Cultivation of microalgae for oil production with a cultivation strategy of urea limitation

The microalgae, Chlorella sp., were cultivated in various culture modes to assess biomass and lipid productivity in this study. In the batch mode, the biomass concentrations and lipid content of Chlorella sp. cultivated in a medium containing 0.025–0.200 g L⁻¹ urea were 0.464–2.027 g L⁻¹ and 0.661–0.326 g g⁻¹, respectively. The maximum lipid productivity of 0.124 g d⁻¹ L⁻¹ occurred in a medium containing 0.100 g L⁻¹ urea. In the fed-batch cultivation, the highest lipid content was obtained by feeding 0.025 g L⁻¹ of urea during the stationary phase, but the lipid productivity was not significantly increased. However, a semi-continuous process was carried out by harvesting the culture and renewing urea at 0.025 g L⁻¹ each time when the cultivation achieved the early stationary phase. The maximum lipid productivity of 0.139 g d⁻¹ L⁻¹ in the semi-continuous culture was highest in comparison with those in the batch and fed-batch cultivations.     

Production cost of a real microalgae production plant and strategies to reduce it

The cost analysis of a real facility for the production of high value microalgae biomass is presented. The facility is based on ten 3 m³ tubular photobioreactors operated in continuous mode for 2 years, data of Scenedesmus almeriensis productivity but also of nutrients and power consumption from this facility being used. The yield of the facility was close to maximum expected for the location of Almería, the annual production capacity being 3.8 t/year (90 t/ha·year) and the photosynthetic efficiency being 3.6%. The production cost was 69 €/kg. Economic analysis shows that labor and depreciation are the major factors contributing to this cost. Simplification of the technology and scale-up to a production capacity of 200 t/year allows to reduce the production cost up to 12.6 €/kg. Moreover, to reduce the microalgae production cost to approaches the energy or commodities markets it is necessary to reduce the photobioreactor cost (by simplifying its design or materials used), use waste water and flue gases, and reduce the power consumption and labor required for the production step. It can be concluded that although it has been reported that production of biofuels from microalgae is relatively close to being economically feasible, data here reported demonstrated that to achieve it by using the current production technologies, it is necessary to substantially reduce their costs and to operate them near their optimum values.     

The inter-monomer interface of the major light-harvesting chlorophyll a/b complexes of photosystem II (LHCII) influences the chlorophyll triplet distribution

Under strong light conditions, long-lived chlorophyll triplets (³Chls) are formed, which can sensitize singlet oxygen, a species harmful to the photosynthetic apparatus of plants. Plants have developed multiple photoprotective mechanisms to quench ³Chl and scavenge singlet oxygen in order to sustain the photosynthetic activities. The lumenal loop of light-harvesting chlorophyll a/b complex of photosystem II (LHCII) plays important roles in regulating the pigment conformation and energy dissipation. In this study, site-directed mutagenesis analysis was applied to investigate triplet–triplet energy transfer and quenching of ³Chl in LHCII. We mutated the amino acid at site 123 located in this region to Gly, Pro, Gln, Thr and Tyr, respectively, and recorded fluorescence excitation spectra, triplet-minus-singlet (TmS) spectra and kinetics of carotenoid triplet decay for wild type and all the mutants. A red-shift was evident in the TmS spectra of the mutants S123T and S123P, and all of the mutants except S123Y showed a decrease in the triplet energy transfer efficiency. We propose, on the basis of the available structural information, that these phenomena are related to the involvement, due to conformational changes in the lumenal region, of a long-wavelength lutein (Lut2) involved in quenching ³Chl.     

Benthic microalgae: comparisons of chlorophyll a in mesocosms and field sites

A mesocosm facility is being developed by the CSIRO Division of Fisheries to study the movement, fate and impact of pollutants in coastal marine environments. Initially, we are studying coastal habitats in S.W Australia that are subject to wave action. A 12 week experiment assessed changes in biotic and abiotic components of sediment brought from the field into mesocosms. In these sediments, benthic microalgae are the major primary producers. Microalgal standing stocks were measured as the concentration of chlorophyll a and phaeopigments in the top 2 cm of sediment, and these measures were compared among mesocosms and field sites. Significant increases in chlorophyll a and phaeopigments, increased numbers of species and a shift from episammic to periphytic diatoms were observed, possibly caused by decreased water motion in mesocosms or other containment effects. Results from statistical power analyses suggested that our sampling was sufficiently well replicated and successfully incorporated variation at small spatial and temporal scales. Sampling effort used in this experiment should form the basis for future work with benthic microalgae.     

Massive cultivation of microalgae: Results and prospects

An account is given of the development of the utilization of microalgae for food and feed with special emphasis on the advantages of algal technologies for tropical and subtropical countries. The present status of microalgae mass production is characterized with respect to technology, product properties, yields, nutrition, toxicology and economics. As a multipurpose operation, the treatment of liquid wastes with algae-bacteria systems is the most promising microalgal technology. It yields proteinaceous microbial biomass as a comparatively inexpensive by-product of the operation of high-rate algal ponds, either at the simplified rural level or at the technically more elaborate industrial level. The aspect of hard-currency saving by employing algae-bacteria systems in sewage treatment for animal feed production is stressed.     

Isolation of thermo-tolerant and high lipid content green microalgae: oil accumulation is predominantly controlled by photosystem efficiency during stress treatments in Desmodesmus

Discoveries of new microalgae with thermo-tolerance, high growth rate, and high lipid content are crucial to algal biodiesel production in tropical and subtropical zones. Four new green microalgae were isolated in southern Taiwan. All four species are members of the genus Desmodesmus under the family Scenedesmaceae based on molecular and morphological analyses. Two of the four species survived at 45 °C for 24 h, with 5-13% of mortality rates caused by the heat. Total lipid contents of the two species reached over 50% in dry biomass under nitrogen starvation, and their triacylglycerols constituted around 75% of the total lipids. Thus the two species are good potential feedstocks for biodiesel production. Oil accumulation in the four species positively correlates with their photosystem II efficiencies during stress treatments (R2=0.90). This finding further supports that photosynthesis is essential for oil body formation under nitrogen starvation in green microalgae.     

Assessment of a dry and a wet route for the production of biofuels from microalgae: energy balance analysis

In this study, the energy balance of two microalgae-to-biofuel concepts, one via a so called “dry route” (oil extraction from dried algae) and one via a “wet route” (oil extraction in the water phase), are assessed. Both routes are intended to convert the chemical energy contained in the microalgae into high-value biofuels with minimal fossil energy consumption. The analysis shows that the drying process in the dry route and the oil extraction process in the wet route consume a significant amount of energy. By coupling waste heat from a nearby power plant to the process, the energy balance can be improved and a potential fossil energy ratio (FER) up to 2.38 and 1.82 can be reached for the dry and wet route, respectively. The results indicate that based on current available technologies, the dry route has higher FER and the wet route has more potential in producing high valuable biofuels.     

The Stratiotes aloides L. stand as a habitat in oxbow lake Bużysko

The impact of Stratiotes aloides L. (water soldier) on the accumulation and concentration of nitrogen and phosphorus, seston concentrations and provision a habitat for planktonic crustaceans was studied in a Polish oxbow lake during 2002–2004. The Stratiotes stand and the open water differed in the concentrations of mineral nitrogen and chlorophyll a. Stratiotes accumulated significantly more nitrogen in spring. The lower TN:TP ratio, concentrations of mineral nitrogen and chlorophyll a in stands of the water soldier may suggest nitrogen limitation in the littoral of the oxbow lake. The decrease of phytoplankton biomass in the Stratiotes stands may also result from shading and/or allelopathy. The plants did not reduce seston concentrations in the littoral but did affect the abundance and diversity of planktonic and bentho-planktonic crustaceans and probably provided a daytime refuge for Ceriodaphnia and older stages of Cyclopoida but not for Daphnia.     

A closed solar photobioreactor for cultivation of microalgae under supra-high irradiance: basic design and performance

An account is given of the setting up and use of a novel type of closed tubular photobioreactor at the Academic and University Centre in Nove Hrady, Czech Republic. This "penthouse-roof" photobioreactor was based on solar concentrators (linear Fresnel lenses) mounted in a climate-controlled greenhouse on top of the laboratory complex combining features of indoor and outdoor cultivation units. The dual-purpose system was designed for algal biomass production in temperate climate zone under well-controlled cultivation conditions and with surplus solar energy being used for heating service water. The system was used to study the strategy of microalgal acclimation to supra-high solar irradiance, with values as much as 3.5 times the ambient value, making the approach unique. The cultivation system proved to be fully functional with sufficient mixing and cooling, efficient oxygen stripping and light tracking. Experimental results (measurement of the maximum photochemical yield of PSII and non-photochemical quenching) showed that the cyanobacterium Spirulina (= Arthrospira) platensis cultivated under sufficient turbulence and biomass density was able to acclimate to irradiance values as high as 7 mmol photon m^–2 s^–1. The optimal biomass concentration of Spirulina cultures in September ranged between 1.2 to 2.2 g L^–1, which resulted in a net productivity of about 0.5 g L^–1 d^–1 corresponding to a biomass yield of 32.5 g m^–2 d^–1 (based on the minimum illuminated surface area of the photobioreactor).     

Antenna ring around trimeric Photosystem I in chlorophyll b containing cyanobacterium Prochlorothrix hollandica

Prochlorothrix hollandica is one of the three known species of an unusual clade of cyanobacteria (formerly called “prochlorophytes”) that contain chlorophyll a and b molecules bound to intrinsic light-harvesting antenna proteins. Here, we report the structural characterization of supramolecular complex consisting of Photosystem I (PSI) associated with the chlorophyll a/b-binding Pcb proteins. Electron microscopy and single particle image analysis of negatively stained preparations revealed that the Pcb-PSI supercomplex consists of a central trimeric PSI surrounded by a ring of 18 Pcb subunits. We conclude that the formation of the Pcb ring around trimeric PSI represents a mechanism for increasing the light-harvesting efficiency in chlorophyll b-containing cyanobacteria.     

Evidence of functional trimeric chlorophyll a/c2-peridinin proteins in the dinoflagellate Symbiodinium

The chlorophyll a-chlorophyll c₂-peridinin-protein (apcPC), a major light harvesting component in peridinin-containing dinoflagellates, is an integral membrane protein complex. We isolated functional acpPC from the dinoflagellate Symbiodinium. Both SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) analysis quantified the denatured subunit polypeptide molecular weight (MW) as 18 kDa. Size-exclusion chromatography (SEC) and blue native gel electrophoresis (BN-PAGE) were employed to estimate the size of native acpPC complex to be 64–66 kDa. We also performed native ESI-MS, which can volatilize and ionize active biological samples in their native states. Our result demonstrated that the native acpPC complex carried 14 to 16 positive charges, and the MW of acpPC with all the associated pigments was found to be 66.5 kDa. Based on these data and the pigment stoichiometry, we propose that the functional light harvesting state of acpPC is a trimer. Our bioinformatic analysis indicated that Symbiodinium acpPC shares high similarity to diatom fucoxanthin Chl a/c binding protein (FCP), which tends to form a trimer. Additionally, acpPC protein sequence variation was confirmed by de novo protein sequencing. Its sequence heterogeneity is also discussed in the context of Symbiodinium eco-physiological adaptations.     

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves: 2. Multi-frequency phase and modulation analysis evidences a loosely connected PSII pigment-protein complex

We report the first direct decomposition of the fluorescence lifetime heterogeneity during multiphasic fluorescence induction in dark-adapted leaves by multi-frequency phase and modulation fluorometry (PMF). A very fast component, assigned to photosystem I (PSI), was found to be constant in lifetime and yield, whereas the two slow components, which are strongly affected by the closure of the reaction centers by light, were assigned to PSII. Based on a modified “reversible radical pair” kinetic model with three compartments, we showed that a loosely connected pigment complex, which is assumed to be the CP47 complex, plays a specific role with respect to the structure and function of the PSII: (i) it explains the heterogeneity of PSII fluorescence lifetime as a compartmentation of excitation energy in the antenna, (ii) it is the site of a conformational change in the first second of illumination, and (iii) it is involved in the mechanisms of nonphotochemical quenching (NPQ). On the basis of the multi-frequency PMF analysis, we reconciled two apparently antagonistic aspects of chlorophyll a fluorescence in vivo: it is heterogeneous with respect to the kinetic structure (several lifetime components) and homogeneous with respect to average quantities (quasi-linear mean τ-Φ relationship).     

Two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production

A two-stage heterotrophic and phototrophic culture strategy for algal biomass and lipid production was studied, wherein high density heterotrophic cultures of Chlorellasorokiniana serve as seed for subsequent phototrophic growth. The data showed growth rate, cell density and productivity of heterotrophic C.sorokiniana were 3.0, 3.3 and 7.4 times higher than phototrophic counterpart, respectively. Hetero- and phototrophic algal seeds had similar biomass/lipid production and fatty acid profile when inoculated into phototrophic culture system. To expand the application, food waste and wastewater were tested as feedstock for heterotrophic growth, and supported cell growth successfully. These results demonstrated the advantages of using heterotrophic algae cells as seeds for open algae culture system. Additionally, high inoculation rate of heterotrophic algal seed can be utilized as an effective method for contamination control. This two-stage heterotrophic phototrophic process is promising to provide a more efficient way for large scale production of algal biomass and biofuels.     

Correlation between lifetime heterogeneity and kinetics heterogeneity during chlorophyll fluorescence induction in leaves:: 1. Mono-frequency phase and modulation analysis reveals a conformational change of a PSII pigment complex during the IP thermal phase

The relationship between the fluorescence lifetime (τ) and yield (Φ) obtained in phase and modulation fluorometry at 54 MHz during the chlorophyll fluorescence induction in dark-adapted leaves under low actinic light has been investigated. Three typical phases have been identified: (i) linear during the OI photochemical rise, (ii) convex curvature during the subsequent IP thermal rise, and (iii) linear during the PS slow decay. A similar relationship has been obtained in the fluorescence induction for the fluorescence yield measured at 685 nm plotted versus the fluorescence yield measured at 735 nm. A spectrally resolved analysis shows that the curvature of the τ-Φ relationship is not due to chlorophyll fluorescence reabsorption effects. Several other hypotheses are discussed and we conclude that the curvature of the τ-Φ relationship is due to a variable and transitory nonphotochemical quenching. We tentatively propose that this quenching results from a conformational change of a pigment-protein complex of Photosystem II core antenna during the IP phase and could explain both spectral and temporal transitory changes of the fluorescence. A variable blue shift of the 685 nm peak of the fluorescence spectrum during the IP phase has been observed, supporting this hypothesis.     

A novel photobioreactor with transparent rectangular chambers for cultivation of microalgae

An open tank photobioreactor containing transparent rectangular chambers (TRCs) was developed to improve the photosynthetic efficiency of microalgal cultivation. The TRCs, made of transparent acrylic, conducted light deep into the photobioreactor, especially at high cell concentrations. The average irradiance, I_av, was calculated by Lambert–Beer's law, and was used to determine the light conditions in the cultivation system. The photobioreactor provided large areas of illumination that improved the effective utilization of light energy for microalgae growth and created a good artificial environment for a high rate of cell growth, even at low I_av. The biomass concentration of Chlorella sp. reached 3.745 g L⁻¹ on the 13th day, with biomass productivity of 0.340 g L⁻¹ d⁻¹. The total biomass obtained was 56% more than that of similar culture systems without TRCs.     

Extraction of chlorophyll a from freshwater phytoplankton for spectrophotometric analysis

The efficiency of pigment extraction forms the crux of the spectrophotometric analysis of chlorophyll a. The alcoholic solvents, methanol and ethanol, proved to be superior to acetone and acetone with DMSO. Homogenisation and sonication did not improve the extraction in the alcoholic solvents. Boiling at 100°C had an adverse effect whereas complete extraction of the pigments was obtained at the solvents boiling point and allowing the samples to stand for 24 h in the dark.     

Reuse of effluent water from a municipal wastewater treatment plant in microalgae cultivation for biofuel production

This study assessed the usability of effluent water discharged from a secondary municipal wastewater treatment plant for mass cultivation of microalgae for biofuel production. It was observed that bacteria and protozoa in the effluent water exerted a negative impact on the growth of Chlorella sp. 227. To reduce the effect, filtration or UV-radiation were applied on the effluent water as pre-treatment methods. Of all the pretreatment options tested, the filtration (by 0.2 μm) resulted in the highest biomass and lipid productivity. To be comparable with the growth in the autoclaved effluent water, the filtration with a proper pore size filter (less than 0.45 μm) or UV-B radiation of a proper dose (over 1620 mJ cm⁻²) are proposed. These findings led us to conclude that the utilization can be realized only when bacteria and other microorganisms are greatly reduced or eliminated from the effluent prior to its use.     

Plastid structure and chlorophyll content in leaf primordia of onion bulbs

Angiosperms do not synthesize chlorophyll in the dark. Here we show that leaf primordia in onion bulbs are green, though they developing in dark conditions. We present results that show plastids in green primordia are chloroplasts, and that they contain chlorophyll as well as embryos in seeds of certain angiosperms.     

p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16(INK4a) and p14(ARF), genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16(INK4a) and p14(ARF) in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0-100.1 months). The methylation rate for p16(INK4a) and p14(ARF) was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16(INKa) and p14(ARF) methylation was significantly higher in patients with invasive (>or=pT2) than superficial bladder cancer (pT1) (p=0.006 and p=0.001, respectively). No MSP bands for p16(INK4a) and p14(ARF) were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16(INK4a) and p14(ARF) methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16(INKa) methylation; p16(INK4a) was not methylated in 23 patients without cystectomy (p=0.002). Kaplan-Meier analysis revealed that patients with p14(ARF) methylation had a significantly poorer prognosis than those without (p=0.029). This is the first study indicating that MSP analysis of p16(INK4a) and p14(ARF) genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.     

Changes in chlorophyll fluorescence and chlorophyll content in suppressed Norway spruce [Picea abies (L.) Karst.] in response to release cutting

Summary In order to study physiological strain caused by release cutting, suppressed Norway spruce on mesic and moist sites was completely released from overstory birch, or 500 birches per hectare were left as a shelter. The treatments were conducted in late June in 1988 and in 1989. The spruce's reaction to the environmental change was monitored by measurements of fast chlorophyll fluorescence kinetics and analysis of chlorophyll content. This was done before treatment, 1 week after treatment, 2 months after treatment, and twice during the following growth period. Complete release resulted in a more pronounced decrease in the ratio of variable fluorescence to maximal fluorescence (F_v/F_m) than partial release. There was also a tendency for the build-up of chlorophyll content in needles to be more affected when the spruce was completely released. Released spruces on moist sites tended to be more affected by the release than released spruces on mesic sites. The results suggest that in this kind of stand the risk of damage to the spruces is greatest when the spruces are completely released on moist sites. Furthermore, it is shown that the weather conditions prevailing shortly before and after the release have a large influence on the spruce's reaction to the release. The results also indicate some adjustment to the new environment in mature needles.