., CO2 and NaHCO3, caused aberrant pH, which restricts the biomass and starch production. The present study applied an in situ CO2-NaHCO3 system to regulate photosynthetic biomass and starch production along with starch quality in a marine green microalga Tetraselmis subcordiformis under nitrogen-depletion (-N) and nitrogen-limitation (±N) conditions. Results:The CO2 (2%)-NaHCO3 (1 g L-1) system stabilized the pH at 7.7 in the -N cultivation, under which the optimal biomass and starch accumulation were achieved. The biomass and starch productivity under -N were improved by 2.1-fold and 1.7-fold, respectively, with 1 g L-1 NaHCO3 addition compared with the one without NaHCO3 addition. NaHCO3 addition alleviated the high-dCO2 inhibition caused by the single CO2 aeration, and provided sufficient effective carbon source HCO3 - for the maintenance of adequate photosynthetic efficiency and increase in photoprotection to facilitate the biomass and starch production. The amylose content was also increased by 44% under this CO2-bicarbonate system compared to the single use of CO2. The highest starch productivity of 0.73 g L-1 day-1 under -N cultivation and highest starch concentration of 4.14 g L-1 under ±N cultivation were both achieved with the addition of 1 g L-1 NaHCO3. These levels were comparable to or exceeded the current achievements reported in studies. The addition of 5 g L-1 NaHCO3 under ±N cultivation led to a production of high-amylose starch (59.3% of total starch), which could be used as a source of functional food. Conclusions:The in situ CO2-NaHCO3 system significantly improved the biomass and starch production in T. subcordiformis. It could also regulate the starch quality with varied relative amylose content under different cultivation modes for diverse downstream applications that could promote the economic feasibility of microalgal starch-based biofuel production. Adoption of this system in T. subcordiformis would facilitate the CO2 mitigation couple with its starch-based biorefinery.
Project description:A high-quality draft genome sequence of the microalgal species Tetraselmis striata was generated using PacBio sequencing. The assembled genome is 228?Mb, derived from 3,613 polished contigs at 84× coverage depth. This genome contains an average GC content of 57.9% and 48,906 predicted genes.
Project description:The challenges which the large scale microalgal industry is facing are associated with the high cost of key operations such as harvesting, nutrient supply and oil extraction. The high-energy input for harvesting makes current commercial microalgal biodiesel production economically unfeasible and can account for up to 50% of the total cost of biofuel production. Co-cultivation of fungal and microalgal cells is getting increasing attention because of high efficiency of bio-flocculation of microalgal cells with no requirement for added chemicals and low energy inputs. Moreover, some fungal and microalgal strains are well known for their exceptional ability to purify wastewater, generating biomass that represents a renewable and sustainable feedstock for biofuel production. We have screened the flocculation efficiency of the filamentous fungus A. fumigatus against 11 microalgae representing freshwater, marine, small (5 µm), large (over 300 µm), heterotrophic, photoautotrophic, motile and non-motile strains. Some of the strains are commercially used for biofuel production. Lipid production and composition were analysed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources contained in wheat straw and swine wastewater, respectively. Co-cultivation of algae and A. fumigatus cells showed additive and synergistic effects on biomass production, lipid yield and wastewater bioremediation efficiency. Analysis of fungal-algal pellet's fatty acids composition suggested that it can be tailored and optimised through co-cultivating different algae and fungi without the need for genetic modification.
Project description:Background:Euglena gracilis, a unicellular flagellated microalga, is regarded as one of the most promising species as microalgal feedstock for biofuels. Its lipids (mainly wax esters) are suitable for biodiesel and jet fuel. Culture of E. gracilis using wastewater effluent will improve the economics of E. gracilis biofuel production. Enhancement of the productivity of E. gracilis biomass is critical to creating a highly efficient biofuels production system. Certain bacteria have been found to promote microalgal growth by creating a favorable microenvironment. These bacteria have been characterized as microalgae growth-promoting bacteria (MGPB). Co-culture of microalgae with MGPB might offer an effective strategy to enhance microalgal biomass production in wastewater effluent culture systems. However, no MGPB has been identified to enhance the growth of E. gracilis. The objectives of this study were, therefore, to isolate and characterize the MGPB effective for E. gracilis and to demonstrate that the isolated MGPB indeed enhances the production of biomass and lipids by E. gracilis in wastewater effluent culture system. Results:A bacterium, Emticicia sp. EG3, which is capable of promoting the growth of microalga E. gracilis, was isolated from an E. gracilis-municipal wastewater effluent culture. Biomass production rate of E. gracilis was enhanced 3.5-fold and 3.1-fold by EG3 in the co-culture system using a medium of heat-sterilized and non-sterilized wastewater effluent, respectively, compared to growth in the same effluent culture but without EG3. Two-step culture system was examined as follows: E. gracilis was cultured with or without EG3 in wastewater effluent in the first step and was further grown in wastewater effluent in the second step. Production yields of biomass and lipids by E. gracilis were enhanced 3.2-fold and 2.9-fold, respectively, in the second step of the system in which E. gracilis was co-cultured with EG3 in the first step. Conclusion:Emticicia sp. EG3 is the first MGPB for E. gracilis. Growth-promoting bacteria such as EG3 will be promising agents for enhancing E. gracilis biomass/biofuel productivities.
Project description:BACKGROUND: Microalgae are touted as an attractive alternative to traditional forms of biomass for biofuel production, due to high productivity, ability to be cultivated on marginal lands, and potential to utilize carbon dioxide (CO2) from industrial flue gas. This work examines the fossil energy return on investment (EROIfossil), greenhouse gas (GHG) emissions, and direct Water Demands (WD) of producing dried algal biomass through the cultivation of microalgae in Open Raceway Ponds (ORP) for 21 geographic locations in the contiguous United States (U.S.). For each location, comprehensive life cycle assessment (LCA) is performed for multiple microalgal biomass production pathways, consisting of a combination of cultivation and harvesting options. RESULTS: Results indicate that the EROIfossil for microalgae biomass vary from 0.38 to 1.08 with life cycle GHG emissions of -46.2 to 48.9 (g CO2 eq/MJ-biomass) and direct WDs of 20.8 to 38.8 (Liters/MJ-biomass) over the range of scenarios analyzed. Further anaylsis reveals that the EROIfossil for production pathways is relatively location invariant, and that algae's life cycle energy balance and GHG impacts are highly dependent on cultivation and harvesting parameters. Contrarily, algae's direct water demands were found to be highly sensitive to geographic location, and thus may be a constraining factor in sustainable algal-derived biofuel production. Additionally, scenarios with promising EROIfossil and GHG emissions profiles are plagued with high technological uncertainty. CONCLUSIONS: Given the high variability in microalgae's energy and environmental performance, careful evaluation of the algae-to-fuel supply chain is necessary to ensure the long-term sustainability of emerging algal biofuel systems. Alternative production scenarios and technologies may have the potential to reduce the critical demands of biomass production, and should be considered to make algae a viable and more efficient biofuel alternative.
Project description:Increasing petroleum costs and climate change have resulted in microalgae receiving attention as potential biofuel producers. Little information is available on the diversity and functions of bacterial communities associated with biofuel-producing algae. A potential biofuel-producing microalgal strain, Nannochloropsis oceanica IMET1, was grown in Permian groundwater. Changes in the bacterial community structure at three temperatures were monitored by two culture-independent methods, and culturable bacteria were characterized. After 9 days of incubation, N. oceanica IMET1 began to aggregate and precipitate in cultures grown at 30°C, whereas cells remained uniformly distributed at 15°C and 25°C. The bacterial communities in cultures at 30°C changed markedly. Some bacteria isolated only at 30°C were tested for their potential for aggregating microalgae. A novel bacterium designated HW001 showed a remarkable ability to aggregate N. oceanica IMET1, causing microalgal cells to aggregate after 3 days of incubation, while the total lipid content of the microalgal cells was not affected. Direct interaction of HW001 and N. oceanica is necessary for aggregation. HW001 can also aggregate the microalgae N. oceanica CT-1, Tetraselmis suecica, and T. chuii as well as the cyanobacterium Synechococcus WH8007. 16S rRNA gene sequence comparisons indicated the great novelty of this strain, which exhibited only 89% sequence similarity with any previously cultured bacteria. Specific primers targeted to HW001 revealed that the strain originated from the Permian groundwater. This study of the bacterial communities associated with potential biofuel-producing microalgae addresses a little-investigated area of microalgal biofuel research and provides a novel approach to harvest biofuel-producing microalgae by using the novel bacterium strain HW001.
Project description:Algal cultures are usually co-cultures of algae and bacteria, especially when considering outdoor mass cultivation. The influence of associated bacteria on algal culture performance has been poorly investigated, although bacteria may strongly affect biomass (or derived product) yield and quality. In this work, the influence on growth and productivity of Tetraselmis suecica F&M-M33 of bacterial communities and single bacterial isolates from the algal phycosphere was investigated. Xenic laboratory and outdoor cultures were compared with an axenic culture in batch. The presence of the bacterial community significantly promoted culture growth. Single bacterial isolates previously found to be strictly associated with T. suecica F&M-M33 also increased growth compared with the axenic culture, whereas loosely associated and common seawater bacteria induced variable growth responses, from positive to detrimental. The increased growth was mainly evidenced as increased algal biomass production and cell size, and occurred after exhaustion of nutrients. This finding is of interest for biofuel production from microalgae, often attained through nutrient starvation processes leading to oil or carbohydrate accumulation. As axenic T. suecica F&M-M33 showed a similar growth with or without vitamins, the most probable mechanism behind bacterial positive influence on algal growth seems nutrient recycling.