Enhanced Microalgal Biomass Research Articles

Enhanced production of biomass and lipids by Euglena gracilis via co-culturing with a microalga growth-promoting bacterium, Emticicia sp. EG3

BackgroundEuglena 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.ResultsA 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.ConclusionEmticicia 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.

Enhancing Microalgal Biomass Productivity in Floating Photobioreactors with Semi-Permeable Membranes Grafted with 4-Hydroxyphenethyl Bromide

Microalgal biomass productivity of semi-permeable membrane photobioreactors (SPM-PBRs) highly depends on diffusion of nutrient ions from surrounding environment through SPMs. The objective of this study was to enhance microalgal biomass productivity in floating photobioreactors (PBRs) with semi-permeable membranes (SPMs) grafted with 4-hydroxyphenethyl bromide (4-HPB) by increasing ion permeabilities and reducing biofouling of SPMs. In this study, we investigated the reduction of biofouling and improvement of biomass productivity by grafting 4-HPB onto SPMs which could decrease hydrophobicity. The degree of alkylation could be increased by up to 4.9%, with water contact angles on alkylated SPM surfaces decreased from 102.5° to 58.2°, indicating decreased surface hydrophobicity. Ion and water permeabilities of SPMs were increased in alkylated SPMs by 148% and 200%, respectively, compared to non-treated SPM control. Moreover, biomass productivity of PBRs with alkylated SPMs was 75% (740 mg m−2 day−1) higher than that of the control. Biofouling was also remarkably reduced by up to 40% in 4-HPB grafted SPMs after cultivation of microalgae. These results suggest that decreasing hydrophobicity of SPMs by grafting 4-HPB could enhance biomass productivity in SPM-PBRs through improving ion permeability and reducing biofouling.

Multilevel heuristic LED regime for stimulating lipid and bioproducts biosynthesis in Haematococcus pluvialis under mixotrophic conditions

Enhanced biosynthesis and overproduction of lipid and high-value bioproducts endows microalgae as a promising feedstock for the cost-effective development of advanced bioenergy currently. In this study, multilevel heuristic LED regimes, designed with the coordination of light, nitrogen and carbons in terms of cell physiology and morphology, have been implemented to significantly induce the growth and bioproducts accumulation of Haematococcus pluvialis under mixotrophic conditions. The white-red regime with C5 organic carbon showed a good potential for enhancing microalgal biomass and lipid synthesis, especially for saturated fatty acids. The astaxanthin biosynthesis has been significantly enhanced and the highest content of 3.3% was achieved with gluconate at the white-blue regime. Additionally, white-blue regime enhanced the protein content of H. pluvialis up to 45%, increased by 40% over the white-red culture. The multilevel LED protocol can be developed synergistically with the different adaptation and tolerance of algal cell physiology and morphology for target bioproducts.

Quercetin potentiates the concurrent hyper-accumulation of cellular biomass and lipids in Chlorella vulgaris

Provision of chemical modulators has emerged as an effective strategy to govern cell growth and development. Here, the impact of flavonoid quercetin on algal growth, lipid accumulation and transcriptional patterns was investigated in the green microalga Chlorella vulgaris. These results demonstrated that quercetin (15 μg/l) significantly enhanced the cellular biomass and photosynthetic efficiency, with up to 2.5-fold in the biomass in the stationary phase. Lipidomic analyses revealed that lipid content was increased by 1.8-fold. Furthermore, the functional mechanism of quercetin on the molecular level was dissected by transcriptomic analysis. Results revealed that quercetin upregulated the expression pattern of key genes involved in cellular signaling mechanisms such as phosphatidylinositol 4-kinase α, thus consequently enhanced cell growth. Altogether, the data present in this study demonstrate the dramatic role of quercetin on enhancing microalgal biomass and lipid accumulation by unprecedented regulation, of key metabolic nodes, for the first time and provide a novel insight into microalgal metabolism and regulation.

Glycerol and nitrate utilisation by marine microalgae Nannochloropsis salina and Chlorella sp. and associated bacteria during mixotrophic and heterotrophic growth

Mixotrophic growth is an ability of microalgae to concurrently assimilate CO2 and organic carbon. In the present study, the use of glycerol to enhance microalgal biomass and lipid productivities was investigated in relation to nitrate availability. Under nitrate sufficient conditions, the biomass productivities of Nannochloropsis salina (CCMP 1776) and a marine Chlorella sp. were 1.7 and 1.9 times higher in mixotrophic culture than under strictly photoautotrophic conditions, respectively. Both algae required light to assimilate glycerol. No significant algae growth was observed under heterotrophic conditions, despite apparent utilisation of both nitrate and glycerol. Under nitrate deplete conditions both species took up only minimal amounts of glycerol thereby indicating the importance of the combined effects of carbon and nitrogen. Additionally, a carbon mass balance was conducted based on glycerol utilisation and biomass production in N. salina cultures, which revealed a net release of CO2 during mixotrophic growth. In the presence of glycerol, the proliferation of bacteria was consistently observable in these non-axenic cultures. 16S rRNA sequencing confirmed the presence of two phylogenetically distinct Gram-negative bacterial isolates belonging to α- and γ-subclass of Proteobacteria (Paracoccus sp. MBIC4036, and Marinobacter alkaliphilus NBSL03 and Marinobacter lipolyticus SM-19, respectively) in the cultures of N. salina. Among these strains, M. alkaliphilus was shown to have denitrifying capabilities, which allowed it to oxidise glycerol using nitrate as a terminal electron acceptor. This provides an explanation for the utilisation of nitrate and glycerol that was observed in heterotrophic cultures despite the absence of microalgal growth. These results provide a better understanding of the interactions between bacteria and algae in mixotrophic growth that may enable the development of strategies to better utilise nitrogen and carbon for algae production. The present study has demonstrated the possibility of reutilisation of glycerol, a biodiesel by-product, to promote algal growth and lipid accumulation.

Combined yeast and microalgal cultivation in a pilot-scale raceway pond for urban wastewater treatment and potential biodiesel production.

A mixed culture of oleaginous yeast Lipomyces starkeyi and wastewater native microalgae (mostly Scenedesmus sp. and Chlorella sp.) was performed to enhance lipid and biomass production from urban wastewaters. A 400 L raceway pond, operating outdoors, was designed and used for biomass cultivation. Microalgae and yeast were inoculated into the cultivation pond with a 2:1 inoculum ratio. Their concentrations were monitored for 14 continuous days of batch cultivation. Microalgal growth presented a 3-day initial lag-phase, while yeast growth occurred in the first few days. Yeast activity during the microalgal lag-phase enhanced microalgal biomass productivity, corresponding to 31.4 mgTSS m-2 d-1. Yeast growth was limited by low concentrations in wastewater of easily assimilated organic substrates. Organic carbon was absorbed in the first 3 days with a 3.7 mgC L-1 d-1 removal rate. Complete nutrient removal occurred during microalgal linear growth with 2.9 mgN L-1 d-1 and 0.96 mgP L-1 d-1 removal rates. Microalgal photosynthetic activity induced high pH and dissolved oxygen values resulted in natural bactericidal and antifungal activity. A 15% lipid/dry weight was measured at the end of the cultivation time. Fatty acid methyl ester (FAME) analysis indicated that the lipids were mainly composed of arachidic acid.

Isolation, phenotypic characterization and genome wide analysis of a Chlamydomonas reinhardtii strain naturally modified under laboratory conditions: towards enhanced microalgal biomass and lipid production for biofuels

BackgroundMicroalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing.ResultsIn this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation.ConclusionOur phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production.

Mixed cultivation as an effective approach to enhance microalgal biomass and triacylglycerol production in domestic secondary effluent

Compared with monoculture, the mixed culture of certain microalgal strains showed important advantages, including higher biomass production, better resistance against sudden disruptions, and so on. In this study, the growth and lipid accumulation properties of Scenedesmus sp. LX1 (S. LX1) and Haematococcus pluvialis (H. pluvialis) were investigated in mixed culture using domestic secondary effluent as medium. In the mixed culture with initial biomass ratio of 4:1, 1:1 and 1:10 (S. LX1:H. pluvialis, w:w, dry weight), the total biomass production and triacylglycerol (TAG) production were enhanced significantly compared with the monocultures, and reached 0.51–0.54g·L−1 and 6.9–9.3mg·L−1 after 13-day cultivation, respectively. Further analysis revealed that interspecific nutrient competition between S. LX1 and H. pluvialis caused lower total nitrogen (TN) concentration in the medium of mixed culture, serving as an environmental stress to enhance the TAG accumulation. Furthermore, the soluble algal products (SAPs) released by H. pluvialis could improve the photosynthetic activity of S. LX1; while the SAPs released by S. LX1 could be assimilated by H. pluvialis for heterotrophic growth. The mutually beneficial relationship based on the effects of SAPs was the main mechanism of enhancing the total biomass produciton in the mixed culture.

Enhanced microalgal biomass and lipid production from a consortium of indigenous microalgae and bacteria present in municipal wastewater under gradually mixotrophic culture conditions

The goal of this study was to investigate the influences of gradually mixotrophic culture conditions on microalgal biomass and lipid production by a consortium of indigenous microalgae and bacteria present in raw municipal wastewater. Lab-scale photobioreactors containing the consortium were operated in repeated batch mode. Initial cultivation (phase I) was performed using only the municipal wastewater, then 10% and 25% of the reactor volumes were replaced with the effluent from a sewage sludge fermentation system producing volatile fatty acids (SSFV) at the beginnings of phase II and phase III, respectively. The highest biomass productivity (117.1±2.7mg/L/d) was attained during phase II, but the lipid productivity (17.2±0.2mg/L/d) was attained during phase III. The increase in the effluent from the SSFV influenced microalgal diversity with a preference for Chlorella sp., but bacterial diversity increased significantly during phase III.

Enzymatic pretreatment of microalgae using fungal broth from Trametes versicolor and commercial laccase for improved biogas production

Coupling microalgae production to wastewater treatment can reduce the costs of microalgae production for non-food bioproducts and energy consumption for wastewater treatment. Furthermore, microalgae anaerobic digestion can be enhanced by applying pretreatment techniques. The aim of this study is to improve the biogas production from microalgal biomass grown in urban wastewater treatment systems by applying an enzymatic pretreatment with crude fungal broth and commercial laccase. To this end, the fungus Trametes versicolor was cultured, and the enzymatic activity of the culture broth analysed by measuring laccase concentration. The results showed that both the fungal broth and commercial laccase pretreatment (100UL−1) over an exposure time of 20min increased the methane yield in batch tests. Indeed, the fungal broth pretreatment increased the methane yield by 74%, while commercial laccase increased the methane yield by 20% as compared to non-pretreated microalgal biomass. In this manner, laccase addition enhanced microalgal biomass anaerobic biodegradability, and addition of T. versicolor broth further improved the results. This fact may be attributed to the presence of other molecules excreted by the fungus.

Mixotrophic cultivation of Nephroselmis sp. using industrial wastewater for enhanced microalgal biomass production

Effect of liquid crystal display (LCD) wastewater on the biomass production and biochemical composition of a green microalga Nephroselmis sp. cultivated in Bold’s Basal Medium (BBM) was investigated. Different dilutions (1/250-1/10,000) of LCD wastewater with BBM were tested in the presence of 5% flue gas CO2. Nephroselmis sp. showed the highest growth (0.37gL−1), lipid productivity (14.2mgL−1day−1), carbohydrate productivity (12.5mgL−1day−1), β-carotene+lutein productivity (74.2μgL−1day−1) and nutrient removal (35.0mg TNL−1 and 11.6mg TPL−1) with 1/1000 diluted LCD wastewater after 6days of cultivation. Cultivation with LCD wastewater increased the microalgal saturated and monounsaturated fatty acid contents up to 9 and 24%, respectively. Application of LCD wastewater improved the growth, lipid/carbohydrate/carotenoids productivity and nutrient removal efficiency of Nephroselmis sp., which can be a cost effective strategy for microalgal biomass production.

Microalgae as Future Fuel: Real Opportunities and Challenges

Depleting reserves and souring prices of petroleum and oil have daunting effect on the economy of many developed and developing nations and forced researchers, government and federal agencies to opt for development of alternate fuels. In recent years, there is renewed interest in the field of algal biofuel production owing to its ability to grow in non-agriculturalable waste land and municipal wastewater/agricultural runoff water. Previous decade was attributed in developing new open culture/ reactor design for enhanced microalgal biomass production, efficient harvesting and pretreatment systems for commercial algal biofuel production. The true potential of algal biofuel spurs debate between algae experts and external observer over economics and environmental sustainability. Currently, no commercial algal energy generation system, which can surpass crop based energy content, is available. In present review, efficient culturing, operational strategies, reactor design and technological advancement in processing of algal fuel have been reviewed in graphical abstract

Green integrated process for mitigation of municipal and industrial liquid and solid waste mixes for enhanced microalgal biomass and lipid synthesis for biodiesel

We herein report: a sustainable green integrated process for effective waste mitigation and microalgal biomass production for biodiesel feedstock generation in a biorefinery model.

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.

Role of Rhizobium, a plant growth promoting bacterium, in enhancing algal biomass through mutualistic interaction

Rhizobium plays the pivotal role in mutualistic interactions with plants and this study extends this mutualism to several species of green algae. Denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library experiments of Chlamydomonas reinhardtii, Chlorella vulgaris, Scenedesmus sp., and Botryococcus braunii revealed that the dominant phycosphere bacteria hosted by these green algae were Rhizobium, Mesorhizobium, and Shinella within Rhizobiales, Flavobacterium within Flavobacteriales, and Pseudomonas within Pseudomonadales. When Rhizobium sp., most prevalent and dominant bacterium isolated from C. vulgaris, was co-cultured with green algae, it promoted algal cell count by ∼72%. The qPCR analysis of 16S rRNA, Rhizobium specific rirA and rhtA genes, performed to understand the effect of green algae on growth of Rhizobium sp., showed a significant increase in copy numbers indicating sustained growth of Rhizobium upon co-culture with green algae. Likewise, growth rates of algae and Rhizobium increased by an average of ∼11% and ∼110%, respectively, confirming mutualistic interaction. Considering the presence of Rhizobium sp. in a variety of green algae, this must be a major mutualistic relationship among green algae and this ubiquitous association might serve as a model for elucidating the mutualistic mechanism between green algae and rhizobacter. This interaction could be also utilized in enhancing microalgal biomass, especially slow growing organisms like B. braunii, to augment their bioenergy productivity.