Characterization and evaluation of substratum material selection for microalgal biofilm cultivation.

Biofilm cultivation is considered a promising method to achieve higher microalgae biomass productivity with less water consumption and easier harvest compared to conventional suspended cultivation. However, studies focusing on the selection of substratum material and optimization of the growth of certain microalgae species on specific substratum are limited. This study investigated the selection of membranous and fabric fiber substrata for the attachment of unicellular microalgae Scenedesmus dimorphus and filamentous microalgae Tribonema minus in biofilm cultivation. The results indicated that both algal species preferred hydrophilic membranous substrata and nitrate cellulose/cellulose acetate membrane (CN-CA) was selected as a suitable candidate on which the obtained biomass yields were up to 10.24 and 7.81 g m-2 day-1 for S. dimorphus and T. minus, respectively. Furthermore, high-thread cotton fiber (HCF) and low-thread polyester fiber (LPEF) were verified as the potential fabric fiber substrata for S. dimorphus (5.42 g m-2 day-1) and T. minus (5.49 g m-2 day-1) attachment, respectively. The regrowth of microalgae biofilm cultivation strategy was applied to optimize the algae growth on the fabric fiber substrata, with higher biomass density and shear resistibility achieved for both algal species. The present data highlight the importance to establish the standards for selection the suitable substratum materials in ensuring the high efficiency and sustainability of the attached microalgal biomass production. KEY POINTS: • CN-CA was suitable membranous substratum candidate for algal biofilm cultivation. • HCF and LPEF were potential fabric fiber substrata for S. dimorphus and T. minus. • Regrowth biofilm cultivation was effective in improving algal biomass and attachment.

Producing high value unsaturated fatty acid by whole-cell catalysis using microalga: A case study with Tribonema minus.

High value unsaturated fatty acids can be produced by de novo synthesis in microalgal cells, especially via heterotrophic cultivation. Unfortunately, the lipid accumulation of heterotrophic microalgae cannot be improved efficiently in conventional ways. Here we reported heterotrophic Tribonema minus, a promising resource for the production of palmitoleic acid which has increasing demands in health service for patients with metabolic syndrome, as whole-cell biocatalyst to develop a novel way of shifting low value exogenous saturated fatty acids to high value ones. Results showed that myristic acid is the best precursor for whole-cell catalysis; it elevated the lipid content of T. minus to 42.2%, the highest among the tried precursors. The influences of cultivation condition on the utilization of extrinsic myristic acid and lipid accumulation were also determined. Under the optimized condition, the lipid content reached as high as 48.9%. In addition, our findings showed that ~13.0% of C16:1 in T. minus is derived from extrinsic myristic acid, and 30.1% of metabolized precursor is converted into heterologous fatty acids. Thus, a feasible approach for both increasing the value of low value saturated fatty acid by bioconversion and enhancing the lipid accumulation in microalgae is proposed by supplementing extrinsic myristic acid.

Genetic Transformation of Tribonema minus, a Eukaryotic Filamentous Oleaginous Yellow-Green Alga.

Eukaryotic filamentous yellow-green algae from the Tribonema genus are considered to be excellent candidates for biofuels and value-added products, owing to their ability to grow under autotrophic, mixotrophic, and heterotrophic conditions and synthesize large amounts of fatty acids, especially unsaturated fatty acids. To elucidate the molecular mechanism of fatty acids and/or establish the organism as a model strain, the development of genetic methods is important. Towards this goal, here, we constructed a genetic transformation method to introduce exogenous genes for the first time into the eukaryotic filamentous alga Tribonema minus via particle bombardment. In this study, we constructed pSimple-tub-eGFP and pEASY-tub-nptⅡ plasmids in which the green fluorescence protein (eGFP) gene and the neomycin phosphotransferase Ⅱ-encoding G418-resistant gene (nptⅡ) were flanked by the T. minus-derived tubulin gene (tub) promoter and terminator, respectively. The two plasmids were introduced into T. minus cells through particle-gun bombardment under various test conditions. By combining agar and liquid selecting methods to exclude the pseudotransformants under long-term antibiotic treatment, plasmids pSimple-tub-eGFP and pEASY-tub- nptⅡ were successfully transformed into the genome of T. minus, which was verified using green fluorescence detection and the polymerase chain reaction, respectively. These results suggest new possibilities for efficient genetic engineering of T. minus for future genetic improvement.

Comparison of Lipid and Palmitoleic Acid Induction of Tribonema minus under Heterotrophic and Phototrophic Regimes by Using High-Density Fermented Seeds.

Palmitoleic acid, one scarce omega-7 monounsaturated fatty acid, has important applications in the fields of medicine and health products. Tribonema has been considered as a promising candidate for the production of palmitoleic acid due to its high lipid and palmitoleic acid content and remarkable heterotrophic ability. The high-density heterotrophic cultivation of Tribonema minus was conducted in this work, and the highest biomass of 42.9 g L-1 and a relatively low lipid content of 28.7% were observed. To further enhance the lipid and palmitoleic acid accumulation, induction strategies under two regimes of phototrophy and heterotrophy with different conditions were investigated and compared. Results demonstrated encouraging promotions both by heterotrophic and phototrophic ways, and the final lipid contents reached 41.9% and 49.0%, respectively. In consideration of the time cost, however, the induction under heterotrophic conditions was much more advantageous, by which the highest lipid and palmitoleic acid productivities of 1.77 g L-1 d-1 and 924 mg L-1 d-1 were obtained respectively, with the lipid yield on glucose of 0.26 g g-1.

Partial Characterization, the Immune Modulation and Anticancer Activities of Sulfated Polysaccharides from Filamentous Microalgae Tribonema sp.

Recently, Tribonema sp., a kind of filamentous microalgae, has been studied for biofuel production due to its accumulation of triacylglycerols. However, the polysaccharides of Tribonema sp. and their biological activities have rarely been reported. In this paper, we extracted sulfated polysaccharides from Tribonema sp. (TSP), characterized their chemical composition and structure, and determined their immunostimulation and anticancer activities on RAW264.7 macrophage cells and HepG2 cells. The results showed that TSP is a sulfated polysaccharide with a Mw of 197 kDa. TSP is a heteropolysaccharide that is composed mainly of galactose. It showed significant immune-modulatory activity by stimulating macrophage cells, such as upregulating interleukin 6 (IL-6), interleukin 10 (IL-10), and tumor necrosis factor α (TNF-α). In addition, TSP also showed significant dose-dependent anticancer activity (with an inhibition rate of up to 66.8% at 250 µg/mL) on HepG2 cells as determined by the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cycle analysis indicated that the anticancer activity of TSP is mainly the result of induced cell apoptosis rather than affecting the cell cycle and mitosis of HepG2 cells. These findings suggest that TSP might have potential as an anticancer resource, but further research is needed, especially in vivo experiments, to explore the anticancer mechanism of TSP.

Development of nitrogen supply strategy for Scenedesmus rubescens attached cultivation toward growth and lipid accumulation.

In this study, the microalgae Scenedesmus rubescens were cultivated under the following nitrogen sources, nitrogen concentrations, and nitrogen feeding times (NFTs). This was to help assess biomass and lipid productivity. Scenedesmus rubescens can grow well by adhering to the cellulose acetate membrane in five kinds of nitrogen medium: KNO3, urea, NaNO3, (NH4)2CO3, and NH4NO3. Under the criteria of bio-productivity and lipid productivity, urea was the optimal nitrogen source. Among different urea concentrates, biomass productivity and lipid content of S. rubescens cultivated in 0.27 g/L urea medium were optimized at 8.8 g/(m2 day) and 31.1%, respectively. With attached cultivation, the highest biomass of 9.4 g/m2 was obtained at NFTs of 4 days. These results showed that culturing S. rubescens using urea as sole nitrogen source by improving nitrogen uptake with attached cultivation is more efficient.

Combined production of fucoxanthin and EPA from two diatom strains Phaeodactylum tricornutum and Cylindrotheca fusiformis cultures.

Fucoxanthin and eicosapentaenoic acid (EPA) provide significant health benefits for human population. Diatom is a potential natural livestock for the combined production of EPA and fucoxanthin. In this study, first, the effects of three important parameters including light intensity, nitrogen concentration and salinity were evaluated for the production of EPA and fucoxanthin in two diatom strains Phaeodactylum tricornutum and Cylindrotheca fusiformis. And then, two steps method based on light intensity were applied to produce EPA and fucoxanthin in large scale. Higher light intensity was first adopted for the high growth rate and lipid content of diatom, and after a period of time, light intensity was lowered to enhance the accumulation of fucoxanthin and EPA. In final, the highest EPA yields were 62.55 and 27.32 mg L-1 for P. tricornutum and C. fusiformis, and the fucoxanthin yield reached 8.32 and 6.05 mg L-1, respectively.

Modeling of carbon dioxide mass transfer behavior in attached cultivation photobioreactor using the analysis of the pH profiles.

The CO2 mass transfer model associated with growth kinetics of microalgal biofilm in attached cultivation photobioreactor was developed and verified by using the analysis of pH profiles which were in equilibrium with inorganic carbon components concentrations (CO2, H2CO3, HCO3- and CO32-) in medium. Model simulation results showed that the model well presented the biofilm growth process. The overall volumetric mass transfer coefficient of CO2 was more influenced by CO2 concentration in aerated gas but less by gas aeration rate and medium circulation rate. Other bio-kinetic parameters related with the microalgal biofilm such as CO2 diffusion coefficient in biofilm, Monod maximum utilization rate of CO2, lag phase duration of biofilm and half-saturation CO2 concentration in the biofilm were independent on operational conditions. The pH profiles provided a way to monitor the variations of inorganic carbon concentrations of medium and to regulate the cultivation of attached microalgal biofilm by CO2 supplement.

Lipid accumulation and metabolic analysis based on transcriptome sequencing of filamentous oleaginous microalgae Tribonema minus at different growth phases.

Filamentous oleaginous microalgae specie Tribonema minus is a promising feedstock for biodiesel production. However, the metabolic mechanism of lipid production in this filamentous microalgal specie remains unclear. Here, we compared the lipid accumulation of T. minus at different growth phases, and described the de novo transcriptome sequencing and assembly and identified important pathways and genes involved in TAG production. Total lipid increased by 2.5-fold and its TAG level in total lipid reached 81.1% at stationary phase. Using the genes involved in the lipid metabolism, the TAG biosynthesis pathways were generated. Moreover, results also demonstrated that, in addition to the observed overexpression of the fatty acid synthesis pathway, TAG production at stationary growth phase was bolstered by repression of the ß-oxidation pathway, up-regulation of genes that funnels acetyl-CoA to lipid biosynthesis, especially gene encoding for phospholipid:diacylglycerol acyltransferase (PDAT) which funnels DAG to TAG biosynthesis.

Feasibility of attached cultivation for polysaccharides production by Porphyridium cruentum.

Porphyridium cruentum is one of the most valued microalgae species able to produce both pigments and exopolysaccharides. Conventional liquid suspended cultivation in ponds and photobioreactors show its disadvantages in lower cultivation efficiency and higher stirring power consumption due to the high viscosity of the medium by the accumulation of polysaccharides. In this work, a new method of culture (called attached cultivation) based on the growth of microalgae using a supporting surface was successfully applied to the cultivation of P. cruentum and the effect of the main influential parameters on its growth rate and polysaccharides production has been investigated. Higher values of these factors resulted in a faster growth rate and, in particular, optimum values of 6.98 g m-2 for initial biomass density, 100 µmol m-2 s-1 for light intensity, continuous illumination, 2.0 % for CO2 concentration, and 0.1 v v-1 min-1 for aeration rate produced the best polysaccharide production of 42 % dry weight. The nutrition profile of P. cruentum obtained in attached and suspended cultivations was similar. Overall these results demonstrate that the attached cultivation is a promising technique which greatly improves the growth rate of P. cruentum as well as its production of polysaccharides and, therefore, it is worth enhancing to be exploited for commercial application.

Growth and palmitoleic acid accumulation of filamentous oleaginous microalgae Tribonema minus at varying temperatures and light regimes.

Palmitoleic acid (C16:1Δ9), contributes greatly to human health, industrial chemicals and biodiesel. The filamentous oleaginous microalgae Tribonema sp. has been identified as a highly efficient producer of palmitoleic acid. Temperature and light regime were adapted to regulate the palmitoleic acid content in this study. Strain T. minus was able to grow well at all the tested temperatures, even at 5 °C. The optimum temperature for palmitoleic acid accumulation (54.25 % of total fatty acid) was 25 °C. Moreover, both light intensity and photoperiod affect the growth, lipid content and fatty acid files of T. minus. The culture exposed to 240 µmol photons m(-2) s(-1) with a photoperiod of 24:0 showed the highest biomass (6.87 g L(-1)) and biggest lipid content (61.27 % of dry weight), whereas the most amount of palmitoleic acid (50.47 % of total fatty acid) was detected at 120 µmol photons m(-2) s(-1). These findings make tangible contributions to culture T. minus for commercial production of lipid or palmitoleic acid.

Aeration strategy for biofilm cultivation of the microalga Scenedesmus dimorphus.

OBJECTIVE: Biofilm cultivation of microalgae may be useful for biofuel production. However, many aspects for this cultivation method have not been well assessed. Accordingly, aeration strategy for biofilm cultivation of Scenedesmus dimorphus has been explored. RESULTS: Biomass, lipid and triacylglycerol (TAG) productivity in increased S. dimorphus as the CO2 concentration increased within 0.038-0.5% and kept constant with further increases. The biomass, lipid and TAG productivity increased with the speed increasing and an obvious threshold point was observed at 6.6 ml(-2) min(-1). The lipid and TAG content was unaffected by the aeration rate. CONCLUSIONS: Both the CO2 concentration as well as aeration speed affected the growth of S. dimorphus in biofilm cultivation. The optimized aeration strategy for biofilm cultivation was continuous air flow enriched with 1% CO2 (v/v) at 6.6 ml(-2) min(-1).

The water footprint of biofilm cultivation of Haematococcus pluvialis is greatly decreased by using sealed narrow chambers combined with slow aeration rate.

OBJECTIVE: Biofilm cultivation of microalgae has great potential in many applications. However, the water footprint for this method has not been well assessed. This issue was explored with the microalga Haematococcus pluvialis. RESULTS: Only 1.25 l water is sufficient to support 1 m(2) biofilm cultivation surface. To produce 1 kg Haematococcus biomass and astaxanthin, the water footprint could be as low as 35.7 and 1440 l, respectively, by sealing the biofilm in a narrow chamber and supplying the proper amount of nutrients if the evaporation water loss was not considered. However, when loss of water by evaporation was considered, the water footprint was as low as 66.9 and 2700 l, respectively, if the chamber was aerated with CO2 at 0.014 vvm. These water footprint values are much lower than values obtained in other research work. CONCLUSIONS: The water footprint of biofilm microalgal cultivation can be potentially reduced by more than 90% if the biofilm is sealed in a narrow chamber and supplied with a slow aeration of CO2 as carbon source.

The growth of Scenedesmus sp. attachment on different materials surface.

BACKGROUND: Microalgae has been concerned as a potential source of biodiesel in the recent years. However, it is costly to harvest microalgae as it is commonly cultured in water and the cells are too small to harvest. In order to reduce the cost of cultivation and harvesting, it is important to improve the biomass productivity of microalgae. Here, we utilized the attachment method to culture microalgae to cut off the cost of culture and harvest. RESULTS: In this paper, various supporting surface with different hydrophility including polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN), polysulfone (PS), which are not easy to be degraded in the culture medium, were used for microalgae culture by the attachment method. The results showed that PVDF supporting cloth was suitable for the algae growth, and its average biomass productivity was to 4.0 g/m2/day. Furthermore, a series of PVDF concentrations were tested, and cloth treated with 3% or 5% PVDF solution was better for the algae culture. In addition, Polyvinylpyrrolidone (PVP) with different molecular weight was added to the PVDF solution as porogens to produce rough surface. And the addition of PVP resulted in better growth with 6.0 g/m2/day of average biomass productivity. CONCLUSION: This attachment method makes the harvest of microalgae easy and energy-saving, because the microalgae grow on the supporting material and is easy to be scraped. The results indicate that the PVDF-treated cloth is a potential alternative for the microalgae attachment culture.

Biofilm cultivation of the oleaginous microalgae Pseudochlorococcum sp.

The cultivation of microalgae in biofilm has been a potential way to overcome the shortcoming of conventional algal culture modes of open pond and photobioreactors in liquid suspension. However, the growth characteristics and related effect factors of the biofilm are still far from being understood. In this work, oleaginous microalgae species Pseudochlorococcum was cultured in an attached biofilm and influential factors on the growth rate of biofilm were investigated. The results showed that Pseudochlorococcum sp. preferred to accumulate more biomass on hydrophilic substrata than on hydrophobic one. The photon flux density of 100 µmol m(-2 )s(-1) was its light saturation point. The optimal inoculum density was about 3-5 g m(-2). The appropriate concentrations of nitrogen, phosphorus in medium and CO(2) in aerated gas were determined as 8.8, 0.22 mmol L(-1) and 1 %, respectively.

Mutagenesis and fluorescence-activated cell sorting of oleaginous Saccharomyces cerevisiae and the multi-omics analysis of its high lipid accumulation mechanisms.

Acquiring lipid-producing strains of Saccharomyces cerevisiae is necessary for producing high-value palmitoleic acid. This study sought to generate oleaginous S. cerevisiae mutants through a combination of zeocin mutagenesis and fluorescence-activated cell sorting, and then to identify key mutations responsible for enhanced lipid accumulation by multi-omics sequencing. Following three consecutive rounds of mutagenesis and sorting, a mutant, MU310, with the lipid content of 44%, was successfully obtained. Transcriptome and targeted metabolome analyses revealed that a coordinated response involving fatty acid precursor biosynthesis, nitrogen metabolism, pentose phosphate pathway, ethanol conversion, amino acid metabolism and fatty acid ß-oxidation was crucial for promoting lipid accumulation. The carbon fluxes of acetyl-CoA and NADPH in lipid biosynthesis were boosted in these pathways. Certain transcriptional regulators may also play significant roles in modulating lipid biosynthesis. Results of this study provide high-quality resource for palmitoleic acid production and deepen the understanding of lipid synthesis in yeast.

Identification and Functional Analysis of Acyl-Acyl Carrier Protein Δ9 Desaturase from Nannochloropsis oceanica.

The oleaginous marine microalga Nannochloropsis oceanica strain IMET1 has attracted increasing attention as a promising photosynthetic cell factory due to its unique excellent capacity to accumulate large amounts of triacylglycerols and eicosapentaenoic acid. To complete the genomic annotation for genes in the fatty acid biosynthesis pathway of N. oceanica, we conducted the present study to identify a novel candidate gene encoding the archetypical chloroplast stromal acyl-acyl carrier protein Δ9 desaturase. The full-length cDNA was generated using rapid-amplification of cDNA ends, and the structure of the coding region interrupted by four introns was determined. The RT-qPCR results demonstrated the upregulated transcriptional abundance of this gene under nitrogen starvation condition. Fluorescence localization studies using EGFP-fused protein revealed that the translated protein was localized in chloroplast stroma. The catalytic activity of the translated protein was characterized by inducible expression in Escherichia coli and a mutant yeast strain BY4389, indicating its potential desaturated capacity for palmitoyl-ACP (C16:0-ACP) and stearoyl-ACP (C18:0-ACP). Further functional complementation assay using BY4839 on plate demonstrated that the expressed enzyme restored the biosynthesis of oleic acid. These results support the desaturated activity of the expressed protein in chloroplast stroma to fulfill the biosynthesis and accumulation of monounsaturated fatty acids in N. oceanica strain IMET1.

Pretreatment and enzymatic hydrolysis optimization of lignocellulosic biomass for ethanol, xylitol, and phenylacetylcarbinol co-production using Candida magnoliae.

Cellulosic bioethanol production generally has a higher operating cost due to relatively expensive pretreatment strategies and low efficiency of enzymatic hydrolysis. The production of other high-value chemicals such as xylitol and phenylacetylcarbinol (PAC) is, thus, necessary to offset the cost and promote economic viability. The optimal conditions of diluted sulfuric acid pretreatment under boiling water at 95°C and subsequent enzymatic hydrolysis steps for sugarcane bagasse (SCB), rice straw (RS), and corn cob (CC) were optimized using the response surface methodology via a central composite design to simplify the process on the large-scale production. The optimal pretreatment conditions (diluted sulfuric acid concentration (% w/v), treatment time (min)) for SCB (3.36, 113), RS (3.77, 109), and CC (3.89, 112) and the optimal enzymatic hydrolysis conditions (pretreated solid concentration (% w/v), hydrolysis time (h)) for SCB (12.1, 93), RS (10.9, 61), and CC (12.0, 90) were achieved. CC xylose-rich and CC glucose-rich hydrolysates obtained from the respective optimal condition of pretreatment and enzymatic hydrolysis steps were used for xylitol and ethanol production. The statistically significant highest (p ≤ 0.05) xylitol and ethanol yields were 65% ± 1% and 86% ± 2% using Candida magnoliae TISTR 5664. C. magnoliae could statistically significantly degrade (p ≤ 0.05) the inhibitors previously formed during the pretreatment step, including up to 97% w/w hydroxymethylfurfural, 76% w/w furfural, and completely degraded acetic acid during the xylitol production. This study was the first report using the mixed whole cells harvested from xylitol and ethanol production as a biocatalyst in PAC biotransformation under a two-phase emulsion system (vegetable oil/1 M phosphate (Pi) buffer). PAC concentration could be improved by 2-fold compared to a single-phase emulsion system using only 1 M Pi buffer.

Impacts of surface wettability and roughness of styrene-acrylic resin films on adhesion behavior of microalgae Chlorella sp.

Biofilm attached cultivation is a promising method for efficient production of microalgae. Determining the surface property index to select an appropriate substrate benefiting the algae adhesion and biofilm formation is very important for the cultivation method. This work focused on elucidating and quantifying the influence of surface wettability and roughness of substrate on Chlorella vulgaris adhesion. Firstly, surface modified styrene-acrylic (SA) resin films by adding different dosage of perfluoroalkyl ethyl acrylate (FM) were prepared. Property characterization shows that the surface contact angle in water, formamide and diiodomethane of FM modified SA films is significantly associated with the FM dosage, while the other surface properties including zeta potential, surface potential and surface roughness have insignificant difference. The calculated surface free energy parameters show that the SA films belong to the non-polar substrata. A well quantitative correlation that the adhesion capacity of C. vulgaris linearly declines with the increase of water contact angle was obtained. And a near linear relationship between the adhesion capacity and the surface free energy (γ), or the cohesion free energy (ΔGcoh) was also observed. Secondly, the surface roughness solely changed SA films were prepared by replicating the morphology of stainless steel sieves through the PDMS template method. The patterned SA films have alternately arranged rectangular "valleys" and "ridges". A well linear correlation between the microalgae adhesion capacity and the surface roughness was also obtained.

Modeling and improving arrayed microalgal biofilm attached culture system.

A microalgal biofilm-attached-system is an alternative cultivation method, that offers potential advantages of improved biomass productivity, efficient harvesting, and water saving. These biofilm systems have been widely tested and utilized for microalgal biomass production and wastewater treatment. This research a microalgal growth model for the biofilm attached culture system has been developed and experimentally validated, both, in single and arrayed biofilm systems. It has been shown that the model has the capability to accurately describe microalgae growth. Moreover, via the model simulation, it was observed that system structural parameters, light dilution rate, and light intensity significantly affected the culture performance. The limitations, and improvement aspects of the model, are also discussed in this study. To our knowledge, this is the first time that a mathematical model for an arrayed-biofilm-attached-system has been developed and validated. This model will certainly be helpful in the design, improvement, optimization, and evaluation of the biofilm-attached-systems.