Growth of Dunaliella tertiolecta and associated bacteria in photobioreactors

The aim of this study was to test three flat-plate photobioreactor configurations for cultivation of marine green alga Dunaliella tertiolecta under non-axenic growth conditions and to characterize and quantify the associated bacteria. The photobioreactor cultivations were conducted using tap water-based media. Static mixers intended to enhance mixing and light utilization did not generally increase algal growth at the low light intensities used. The maximum biomass concentration (measured as volatile suspended solids) and maximum specific growth rate achieved in the flat plate with no mixer were 2.9?g?l(-1) and 1.3?day(-1), respectively. Based on quantitative polymerase chain reaction, bacterial growth followed the growth of D. tertiolecta. Based on 16S rDNA amplification and denaturing gradient gel electrophoresis profiling, heterotrophic bacteria in the D. tertiolecta cultures mainly originated from the non-axenic algal inocula, and tap water heterotrophs were not enriched in high chloride media (3?% salinity). Bacterial communities were relatively stable and reproducible in all flat-plate cultivations and were dominated by Gammaproteobacteria, Flavobacteria, and Alphaproteobacteria.     

Enhanced Chlorella vulgaris Buitenzorg growth by photon flux density alteration in serial photobioreactors

Microalgae perform oxygenic photosynthesis and are capable of taking up a large amount of CO₂, using an inducible CO₂ concentrating mechanism (CCM), and fixing CO₂ into higher compounds. These characteristics make the microalgae potentially useful for removal and utilization of CO₂ emitted from industrial plants and, generally, the usage of photosynthetic microorganisms has increased and significantly improved as a solution for CO₂ emissions. In this light and based on previous research using Anabaena cylindrica IAM M1 and Spirulina platensis IAM M 135, enhancement was sought for CO₂ fixation and biomass production by Chlorella vulgaris Buitenzorg by increasing the photon flux density concurrent with increases in culture biomass during the cellular growth phase and was compared to cultures of Chlorella grown at optimal constant illumination, with all cultures grown using Bennick basal medium, 29°C, and a flow of 1.0 atm. 10% CO₂ enriched air delivered to three in serial photobioreactors of 0.200 dm³ capacity each. The results showed that increasing illumination during culture increased biomass production of Chlorella by ∼60% as well as increased CO₂ fixation ability by ∼7.0%. It was also demonstrated that the non-competitive inhibition of [HCO₃ ⁻] as a carbon source significantly affected the cultivation in both the increasing and constant photon flux density regimes.     

Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production

Chlorella vulgaris was cultivated in two different 2.0 L-helicoidal and horizontal photobioreactors at 5 klux using the bicarbonate contained in the medium and ambient air as the main CO₂ sources. The influence of bicarbonate concentration on biomass growth as well as lipid content and profile was first investigated in shake flasks, where the stationary phase was achieved in about one half the time required by the control. The best NaHCO₃ concentration (0.2 g L⁻¹) was then used in both photobioreactors. While the fed-batch run performed in the helicoidal photobioreactor provided the best result in terms of biomass productivity, which was (84.8 mg L⁻¹ d⁻¹) about 2.5-fold that of the batch run, the horizontal configuration ensured the highest lipid productivity (10.3 mg L⁻¹ d⁻¹) because of a higher lipid content of biomass (22.8%). These preliminary results suggest that the photobioreactor configuration is a key factor either for the growth or the composition of this microalga. The lipid quality of C. vulgaris biomass grown in both photobioreactors is expected to meet the standards for biodiesel, especially in the case of the helicoidal configuration, provided that further efforts will be made to optimize the conditions for its production as a biodiesel source.     

Investigation and modeling of the effects of light spectrum and incident angle on the growth of Chlorella vulgaris in photobioreactors

An in‐depth investigation of how various illumination conditions influence microalgal growth in photobioreactors (PBR) has been presented. Effects of both the light emission spectrum (white and red) and the light incident angle (0° and 60°) on the PBR surface were investigated. The experiments were conducted in two fully controlled lab‐scale PBRs, a torus PBR and a thin flat‐panel PBR for high cell density culture. The results obtained in the torus PBR were used to build the kinetic growth model of Chlorella vulgaris taken as a model species. The PBR model was then applied to the thin flat‐panel PBR, which was run with various illumination conditions. Its detailed representation of local rate of photon absorption under various conditions (spectral calculation of light attenuation, incident angle influence) enabled the model to take into account all the tested conditions with no further adjustment. This allowed a detailed investigation of the coupling between radiation field and photosynthetic growth. Effects of all the radiation conditions together with pigment acclimation, which was found to be relevant, were investigated in depth. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:247–261, 2016     

Growth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris

Structural and functional alterations to the photosynthetic apparatus after growth at low temperature (5[deg]C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5[deg]C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27[deg]C even though growth irradiance was kept constant at 150 [mu]mol m-2 s-1. Concomitant with the increase in the chlorophyll a/b ratio was a lower abundance of light-harvesting polypeptides in 5[deg]C-grown cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and confirmed by western blotting.The differences in pigment composition were found to be alleviated within 12 h of transferring 5[deg]C-grown cells to 27[deg]C. Furthermore, exposure of 5[deg]C-grown cells to a 30-fold lower growth irradiance (5 [mu]mol m-2 s-1) resulted in pigment content and composition similar to that in cells grown at 27[deg]C and 150 [mu]mol m-2 s-1. Although both cell types exhibited similar measuring-temperature effects on CO2-saturated O2 evolution, 5[deg]C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8-and 3.9-fold higher than 27[deg]C-grown cells measured at 27[deg]C and 5[deg]C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem II electron transport of 5[deg]C-grown cells was less temperature sensitive than that of 27[deg]C-grown cells. This appears to be due to an increased capacity to keep the primary, stable quinone electron acceptor of photosystem II (QA) oxidized at low temperature in 5[deg]C- compared with 27[deg]C-grown cells regardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts its photosynthetic apparatus in response to the excitation pressure on photosystem II and not to the absolute external irradiance. We suggest that the redox state of QA may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.     

Metabolic activity,the chemical composition of biomass and photosynthetic activity of Chlorella vulgaris under different light spectra in photobioreactors

Omitting the far‐red in LED lighting for bioreactors is inexplicable because it affects photosynthetic efficiency and photomorphogenetic activity. Therefore, this work compares three light sources (fluorescent—white light (WL), and LED: blue + red—BRL and blue + red + far‐red—BRFRL) for cultures of Chlorella vulgaris. Metabolic activity was determined by isothermal calorimetry. Changes in the chemical composition of biomass were examined by spectrofluorimetry and Raman spectroscopy. Maximum quantum yield of photosystem II was determined on the basis of chlorophyll a fluorescence parameters. The algae grown under BRL produced significantly more thermal energy than those cultured under BRFRL and WL. The Raman spectra of cultures showed characteristic bands for carotenoids, chlorophyll, phenolics, lipids, aliphatic carbohydrates, pectins, and disaccharides. According to the cluster analysis, the chemical composition of cultures grown under BRL and BRFRL was very similar, whereas the WL represented a distinct group. BRL and BRFRL stimulated the biosynthesis of an unidentified compound(s) with fluorescence maximum at 614 nm. At the beginning of the cultivation, photosystem II had very weak photochemical activity. Under BRFRL, ratios of Fv/Fm reached the maximum after 4 days, whereas under BRL and WL, after 6 days of cultivation. The results point to the favorable influence of the far‐red on the metabolism of microalgae.     

植物激素在小球藻异养培养中的作用

本文研究了几种植物激素对小球藻异养培养的影响。结果表明,IAA、IBA及6-BA三种植物激素均不同程度地促进了小球藻的异养生长,培养≤36h时,IAA或IBA以20mg／L的促进小球藻异养生长的效应最大,100mg／L IAA或IBA则抑制了藻的生长;>36h时,100mg／L IAA或IBA表现出促进小球藻生长的效应,并最终获最大净A540增长量;6-BA以0.1 mg／L的促进作用最大。IBA与6-BA组合同样表现出促进小球藻异养生长的效应,但并非IBA和6-BA简单的加合效应,5 mg／LIBA与6-BA组合的效应维持6-BA单因子的作用趋势,20mg／LIBA与lmg/L6-BA组合的效应大于与0.1mg／L6-BA组合的,100mg／LIBA与0.1mg／L6-BA组合的效应在≤36h时大于与1mg／L6-BA组合的,>36h时则相反。另外,高浓度IBA(≥20mg／L)与6-BA组合抑制了前中期异养藻对葡萄糖的吸收,但加速了中后期葡萄的吸收。再者,IBA与6-BA组合加速了异养小球藻对No-3的吸收。     

Morphometric and Stereologic Analysis of Chlorella vulgaris Under Heterotrophic Growth Conditions

A computer-supported determination of stereological parameterswas used to study the possible ultrastructural changes of Chlorellavulgaris UAM 101 under photolithotrophic, mixotrophic and photoheterotrophicconditions of growth. Data recording was carried out througha semi-automatic digitizing image analysis system instead ofthe current method of superimposition of an array of short lines. Glucose promoted drastic physiological changes [Martínezand Orús, 1991. Plant Physiology (in press)], which stronglyaffected the size of the cells and volume densities of storagematerials. However, volumetric ratios of the mitochondrion orchloroplast active fraction were not affected by the presenceof glucose, probably indicating that these ratios are characteristicof each species. Cell wall ultrastructure was also analysed and the presenceof sporopollenin demonstrated, in contrast to the thin and sporopollenin-lackingcell wall generally described for Chlorella vulgaris species. Chlorella vulgaris, photoautotrophic, mixotrophic, photoheterotrophic, image analysis, stereology     

植物激素在小球藻异养培养中的作用

本文研究了几种植物激素对小球藻异养培养的影响。结果表明 ,IAA、IBA及 6_BA三种植物激素均不同程度地促进了小球藻的异养生长 ,培养≤ 36h时 ,IAA或IBA以 2 0mg/L的促进小球藻异养生长的效应最大 ,1 0 0mg/LIAA或IBA则抑制了藻的生长 ;>36h时 ,1 0 0mg/LIAA或IBA表现出促进小球藻生长的效应 ,并最终获最大净A540 增长量 ;6_BA以 0 1mg/L的促进作用最大。IBA与 6_BA组合同样表现出促进小球藻异养生长的效应 ,但并非IBA和 6_BA简单的加合效应 ,5mg/LIBA与 6_BA组合的效应维持 6_BA单因子的作用趋势 ,2 0mg/LIBA与 1mg/L 6_BA组合的效应大于与 0 .1mg/L 6_BA组合的 ,1 0 0mg/LIBA与 0 .1mg/L 6_BA组合的效应在≤ 36h时大于与 1mg/L 6_BA组合的 ,>36h时则相反。另外 ,高浓度IBA(≥ 2 0mg/L)与 6_BA组合抑制了前中期异养藻对葡萄糖的吸收 ,但加速了中后期葡萄的吸收。再者 ,IBA与 6_BA组合加速了异养小球藻对NO_3 的吸收。     

Growth, lipid extraction and thermal degradation of the microalga Chlorella vulgaris

The microalga Chlorella vulgaris was cultured in a combined medium obtained by mixing standard Jaworski medium with a solution from the modified Solvay process that contained only NaHCO(3) and NH(4)Cl. Cell number, pH and nitrogen content were monitored throughout growth. Lipids were extracted from lyophilised biomass using CHCl(3)-MeOH. A combination of grinding, microwave treatment and sonication proved to give the best lipid extract yield. Freeze-dried algal biomass was also utilised for thermal degradation studies. The degradation exhibited three distinct regions - primary cell structure breakage paralleled by evaporation of water, followed by two predominant exothermic degradation processes. The latter were modelled using nth order apparent kinetics. The activation energies of the degradation processes were determined to be 120-126kJ/mol and 122-132kJ/mol, respectively. The degradation model may be readily applied to an assortment of thermal algal processes, especially those relating to renewable energy.     

Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors

Culturing microalgae using natural sunlight is an effective way to reduce the cost of microalgae-based biodiesel production. In order to evaluate the feasibility of culturing Chlorella zofingiensis outdoors for biodiesel production, effects of nitrogen limitation and initial cell concentration on growth and lipid accumulation of this alga were investigated in 60 L flat plate photobioreactors outdoors. The highest μmax and biomass productivity obtained was 0.994 day(-1) and 58.4 mg L(-1)day(-1), respectively. The lipid content was much higher (54.5% of dry weight) under nitrogen limiting condition than under nitrogen sufficient condition (27.3%). With the increasing initial cell concentrations, the lipid contents declined, while lipid concentrations and productivities increased. The highest lipid content, lipid concentration, and lipid productivity obtained was 54.5%, 536 mg L(-1) and 22.3 mg L(-1)day(-1), respectively. This study demonstrated that it was possible to culture C. zofingiensis under outdoor conditions for producing biodiesel feedstock.     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

Methionine Transport in Chlorella vulgaris

Absorption of ³⁵S-l-methionine by Chlorella vulgaris was measured at concentrations that ranged from 0.1 to 10.0 μmoles/ml. A brief, rapid phase of uptake was followed by a more prolonged, slower phase that was linear only at the lowest concentrations. The radioactivity accumulated by the end of 1 hour's incubation at an exogenous level of 0.1 μmole/ml was retained by the cells despite the inclusion of 10 μmoles/ml of nonradioactive methionine in the rinse medium. As the exogenous concentration was raised, the ratio of intracellular soluble radioactivity to exogenous radioactivity decreased. Analysis of the accumulated, soluble radioactivity showed that 90% was in the form of methionine and that about 10% had been converted to a compound with properties of S-adenosylmethionine. Azide and ethionine were the most effective of the inhibitors tested.     

The interactions between Chlorella vulgaris and algal symbiotic bacteria under photoautotrophic and photoheterotrophic conditions

A Chlorella vulgaris ATCC 13482 culture was semi-continuously cultivated for 18 months in a 4-L photobioreactor and formed associated consortia with other symbionts. Three symbiotic bacterial strains were isolated on heterotrophic medium agar plates. Based on 16S rDNA analysis, they were found to show closest similarity to Pseudomonas alcaligenes, Elizabethkingia miricola and Methylobacterium radiotolerans. C. vulgaris was co-cultured with each bacterial strain, and it was found that the symbiotic bacterium Pseudomonas sp. had a growth-promoting effect on C. vulgaris while the other two inhibited algal growth. The interactions between C. vulgaris and Pseudomonas sp. were further investigated under different cultivation conditions. The co-culture resulted in 1.4 times greater algal cell concentration than that of C. vulgaris alone under photoautotrophic condition. In contrast, the algal cell concentration was lower in the co-culture compared with single algal culture when glucose was supplied in the medium (photoheterotrophic). Under both cultivation conditions, the number of Pseudomonas sp. increased at the beginning of experiment, and then decreased. However, the bacterial number decreased to almost zero under photoheterotrophic conditions, while the growth of bacteria went into a stationary phase under photoautotrophic conditions. The chlorophyll content in C. vulgaris cell was higher in co-culture than in single algal culture. Algal cells in photoautotrophic condition showed higher photosynthetic efficiency compared to those in photoheterotrophic condition. Extracellular organic carbon dissolved in the medium continuously increased under photoautotrophic condition. The mutualistic and competing relationships between C. vulgaris and symbiotic bacteria observed in this study could aid our understanding of algae–bacteria interactions in nature as well as broadening its practical applications.