Development of PMA-qPCR assay to accurately and reproducible quantify viable bacteria of Paenibacillus polymyxa.

Paenibacillus polymyxa is an important biocontrol bacterium. The combination of propidium monoazide (PMA) and quantitative polymerase chain reactionq (qPCR) has proven effective in quantifying live bacteria from various microorganisms. The objective was to create a PMA-qPCR assay to precisely and consistently measure the number of living bacteria of biocontrol P. polymyxa. The primers were designed for the spo0A gene of P. polymyxa HY96-2. The optimal conditions for treating the target strain with PMA were a PMA concentration of 15 µg/mL, an incubation time of 5 min, and an exposure time of 10 min. The PMA-qPCR method had a limit of quantification (LOQ) of 1.0 × 103 CFU/mL for measuring the amount of viable P. polymyxa bacteria. The PMA-qPCR method is more sensitive than the qPCR method in detecting viable bacteria in the mixtures of viable and dead bacteria. The accuracy and reproducibility of quantifying viable P. polymyxa bacteria using the PMA-qPCR method were higher compared to the plate count method.

Controlling average number of photons received per biomass to promote the growth of Synechocystis sp. PPC 6803.

To investigate the actually received light of cells in the photo bioreactor, a light attenuation model of Synechocystis sp. PCC 6803 was established. The relationship between the average number of photons received per biomass (APRPB) and the growth of cell was analyzed. The results demonstrated, Cornet model was accurately fitted with the light attenuation of Synechocystis sp. PCC 6803 and the cell growth rate was affected by APRPB. When the value of APRPB is 3.2 µmol g-1 s-1, the cell have the maximum light efficiency. A maximum specific growth rate of 0.05 h-1 was achieved with APRPB from 3.2 to 12.8 µmol g-1 s-1. After 156 h cultivation, compared to cells cultured under constant light [light intensity: 100 and 1800 µmol/(m2 s)], the DCW under controlled light intensity (light intensity increasing with the cell density) was higher by 79.1% and 20.0%, respectively. This study indicated that APRPB could be used as a light intensity regulation criterion to improve cell production despite different types of reactor and cell density, which provided a theoretical basis for improving the biomass yield of Synechocystis sp. PCC 6803 or other photosynthetic auto-trophic organism.

Genomic Foundation of Starch-to-Lipid Switch in Oleaginous Chlorella spp.

The ability to rapidly switch the intracellular energy storage form from starch to lipids is an advantageous trait for microalgae feedstock. To probe this mechanism, we sequenced the 56.8-Mbp genome of Chlorella pyrenoidosa FACHB-9, an industrial production strain for protein, starch, and lipids. The genome exhibits positive selection and gene family expansion in lipid and carbohydrate metabolism and genes related to cell cycle and stress response. Moreover, 10 lipid metabolism genes might be originated from bacteria via horizontal gene transfer. Transcriptomic dynamics tracked via messenger RNA sequencing over six time points during metabolic switch from starch-rich heterotrophy to lipid-rich photoautotrophy revealed that under heterotrophy, genes most strongly expressed were from the tricarboxylic acid cycle, respiratory chain, oxidative phosphorylation, gluconeogenesis, glyoxylate cycle, and amino acid metabolisms, whereas those most down-regulated were from fatty acid and oxidative pentose phosphate metabolism. The shift from heterotrophy into photoautotrophy highlights up-regulation of genes from carbon fixation, photosynthesis, fatty acid biosynthesis, the oxidative pentose phosphate pathway, and starch catabolism, which resulted in a marked redirection of metabolism, where the primary carbon source of glycine is no longer supplied to cell building blocks by the tricarboxylic acid cycle and gluconeogenesis, whereas carbon skeletons from photosynthesis and starch degradation may be directly channeled into fatty acid and protein biosynthesis. By establishing the first genetic transformation in industrial oleaginous C. pyrenoidosa, we further showed that overexpression of an NAD(H) kinase from Arabidopsis (Arabidopsis thaliana) increased cellular lipid content by 110.4%, yet without reducing growth rate. These findings provide a foundation for exploiting the metabolic switch in microalgae for improved photosynthetic production of food and fuels.

A new paradigm for producing astaxanthin from the unicellular green alga Haematococcus pluvialis.

The unicellular green alga Haematococcus pluvialis has been exploited as a cell factory to produce the high-value antioxidant astaxanthin for over two decades, due to its superior ability to synthesize astaxanthin under adverse culture conditions. However, slow vegetative growth under favorable culture conditions and cell deterioration or death under stress conditions (e.g., high light, nitrogen starvation) has limited the astaxanthin production. In this study, a new paradigm that integrated heterotrophic cultivation, acclimation of heterotrophically grown cells to specific light/nutrient regimes, followed by induction of astaxanthin accumulation under photoautotrophic conditions was developed. First, the environmental conditions such as pH, carbon source, nitrogen regime, and light intensity, were optimized to induce astaxanthin accumulation in the dark-grown cells. Although moderate astaxanthin content (e.g., 1% of dry weight) and astaxanthin productivity (2.5 mg L(-1) day(-1) ) were obtained under the optimized conditions, a considerable number of cells died off when subjected to stress for astaxanthin induction. To minimize the susceptibility of dark-grown cells to light stress, the algal cells were acclimated, prior to light induction of astaxanthin biosynthesis, under moderate illumination in the presence of nitrogen. Introduction of this strategy significantly reduced the cell mortality rate under high-light and resulted in increased cellular astaxanthin content and astaxanthin productivity. The productivity of astaxanthin was further improved to 10.5 mg L(-1) day(-1) by implementation of such a strategy in a bubbling column photobioreactor. Biochemical and physiological analyses suggested that rebuilding of photosynthetic apparatus including D1 protein and PsbO, and recovery of PSII activities, are essential for acclimation of dark-grown cells under photo-induction conditions. Biotechnol. Bioeng. 2016;113: 2088-2099. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Investigation on novel raceway pond with inclined paddle wheels through simulation and microalgae culture experiments.

The open raceway ponds are nowadays the most used large-scale reactors for microalgae culture. To avoid the stacking of microalgae, the paddle wheels are the most widely used to circulate and mix the culture medium. In this paper, a numerical simulation using computational fluid dynamics (CFD) was used to investigate the hydrodynamic characteristics of open raceway ponds with different types of paddle wheels (the traditional paddle wheels and the novel paddle wheels with specially inclined angle of the blades). The particle image velocimetry (PIV) was used to validate the reliability of the CFD model. The CFD simulation results showed that the novel raceway pond with 15° inclined angle of the blades had the best mixing efficiency under the same power consumption. Lastly, the results of microalgae culture experiments showed that the growth rates of Chlorella pyrenoidosa in the novel raceway pond with 15° inclined angle of the blades were higher than those in the traditional reactor. The results of the culture experiments and CFD simulations were identical with each other. Therefore, a novel paddle wheel with 15° inclined angle of the blades was obtained for better microalgae cultivation.

Impacts of CO2 concentration on growth, lipid accumulation, and carbon-concentrating-mechanism-related gene expression in oleaginous Chlorella.

Biodiesel production by microalgae with photosynthetic CO2 biofixation is thought to be a feasible way in the field of bioenergy and carbon emission reduction. Knowledge of the carbon-concentrating mechanism plays an important role in improving microalgae carbon fixation efficiency. However, little information is available regarding the dramatic changes of cells suffered upon different environmental factors, such as CO2 concentration. The aim of this study was to investigate the growth, lipid accumulation, carbon fixation rate, and carbon metabolism gene expression under different CO2 concentrations in oleaginous Chlorella. It was found that Chlorella pyrenoidosa grew well under CO2 concentrations ranging from 1 to 20 %. The highest biomass and lipid productivity were 4.3 g/L and 107 mg/L/day under 5 % CO2 condition. Switch from high (5 %) to low (0.03 %, air) CO2 concentration showed significant inhibitory effect on growth and CO2 fixation rate. The amount of the saturated fatty acids was increased obviously along with the transition. Low CO2 concentration (0.03 %) was suitable for the accumulation of saturated fatty acids. Reducing the CO2 concentration could significantly decrease the polyunsaturated degree in fatty acids. Moreover, the carbon-concentrating mechanism-related gene expression revealed that most of them, especially CAH2, LCIB, and HLA3, had remarkable change after 1, 4, and 24 h of the transition, which suggests that Chlorella has similar carbon-concentrating mechanism with Chlamydomonas reinhardtii. The findings of the present study revealed that C. pyrenoidosa is an ideal candidate for mitigating CO2 and biodiesel production and is appropriate as a model for mechanism research of carbon sequestration.

Comparing the performances of circular ponds with different impellers by CFD simulation and microalgae culture experiments.

In this study, a numerical simulation using computational fluid dynamics (CFD) was used to investigate the hydrodynamic characteristics of circular ponds with three different impellers (hydrofoil, four-pitched-blade turbine, and grid plate). The reliability of the CFD model was validated by particle image velocimetry (PIV). Hydrodynamic analyses were conducted to evaluate the average velocity magnitude along the light direction (Uz), turbulence properties, average shear stress, pressure loss and the volume percentage of dead zone inside circular ponds. The simulation results showed that Uz value of hydrofoil was 58.9, 40.3, and 28.8% higher than those of grid plate with single arm, grid plate with double arms and four-pitched blade turbines in small-scale circular ponds, respectively. In addition, hydrofoil impeller with down-flow operation had outstanding mixing characteristics. Lastly, the results of Chlorella pyrenoidosa cultivation experiments indicated that the biomass concentration of hydrofoil impeller with down-flow operation was 65.2 and 88.8% higher than those of grid plate with double arms and four-pitched-blade turbine, respectively. Therefore, the optimal circular pond mixing system for microalgae cultivation involved a hydrofoil impeller with down-flow operation.

Artificial neural networks prediction and optimization based on four light regions for light utilization from Synechocystis sp. PCC 6803.

Light is crucial in microalgae growth. However, dividing the microalgae growth region into light and dark regions has limitations. In this study, the light response of Synechocystis sp. PCC 6803 was investigated to define four light regions (FLRs): light compensation region, light limitation region, light saturation region, and photoinhibition region. The proportions of cells' residence time in the FLRs and the number of times cells (NTC) passed through the FLRs in photobioreactors were calculated by using MATLAB. Based on the FLRs and NTC passed through the FLRs, a growth model was established by using artificial neural network (ANN).The ANN model had a validation R2 value of 0.97, which was 76.36% higher than the model based on light-dark regions. The high accuracy of the ANN model was further verified through dynamic adjustment of light intensity experiments.This study confirmed the importance of the FLRs for studying microalgae growth dynamics.

[Advances of studies on culture and product functions of Euglena gracilis].

Euglena gracilis is a unicellular eukaryote between animal and plant cells, which is widely distributed in nature. E. gracilis has both plant and animal characteristics, and can grow photoautotrophically, heterotrophically and mixotrophically. E. gracilis also features on abundant and various cellular composition. Recently, extensive researches on unique cellular components of E. gracilis have revealed its application in the field of medicine, food, and feedstuff, in terms of improving immunity, fighting inflammation, and lowering uric acid levels. The application prospects of paramylon in biomedical area were also discovered. As food ingredients, food additives, feedstuffs and cosmetic ingredients, E. gracilis has been certified domestically and overseas. A series of products have been developed overseas, especially in Japan. However, the research and development of E. gracilis are still in its infancy in China, and there is huge space for development. At present, the research and potential application of cultivation and product functions of E. gracilis have been rarely reviewed. This review systematically examines both the domestic and abroad research of cultivation and production of E. gracilis, as well as the biological activity of E. gracilis powder and paramylon. The existing problems in the application, exploitation, and possible development direction of E. gracilis in the future are prospected. This review might be useful for establishing and optimizing large-scale and efficient heterotrophic technology, as well as developing related products of E. gracilis with specific functions.

Multi-stage development process and model of steam chamber for SAGD production in a heavy oil reservoir with an interlayer.

Steam-assisted gravity drainage (SAGD) is an efficient thermal recovery technique for oil sands and extra heavy oil exploitation. The development of steam chamber goes through multi-stage physical processes for SAGD production in a heavy oil reservoir with an interlayer. In this study, considering the situation that an interlayer is located directly above a pair of horizontal wells, we analyzed the whole process of steam chamber development. We divided the whole process into stages I-V, which are the first rising stage, the first lateral expansion stage, the second rising stage, the second lateral expansion stage and the confinement stage, respectively. Particularly, we further divided stage II into 2 periods and stage IV into 3 periods. These stages and periods can help us understand the development process of steam chamber dominated by an interlayer more profoundly. Based on the divided stages and periods, we established different models of SAGD production by assuming different geometric shapes of steam chamber in different stages and periods. Oval shape was assumed in stages I and III, and inverse triangle shape was hypothesized in stages II, IV and V. The formulas of the front distance of steam chamber and the oil production rate of SAGD were deduced from the established models for different development stages. At the end, we performed two example applications to SAGD production in heavy oil reservoirs with an interlayer. The real oil production rates were matched very well with the theoretical oil production rates calculated by the deduced formulas, which implies the multi-stage development model of steam chamber is of reliability and utility.

[Effects of directional adaptation on selenium tolerance and accumulation of heterotrophic Chlorella pyrenoidosa].

Selenium (Se) is an essential trace element for organisms. Se deficiency will cause diseases such as Keshan disease and Kashin-Beck in human being, and huge loss to animal husbandry. Currently available Se supplements have such problems as low Se content, poor bioavailability, and poor safety. Chlorella pyrenoidosa can produce bioavailable and safe organic Se under suitable conditions, which is thus a promising Se supplement. Therefore, in this study, we tried to improve the Se tolerance and accumulation of C. pyrenoidosa by directional adaptation. To be specific, we gradually increased the concentration of Na2SeO3 in medium to domesticate C. pyrenoidosa and optimized the adapting time and concentration gradient of Na2SeO3 during the adaptation. The results showed that the adapted C. pyrenoidosa was more tolerant to Se and had stronger Se enrichment ability. In 5 L fermenter, the adapted strains could tolerate 40 mg/L Na2SeO3 and the synthesis rate of organic Se was 175.6% higher. Then, Se addition method in the 5 L fermenter was optimized. The result demonstrated that addition of Na2SeO3 at 40 mg/L during heterotrophic culture achieved the final dry weight of C. pyrenoidosa cells at 106.4 g/L, content of organic Se at 1 227 mg/kg, and synthesis rate of organic Se at 1.36 mg/(L·h). Compared with the reported highest cell density of 75 g/L and the highest organic Se content of 560 mg/kg, the corresponding figures in this study were 41.9% and 119.1% higher, respectively. In conclusion, directional adaptation can remarkably improve the Se tolerance and enrichment of C. pyrenoidosa.

A new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of marine-derived Bacillus velezensis B-9987.

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24-48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 µg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3-4 h and viable B-9987 spores in marine Bacillus WP within 4-6 h.

The optimization of centrifugal pump driving horizontal tubular photobioreactor for enhancing astaxanthin production using heterotrophic Haematococcus pluvialis.

Haematococcus pluvialis is the prime source of natural astaxanthin for commercial exploitation. The large-scale cultivation of H. pluvialis is one of the key technologies for the development of natural astaxanthin industry. So far, horizontal tubular photobioreactor (HTPBR) circulated by a centrifugal pump has been the main PBR for the large-scale cultivation of H. pluvialis. Shear stress is a negative factor in microalgal cultivation at different scales, particularly for large-scale cultivation. To reduce the adverse impact of shear stress, the tolerance of H. pluvialis to the shear stress during the induction stage was first investigated in this study. H. pluvialis aplanospore was not sensitive to stresses between 19.18 and 27.32 Pa, but was resulted in about 30% cell death under shear stress between 27.32 and 63.84 Pa. Accordingly, two centrifugal pumps with different impellers was selected in 400 L HTPBRs to study the outdoor photoinduction for astaxanthin accumulation. The highest astaxanthin productivity and astaxanthin concentration were obtained in HTPBRs using a centrifugal pump equipped with three unshrouded backward-bladed impellers. The HTPBR was then successfully scaled up to 800 L with a similar performance, showing good scalability.

Two new antifungal cyclic lipopeptides from Bacillus marinus B-9987.

Two new cyclic lipopeptides maribasins A (1) and B (2) were isolated from the fermentation broth of the marine microorganism Bacillus marinus B-9987 isolated from Suaeda salsa in Bohai coastline of P. R. China. Both structures were established to be cyclo (D-Pro-L-Gln-L-Asn-L-Ser-D-Asn¹-D-Tyr-D-Asn²-D-ß-aminoisopentadecanoic acid) (1) and cyclo (D-Pro-L-Gln-L-Asn-L-Ser-D-Asn¹-D-Tyr-D-Asn²-D-ß-aminoanteisopentadecanoic acid) (2) by spectroscopic analysis and exhibited broad-spectrum activity against phytopathogens by the antifungal bioassay.

[Antifungal mechanism of Bacillus marinus B-9987].

OBJECTIVE: We studied the antifungal effect of the metabolite BMME-1 from Bacillus marinus B-9987, to reveal its antifungal mechanism. METHODS: The permeability of the Alternaria solani was tested by spectrophotometer after the treating the crude extracts of B-9987. The composition of cell wall and the sterol components of the fungal plasmalemma of Alternaria solani were analyzed with Infrared Spectrum and GC-MS, respectively. RESULTS: We found that the metabolites of B-9987 had strong antifungal activity with MIC50 and MFC value being 6.2mg/L and 50mg/L. The absorbance in extracellular fluid detection showed that the tegument of the fungi was impaired. The detection of glucan and chitin indicated the change in the structure of the cell wall. The absorption peak of the carbon-hydrogen bond, beta-glucosidic bond, carbon--oxygen bond was attenuated but the hydroxyl, carbonyl absorption was enhanced on the contrary. There were only one peak change in chitin chromatogram on the absorption of amide linkage comparing to the control. These changes on the structure may affect the stability of the fungal cell wall. Ergosterol was the predominant component of sterol with the proportion of 62.52 +/- 3.31% in control cells, but showed a decline during treatment with BMME-1 at a concentration of 56.36 +/- 2.52%. Accumulation of coprostanol, the precursor of ergosterol, was found in the test. CONCLUSION: From the result we can conclude that the antifungal mechanism of the crude extracts was interfering of ergosterol synthesis resulting in the change on permeability, and also mainly changed the structure of the cell wall, mainly acting on the glucan synthesis.

Complete Genome Sequence of Industrial Biocontrol Strain Paenibacillus polymyxa HY96-2 and Further Analysis of Its Biocontrol Mechanism.

Paenibacillus polymyxa (formerly known as Bacillus polymyxa) has been extensively studied for agricultural applications as a plant-growth-promoting rhizobacterium and is also an important biocontrol agent. Our team has developed the P. polymyxa strain HY96-2 from the tomato rhizosphere as the first microbial biopesticide based on P. polymyxa for controlling plant diseases around the world, leading to the commercialization of this microbial biopesticide in China. However, further research is essential for understanding its precise biocontrol mechanisms. In this paper, we report the complete genome sequence of HY96-2 and the results of a comparative genomic analysis between different P. polymyxa strains. The complete genome size of HY96-2 was found to be 5.75 Mb and 5207 coding sequences were predicted. HY96-2 was compared with seven other P. polymyxa strains for which complete genome sequences have been published, using phylogenetic tree, pan-genome, and nucleic acid co-linearity analysis. In addition, the genes and gene clusters involved in biofilm formation, antibiotic synthesis, and systemic resistance inducer production were compared between strain HY96-2 and two other strains, namely, SC2 and E681. The results revealed that all three of the P. polymyxa strains have the ability to control plant diseases via the mechanisms of colonization (biofilm formation), antagonism (antibiotic production), and induced resistance (systemic resistance inducer production). However, the variation of the corresponding genes or gene clusters between the three strains may lead to different antimicrobial spectra and biocontrol efficacies. Two possible pathways of biofilm formation in P. polymyxa were reported for the first time after searching the KEGG database. This study provides a scientific basis for the further optimization of the field applications and quality standards of industrial microbial biopesticides based on HY96-2. It may also serve as a reference for studying the differences in antimicrobial spectra and biocontrol capability between different biocontrol agents.

Dolyemycins A and B, two novel cyclopeptides isolated from Streptomyces griseus subsp. griseus HYS31.

Two novel cyclopeptides with special skeleton, namely, dolyemycins A (1) and B (2) were isolated from Streptomyces griseus subsp. griseus HYS31 by bio-guided isolation. Their structures were elucidated by detailed analysis of spectroscopic data. These two compounds were cyclopeptides containing eleven amino acids including five unusual amino acids (hydroxyglycine, 3-hydroxyleucine, 3-phenylserine, ß-hydroxy-O-methyltyrosine, 2,3-diaminobutyric acid) in both of them and an extra nonprotein amino acids (3-methylaspartic acid) in Dolyemycin B only. Dolyemycins A and B performed antiproliferative activity against human lung cancer A549 cells with IC50 values of 1.0 and 1.2 µM, respectively.

[Bioassay-guided isolation of functional components from hot water extract of Chlorella pyrenoidosa].

The main functional ingredients of hot water extract of Chlorella pyrenoidosa (CPE) were investigated through a bioassay-guided fractionation based on free radical scavenging and macrophage proliferation effects. The main functional ingredients of CPE were polysaccharides (PS) that were isolated by high pressure extraction, Sevag method, ethanol precipitation and ultrafiltration separation. Crude polysaccharides were further separated and purified by ion exchange chromatography DEAE52 and size exclusion chromatography Sephadex G-100. The purified fractions were analyzed by gel permeation chromatography. Molecular weights of the purified fractions PS-1-4-2, PS-1-3-2 and PS-2-3-3 were 3.97×104, 2.28×104 and 4.1×10³ Da, respectively. Bioassay-guided fractionation results indicated that CPE could remove free radicals and promote Ana-1 cells proliferation, mainly due to its various components working together. The components of free radicals scavenging mainly concentrated in PS-1-3, PS-1-4, PS-2-3 and PS-2-4. The components of Ana-1 proliferation mainly concentrated in PS-1-3, PS-1-4 and PS-2-3. This study established the activity screening method of main functional component from CPE, and got three new functional ingredients. It can be used to guide the development of high value products, further promote the industrialization process of microalgae energy, and realize microalgae 'high value products, microalgae energy and microalgae carbon' integration of exemplary role.

Ghanamycins A and B, two novel γ-butyrolactones from marine-derived streptomyces ghanaensis TXC6-16.

Two novel γ-butyrolactones ghanamycins A (1) and B (2) were isolated from the fermentation broth of marine-derived Streptomyces ghanaensis TXC6-16. Their structures were elucidated by spectroscopic analysis. These two novel compounds exhibited antimicrobial activities against some phytopathogens. The minimum IC (MIC) of 2 against Pseudomonas syringae and Erwinia sp. were 50 µg ml-1.

Development of a novel multi-column airlift photobioreactor with easy scalability by means of computational fluid dynamics simulations and experiments.

Aiming to culture algae with high efficiency, a novel vertical multi-column airlift photobioreactor (VMAPBR) has been developed. It was constructed with a series of vertically arranged parallel columns with easy scalability. The hydrodynamic, irradiation and shear stress characteristics of the photobioreactor were studied by computational fluid dynamics (CFD). Accordingly, the optimal aeration manner and aeration rate were determined. When the novel airlift PBR was alternately aerated with aeration rate of 0.2vvm, the biomass concentration of Chlorella pyrenoidosa under outdoor condition reached 1.30gL-1 within the prototype PBR and was further increased to 1.56gL-1 within the optimized PBR. The result of cultivation experiment had good agreement with that of CFD prediction.