Three-state mathematical model for the assessment of DCMU-treated photosystem II heterogeneity.

Photosystem II (PSII) is one of the main pigment-protein complexes of photosynthesis which is highly sensitive to unfavorable environmental factors. The heterogeneity of PSII properties is essential for the resistance of autotrophic organisms to stress factors. Assessment of the PSII heterogeneity may be used in environmental monitoring for on-line detection of contamination of the environment. We propose an approach to assess PSII oxygen-evolving complex and light-harvesting antenna heterogeneity that is based on mathematical modeling of the shape of chlorophyll a fluorescence rise of 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated samples. The hierarchy of characteristic times of the processes considered in the model makes it possible to reduce the model to a system of three ordinary differential equations. The analytic solution of the reduced three-state model is expressed as a sum of two exponential functions, and it exactly reproduces the solution of the complete system within the time range from microseconds to hundreds of milliseconds. The combination of several such models for reaction centers with different properties made it possible to use it as an instrument to study PSII heterogeneity. PSII heterogeneity was studied for Chlamydomonas at different intensities of actinic light, for Scenedesmus under short-term heating, and for Chlorella grown in nitrate-enriched and nitrate-depleted media.

Economic co-production of cellulosic ethanol and microalgal biomass through efficient fixation of fermentation carbon dioxide.

An integrated process for the co-production of cellulosic ethanol and microalgal biomass by fixing CO2 generated from bioethanol fermentation is proposed. Specifically, over one-fifth of the fermentative carbon was converted into high-purity CO2 during ethanol production. The optimal concentration of 4 % CO2 was identified for the growth and metabolism of Chlorella sp. BWY-1. A multiple short-term intermittent CO2 supply system was established to efficiently fix and recycle the waste CO2. Using this system, economical co-production of cellulosic ethanol by Zymomonas mobilis and microalgal biomass in biogas slurry wastewater was achieved, resulting in the production of ethanol at a rate of 0.4 g/L/h and a fixed fermentation CO2 of 3.1 g/L/d. Moreover, the amounts of algal biomass and chlorophyll a increased by over 50 % and two-fold, respectively. Through techno-economic analysis, the integrated process demonstrated its cost-effectiveness for cellulosic ethanol production. This study presents an innovative approach to a low-carbon circular bioeconomy.

Applying mixotrophy strategy to enhance biomass production and nutrient recovery of Chlorella pyrenoidosa from biogas slurry: An assessment of the mixotrophic synergistic effect.

Using biogas slurry to cultivate microalgae can simultaneously obtain microalgal biomass and allow nutrient recovery. Mixotrophic microalgae are widely recognized for their high biomass accumulation and low light dependence, making it possible to overcome the drawbacks of photoautotrophy. In this study, three complete metabolic modes of photoautotrophy, heterotrophy, mixotrophy and two incomplete metabolic modes with the addition of diuron and rotenone were applied to investigate Chlorella pyrenoidosa growth in biogas slurry. The results showed that the mixotrophic group obtained 1.15 g/L biomass, 30 % starch content, 99.40 % ammonium removal and 81.69 % total phosphorus removal, which were highly promoted compared to the others. The decline in chlorophyll, the simultaneous downregulation of Rubisco and citrate synthase and the increase in the actual quantum yield of PSII under mixotrophy revealed a synergistic effect: the complementation of photophosphorylation and oxidative phosphorylation greatly contributed to maximizing energy metabolism efficiency and minimizing energy dissipation loss.

Comparative Assessment of Nitrogen Concentration Effect on Microalgal Growth and Biochemical Characteristics of Two Chlorella Strains Cultivated in Digestate.

Microalgae have been recently recognized as a promising alternative for the effective treatment of anaerobic digestion effluents. However, to date, a widely applied microalgae-based process is still absent, due to several constraints mainly attributed to high ammonia concentrations and turbidity, both hindering microalgal growth. Within this scope, the purpose of the present study was to investigate the performance of two Chlorella strains, SAG 211-11b and a local Algerian isolate, under different nitrogen levels, upon ammonia stripping. The experiments were performed on cylindrical photobioreactors under controlled pH (7.8 ± 0.2) and temperature (25 ± 2 °C). Cultures were monitored for biomass production and substrate consumption. After sampling at the beginning of the stationary phase of growth (12th day) and after the maturation of the cells (24th day), an analysis of the produced biomass was conducted, in terms of its biochemical components. The local isolate grew better than C. vulgaris 211-11b, resulting in 1.43 mg L-1 biomass compared to 1.02 mg L-1 under 25 mg NH4-N L-1, while organic carbon and nutrient consumption varied between the two strains and different conditions. Concerning biomass quality, a high initial NH4-N concentration led to high protein content, while low nitrogen levels favored fatty acid (FA) accumulation, though the production of pigments was inhibited. In particular, the protein content of the final biomass was determined close to 45% of the dry weight in all experimental scenarios with adequate nitrogen, while proteins decreased, and the fatty acids approached 20% in the case of the local isolate grown on the substrate with the lowest initial ammonium nitrogen (25 mg NH4-N L-1). The novelty of the present work lies in the comparison of a microalga with industrial applications against a local isolate of the same species, which may prove to be even more robust and profitable.

Toxicity assessment of synthesized titanium dioxide nanoparticles in fresh water algae Chlorella pyrenoidosa and a zebrafish liver cell line.

This study aims to assess the toxicity of the commonly-spread titanium dioxide nanoparticles (TiO2 NPs) by evaluating the exposure impact of the particles on both freshwater algae Chlorella pyrenoidosa and zebrafish liver cell line (ZFL), the two common in vitro models in toxicological studies. To compare the toxic effects of TiO2 NPs with different physiochemical properties, three types of manufactured TiO2 were used: bulk TiO2, Degussa P25 TiO2, and ultrafine TiO2 NPs. Both short and long-term biological responses of green algae, such as the effect on the cell growth rate, pigment autofluorescence, and esterase activity were investigated. The dosage, physical property of TiO2 particles, and their interactions with algal cells affect cellular growth, especially after short-term exposure. The hydrodynamic size plays a critical role in determining the acute toxicity to C. pyrenoidosa in terms of autofluorescence and esterase activity, while all types of TiO2 NPs show toxic effects after exposure for 14 days. However, this observation is not seen when studying the effect of introduced particles in ZFL, for the precipitated Degussa P25 TiO2 showed the highest cellular inhibition. Interestingly, despite the obvious overall toxicity toward C. pyrenoidosa, the photocatalytical properties of TiO2 NPs may contribute to the enhanced photosynthesis in the low concentration range (<40 µg mL-1). Overall, we found that the physical interactions between TiO2 particles and the cells, particles' size and dispersibility play critical role in the cytotoxic effect for both algal and ZFL cells, while the photocatalytical properties of TiO2 particles may produce mixed effects on the cytotoxicity of green algae.

Comparative assessment of pretreatment strategies for production of microalgae-based biodiesel from locally isolated Chlorella homosphaera.

The yield and quality of lipids extracted from microalgal biomass are critical factors in the production of microalgae-based biodiesel. The green microalga Chlorella homosphaera, isolated from Beira Lake, Colombo, Sri Lanka was employed in the present study to identify the effect of chlorophyll removal and cell disruption methods on lipid extraction yield, fatty acid methyl ester (FAME) profile and quality parameters of biodiesel; including cetane number (CN), iodine value (IV), degree of unsaturation (DU) and high heating value (HHV). In the first section of this study, chlorophyll was removed from dry microalgae biomass prior to lipid extraction. Through the analysis of FAME profiles, it was observed that chlorophyll removal yielded biodiesel of enhanced quality, albeit with a lipid loss of 44.2% relative to the control. In the second section of the study, mechanical cell disruption strategies including grinding, autoclaving, water bath heating and microwaving were employed to identify the most effective method to improve lipid recovery from chlorophyll-removed microalgae biomass. Autoclaving (121 °C, 20 min sterilization time, total time 2 h) was the most effective cell disruption technique of the methods tested, in terms of lipid extraction yield (39.80%) and also biodiesel quality. Moreover, it was observed that employing cell disruption subsequent to chlorophyll removal has a significant impact on the FAME profile of microalgae-based biodiesel, and consequently served to increase HHV and CN although IV and DU did not vary significantly.

Optimization of heterotrophic cultivation of Chlorella sp. HS2 using screening, statistical assessment, and validation.

The heterotrophic cultivation of microalgae has a number of notable advantages, which include allowing high culture density levels as well as enabling the production of biomass in consistent and predictable quantities. In this study, the full potential of Chlorella sp. HS2 is explored through optimization of the parameters for its heterotrophic cultivation. First, carbon and nitrogen sources were screened in PhotobioBox. Initial screening using the Plackett-Burman design (PBD) was then adopted and the concentrations of the major nutrients (glucose, sodium nitrate, and dipotassium phosphate) were optimized via response surface methodology (RSM) with a central composite design (CCD). Upon validation of the model via flask-scale cultivation, the optimized BG11 medium was found to result in a three-fold improvement in biomass amounts, from 5.85 to 18.13 g/L, in comparison to a non-optimized BG11 medium containing 72 g/L glucose. Scaling up the cultivation to a 5-L fermenter resulted in a greatly improved biomass concentration of 35.3 g/L owing to more efficient oxygenation of the culture. In addition, phosphorus feeding fermentation was employed in an effort to address early depletion of phosphate, and a maximum biomass concentration of 42.95 g/L was achieved, with biomass productivity of 5.37 g/L/D.

Life cycle assessment of Chlorella species producing biodiesel and remediating wastewater.

Constantly rising energy demands, finite fossil fuel reserves and deteriorating environmental conditions have invoked worldwide interest to explore the sustainable sources of renewable biofuels. Locally adapted photosynthetic oleaginous microalgae with rapid growth on variable temperatures could be an ideal way for bioremediating the wastewater (WW) while producing the feedstock for biodiesel. To test this notion, an unknown strain was isolated from a sewage fed lake (Neela-Hauz). It was discerned as Chlorella sorokiniana-I using the 16S rDNA and 18S rDNA barcodes. The culture conditions such as pH, illumination, different temperature ranges and growth medium were cohesively optimized prior to the assessment of C. sorokiniana-I's efficacy to remediate the WWand biodiesel production. The strain has thrived well up to 40°C when continuously grown for 15 days. The highest lipid accumulation and biomass productivity were recorded in 100% WW. Fatty acid methyl ester (FAME) content was observed to be more than twice in WW (47%), compared to control synthetic media, TAP (20%) and BG11 (10%), which indicate the importance of this new isolate for producing economically viable biodiesel. Moreover, it is highly efficient in removing the total nitrogen (77%), total phosphorous (81%), iron (67%) and calcium (42%) from the WW. The quality of WW was considerably improved by reducing the overall chemical oxygen demand (48%), biological oxygen demand (47%) and alkalinity (15%). Thus, C. sorokiniana-I could be an ideal alga for the tropical countries in the remediation of WW while producing feedstock for biodiesel in a cost-effective manner.

A novel approach using low-cost Citrus limetta waste for mixotrophic cultivation of oleaginous microalgae to augment automotive quality biodiesel production.

The present study reports the use of Citrus limetta (CL) residue for cultivating Chlorella sp. mixotrophically to augment production of biodiesel. The cultivation of Chlorella sp. using CL as media was carried out by employing a fed-batch technique in open tray (open tray+CL) and in software (BioXpert V2)-attached automated photobioreactor (PBR+CL) systems. Data showed the limit of nitrogen substituent and satisfactory organic source of carbon (OSC) in CL, causing > 2-fold higher lipid content in cells, cultivated in both the systems than in control. For the cells grown in both the systems, ≥ 3-fold enhancement in lipid productivity was observed than in control. The total fatty acid methyl ester (FAME) concentrations from lipids extracted from cells grew in PBR+CL and in open tray+CL techniques were calculated as 50.59% and 38.31%, respectively. The PBR+CL system showed improved outcomes for lipid content, lipid and biomass productivity, FAME characteristics and physical property parameters of biodiesel than those obtained from the open tray+CL system. The physical property parameters of biodiesel produced from algal cells grown in PBR+CL were comparable to existing fuel standards. The results have shown lower cold filter plugging point (- 6.57 °C), higher cetane number (58.04) and average oxidative stability (3.60 h). Collectively, this investigation unveils the novel deployment of CL as a cost-effective feedstock for commercialisation of biodiesel production.

Pollutants from fish feeding recycled for microalgae production as sustainable, renewable and valuable products.

Trash fish feeding of cage fish can result in marine pollution. Whole and chopped trash fish can leach pollutants such as ammonia, phosphate and protein into surrounding waters. Reduction of pollution can be achieved by recycling the wastewater generated from trash fish feeding for cultivation of microalgae. Microalgae are potent candidates for the production of renewable and sustainable products such as feed and food, health and pharmaceutical, cosmeceutical, industrial products, and biofuel. Two microalgae, Chlorella saccharophila and Nannochloropsis sp., have the potential to produce high amounts of polyunsaturated fatty acids. Furthermore, high oil content ranging from 10.7 to 13.6% is found in Chlorella saccharophila and up to 9.3% for Nannochloropsis sp. Moreover, these microalgae can also be utilized as a biofuel to give a mean calorific value of 5364 Cal/g which is higher than that of wood for Chlorella saccharophila and 6132 Cal/g which is equivalent to that of coal for Nannochloropsis sp. An alternative biofuel derived from microalgae is feasible due to the fact that they do not compete for arable land for cultivation and land crops for feed and food. This study discusses the synergistic coupling of microalgae mass production with wastewater treatment and carbon sequestration potential for mitigation of environmental impacts and a technically viable alternative energy resource. Additionally, the de-oiled biomass byproduct after oil extraction or its whole biomass can be converted into sustainable and renewal industrial products such as bioplastic, biopaint, bioasphalt, and biobuilding components.

Assessment of municipal wastewaters at various stages of treatment process as potential growth media for Chlorella sorokiniana under different modes of cultivation.

Wastewater utilization for microalgal biomass production is potentially the most economical route for its fuel and feed applications. In this study, suitability of various wastewater streams within a domestic wastewater treatment plant was evaluated for microalgal cultivation. Pre-treatment methods were evaluated to minimize bacterial load. Biomass, cell physiology, nutrient removal efficiencies and biochemical constituents of Chlorella sorokiniana were investigated in influent (INF) and anaerobic tank centrate (AC) under mixotrophic (Mixo) and heterotrophic (Hetero) cultivation. Promising biomass (77.14mgL-1d-1), lipid (24.91mgL-1d-1), protein (22.36mgL-1d-1) and carbohydrate (20.10mgL-1d-1) productivities were observed in Mixo AC with efficient ammonium (94.29%) and phosphate (83.30%) removal. Supplementation of urea at a concentration of 1500mgL-1 further enhanced biomass (162.50mgL-1d-1), lipid (24.91mgL-1d-1), protein (22.36mgL-1d-1) and carbohydrate (20.10mgL-1d-1) productivities in Mixo AC. Urea supplemented mixotrophic cultivation of microalgae in AC is developed as a biomass production strategy.

Evaluation of hydrolysis-esterification biodiesel production from wet microalgae.

Wet microalgae hydrolysis-esterification route has the advantage to avoid the energy-intensive units (e.g. drying and lipid extraction) in the biodiesel production process. In this study, techno-economic evaluation of hydrolysis-esterification biodiesel production process was carried out and compared with conventional (usually including drying, lipid extraction, esterification and transesterification) biodiesel production process. Energy and material balance of the conventional and hydrolysis-esterification processes was evaluated by Aspen Plus. The simulation results indicated that drying (2.36MJ/L biodiesel) and triolein transesterification (1.89MJ/L biodiesel) are the dominant energy-intensive stages in the conventional route (5.42MJ/L biodiesel). By contrast, the total energy consumption of hydrolysis-esterification route can be reduced to 1.81MJ/L biodiesel, and approximately 3.61MJ can be saved to produce per liter biodiesel.

Biomass and lipid enhancement in Chlorella sp. with emphasis on biodiesel quality assessment through detailed FAME signature.

In this study, the concentrations of MgSO4, salinity and light intensity were optimised for maximum biomass productivity and lipid content in Chlorella sp. Lipid synthesized at varied experimental conditions was also assessed in detail for biodiesel properties through FAME analysis. FAMEs mainly composed of C16:0, C16:1(9), C16:3(7, 10, 13), C18:0, C18:1(11), C18:2(9, 12), C18:3(9, 12, 15). The optimum biomass productivity (372.50mgL(-1)d(-1)) and lipid content (32.57%) was obtained at MgSO4-150ppm; salinity-12.5ppm, and light intensity-25µmolm(-2)s(-1). However, at this condition the cetane number, a major biodiesel property was not complying with worldwide biodiesel standard. Therefore, further optimisations were done to check the suitability of biodiesel fuel. The optimum biomass productivity (348.47mgL(-1)d(-1)) and lipid content (12.43%) with suitable biodiesel fuel properties was obtained at MgSO4-50ppm, salinity-25ppm and light intensity-100µmolm(-2)s(-1). The validation experiments confirmed the closeness of predicted and measured response values.

Anaerobic digestion of lipid-extracted Auxenochlorella protothecoides biomass for methane generation and nutrient recovery.

This study evaluated methane production and nutrient recovery from industrially produced, lipid extracted algal biomass (LEA) of Auxenochlorella protothecoides using semi-continuous anaerobic digestion (AD) at different organic loading rates (OLRs) and hydraulic retention times (HRTs). It was shown, that AD can improve biofuel production efficiency and sustainability, especially for scaled processes, through up to 30% increase in energy generation (up to 0.25 L of methane per g of LEA volatile solids) and partial nutrient recovery and recycling. The nutrient recycling with the AD effluent may reduce the cost of the supplied fertilizers by up to 45%. However, methane production was limited to nearly 50% of theoretical maxima potentially due to biomass recalcitrance and inhibition effects from the residual solvent in the LEA. Therefore, further AD optimization is required to maximize methane yield and nutrient recovery as well as investigation and elimination of inhibition from solvent residues.

Granular activated algae for wastewater treatment.

The study used activated algae granules for low-strength wastewater treatment in sequential batch mode. Each treatment cycle was conducted within 24 h in a bioreactor exposed to 235 µmol/m²/s light intensity. Wastewater treatment was performed mostly in aerobic conditions, oxygen being provided by microalgae. High removal efficiency of chemical oxygen demand (COD) was achieved (86-98%) in the first hours of the reaction phase, during which the indicator's removal rate was 17.4 ± 3.9 mg O2/g h; NH(4)(+) was removed during organic matter degradation processes with a rate of 1.8 ± 0.6 mg/g h. After almost complete COD removal, the (O⁺) remaining in the liquor was removed through nitrification processes promoted by the increase of the liquor's oxygen saturation (O2%), the transformation rate of NH4(+) into NO(3)(-) increasing from 0.14 ± 0.05 to 1.5 ± 0.4 mg NH4(+)/g h, along with an O2% increase. A wide removal efficiency was achieved in the case of PO(4)(3)(-) (11-85%), with the indicator's removal rate being 1.3 ± 0.7 mg/g h. In the provided optimum conditions, the occurrence of the denitrifying activity was also noticed. A large pH variation was registered (5-8.5) during treatment cycles. The granular activated algae system proved to be a promising alternative for wastewater treatment as it also sustains cost-efficient microalgae harvesting, with microalgae recovery efficiency ranging between 99.85 and 99.99% after granules settling with a velocity of 19 ± 3.6 m/h.

Assessment of bioavailability of heavy metal pollutants using soil isolates of Chlorella sp.

Biotests conducted with plants are presently used to estimate metal bioavailability in contaminated soils. But when plants are grown in soils, especially the plants with fine roots, root collection is easily biased and tedious. Indeed, at harvest, small amounts of soil can adhere to roots, resulting in overestimation of root metal content, and the finest roots are often discarded from the analysis because of their difficult and almost impossible recovery. This report presents a novel method for assessing the bioavailability of heavy metals in soils using microalgae. Two species of green unicellular microalgae were isolated from two highly contaminated soils and identified by phylogenetic and molecular evolutionary analyses as Chlorella sp. RBM and Chlorella sp. RHM. These two cultures were used to determine the metal uptake from metal-contaminated soils of South Australia as a novel, cost-effective, simple and rapid method for assessing the bioavailability of heavy metals in soils. The suggested method is an attempt to achieve a realistic estimate of bioavailability which overcomes the inherent drawback of root metal contamination in the bioavailability indices so far reported.

Lipid production by microalgae Chlorella protothecoides with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment.

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7%, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.

Optimization of liquid media and biosafety assessment for algae-lysing bacterium NP23.

To control algal bloom caused by nutrient pollution, a wild-type algae-lysing bacterium was isolated from the Baiguishan reservoir in Henan province of China and identified as Enterobacter sp. strain NP23. Algal culture medium was optimized by applying a Placket-Burman design to obtain a high cell concentration of NP23. Three minerals (i.e., 0.6% KNO3, 0.001% MnSO4·H2O, and 0.3% K2HPO4) were found to be independent factors critical for obtaining the highest cell concentration of 10(13) CFU/mL, which was 10(4) times that of the control. In the algae-lysing experiment, the strain exhibited a high lysis rate for the 4 algae test species, namely, Chlorella vulgari, Scenedesmus, Microcystis wesenbergii, and Chlorella pyrenoidosa. Acute toxicity and mutagenicity tests showed that the bacterium NP23 had no toxic and mutagenic effects on fish, even in large doses such as 10(7) or 10(9) CFU/mL. Thus, Enterobacter sp. strain NP23 has strong potential application in the microbial algae-lysing project.

Integrated evaluation of cost, emissions, and resource potential for algal biofuels at the national scale.

Costs, emissions, and resource availability were modeled for the production of 5 billion gallons yr(-1) (5 BGY) of renewable diesel in the United States from Chlorella biomass by hydrothermal liquefaction (HTL). The HTL model utilized data from a continuous 1-L reactor including catalytic hydrothermal gasification of the aqueous phase, and catalytic hydrotreatment of the HTL oil. A biophysical algae growth model coupled with weather and pond simulations predicted biomass productivity from experimental growth parameters, allowing site-by-site and temporal prediction of biomass production. The 5 BGY scale required geographically and climatically distributed sites. Even though screening down to 5 BGY significantly reduced spatial and temporal variability, site-to-site, season-to-season, and interannual variations in productivity affected economic and environmental performance. Performance metrics based on annual average or peak productivity were inadequate; temporally and spatially explicit computations allowed more rigorous analysis of these dynamic systems. For example, 3-season operation with a winter shutdown was favored to avoid high greenhouse gas emissions, but economic performance was harmed by underutilized equipment during slow-growth periods. Thus, analysis of algal biofuel pathways must combine spatiotemporal resource assessment, economic analysis, and environmental analysis integrated over many sites when assessing national scale performance.

Capability of different microalgae species for phytoremediation processes: wastewater tertiary treatment, CO2 bio-fixation and low cost biofuels production.

Scenedesmus obliquus, Chlorella vulgaris, Chlorella kessleri and a natural Bloom were cultivated in batch experiments, under controlled conditions, in urban wastewater (WW) and synthetic wastewater (SW) under 5% CO2 in air, with the object of estimating their capacity for nutrient removal, carbon dioxide biofixation, and generation of valuable biomass. In both culture media, the Bloom (Bl) and Scenedesmus (Sc) showed higher final biomass concentration (dried weight, dw) than the other species; the maximum yield obtained was 1950 ± 243 mg L(-1) for Bl and the minimum 821 ± 88 mg L(-1) for Cv, both in synthetic wastewater. Maximum specific growth rate values do not show significant differences between any of the 4 strains tested (p ≤ 0.05), nor between the 2 culture media. A new homogeneous method of calculating productivities has been proposed. Nitrogen removal in all the reactors was higher than 90%, except for BlSW (79%), and for phosphorus, the removal was higher than 98% in all trials. Maximum CO2 consumption rates reached were 424.4 and 436.7 mg L(-1) d(-1) for ScSW and ScWW respectively.