Strategic implementation of phosphorus repletion strategy in continuous two-stage cultivation of Chlorella sp. HS2: Evaluation for biofuel applications.

Lipid production in microalgae under nitrogen (N) starved condition can be enhanced by excess phosphorus (P) supply in the second stage of two-stage cultivation strategy. However, implementing two-stage cultivation is difficult in large-scale cultivation system as it requires high energy of transferring large algal biomass from first stage to second stage. To address this problem, we have optimized a continuous two-stage (CTS) cultivation strategy using Chlorella sp. HS2, where nitrogen in the growth environment is depleted naturally via consumption. To enhance both biomass and lipid productivity this strategy explored supplementation of additional P from 50% to 2500% of the initial concentration at the start of N-limited second stage of growth. The results of the optimization study in photobioreactor (PBR) showed that supplementing 500% of initial P and 100% of initial other nutrients (O) (N0-P500-O100) on 5th day showed the maximum biomass productivity of 774.4 mg L-1 d-1. It was observed that Chlorella sp. HS2 grown in PBR yielded higher biomass (3.8 times), lipid (6.1 times) and carbohydrate (5.5 times) productivity in comparison to the open raceway ponds (ORP) study, under optimum nutrient and carbon supply condition. The maximum lipid (289.6 mg L-1 d-1) and carbohydrate (219.2 mg L-1 d-1) productivities were obtained in TPBR-3, which were 1.9 and 1.3 times higher than that of TPBR-2 (+ve control) and 9.6 and 3.7 times higher than that of TPBR-1 (-ve control), respectively. Fatty acid mainly composed of C16/C18 (84.5%-85.7%), which makes the microalgal oil suitable for biofuel production. This study concluded that feeding excess amount of P is an effective and scalable strategy to improve the biomass and lipid productivity of CTS cultivation.

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.

Biological Carbon Recovery from Sugar Refinery Washing Water into Microalgal DHA: Medium Optimization and Stress Induction.

Sugar refinery washing water (SRWW) contains abundant levels of carbon sources and lower levels of contaminants than other types of wastewater, which makes it ideal for heterotrophic cultivation of microalgae. Here, carbon sources in SRWW were utilized for conversion into the form of value-added docosahexaenoic acid (DHA) using Aurantiochytrium sp. KRS101. Since SRWW is not a defined medium, serial optimizations were performed to maximize the biomass, lipid, and DHA yields by adjusting the nutrient (carbon, nitrogen, and phosphorus) concentrations as well as the application of salt stress. Optimum growth performance was achieved with 30% dilution of SRWW containing a total organic carbon of 95,488 mg L-1. Increasing the nutrient level in the medium by supplementation of 9 g L-1 KH2PO4 and 20 g L-1 yeast extract further improved the biomass yield by an additional 14%, albeit at the expense of a decrease in the lipid content. Maximum biomass, lipid, and DHA yields (22.9, 6.33, and 2.03 g L-1, respectively) were achieved when 35 g L-1 sea salt was applied on a stationary phase for osmotic stress. These results demonstrate the potential of carbon-rich sugar refinery washing water for DHA production using Aurantiochytrium sp. KRS101 and proper cultivation strategy.

Efficient microalgae removal from aqueous medium through auto-flocculation: investigating growth-dependent role of organic matter.

This study investigated the growth-dependent role of algal organic matters (AOMs) to achieve high removal efficiency (R.E) of microalgae. The results showed that the microalgae cells produced 96 ± 2% of total AOMs as loose bound AOMSS (LB-AOMs) and 4 ± 1% as cell-bound (CB-AOMs) in exponential phase. In stationary phase, LB-AOMs and CB-AOMs were 46 ± 0.7percentage and 54 ± 0.2 percentage, respectively. The R.Es in exponential and stationary phase were 83 ± 2.6% and 66 ± 1.2%, respectively. It is found that the difference of biomass concentration (between exponential and stationary phase) had no significant impact on the R.E (P > 0.01). Further investigations revealed that LB-AOMs inhibit flocculation in exponential and CB-AOMs in stationary phase; however, CB-AOMs showed stronger inhibition than the LB-AOMs (P < 0.01). The provision of calcium (17 ± 0.9 mg/L) to the culture reduced the AOMs inhibition and improved the R.E from 66 ± 1.2% (in control) to 90 ± 4.2%. An increase in R.E was attributed to the interaction of calcium with AOMs and subsequently acting as a flocculant. The findings of this study can be valuable to improve the performance of auto-flocculation technology, which is mainly limited by the presence of AOMs. Graphical Abstract.

Exploration of two-stage cultivation strategies using nitrogen starvation to maximize the lipid productivity in Chlorella sp. HS2.

In this work, the two-stage cultivation of Chlorella sp. HS2 for enhancing the lipid productivity was optimized by adjusting the duration of nitrogen-replete (N+) and -deplete (N-) stages within a 9 day period using urea as nitrogen source. The highest lipid content of 36.7% and productivity of 216.9 mg L-1 d-1 were obtained under five days of N+ followed by four days of N- conditions. Replenishing phosphorus and other nutrients (N-P+O+) at the beginning of the nutrient-starvation resulted in 1.55 and 1.68-folds improvement in lipid productivities compared to the single stage and zero nutrient controls (N-P-O-), respectively. The estimated biodiesel properties based on the fatty acid profiles met all criteria of international standards. The findings of this study indicate that properly adjusting the period of nitrogen availability as well as the presence of other nutrients is highly important in order to maximize the biofuel productivity in two-stage microalgal cultivation.

MAPK/ERK and JNK pathways regulate lipid synthesis and cell growth of Chlamydomonas reinhardtii under osmotic stress, respectively.

Microalgae have great potential for the production of biofuels due to the ability of the organism to accumulate large quantities of storage lipids under stress conditions. Mitogen activated protein kinase (MAPK) signaling cascades are widely recognized for their role in stress response signal transduction in eukaryotes. To assess the correlation between MAPK activation and lipid productivity, Chlamydomonas reinhardtii was studied under various concentrations of NaCl. The results demonstrated that C. reinhardtii exhibits elevated levels of extracellular-signal regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities after undergoing osmotic stress, as well as an increase in cellular lipid content. To establish a more direct causal link between both kinases and lipid productivity, C. reinhardtii was subjected to biochemically induced regulation of ERK and JNK pathways. Activating the MEK-ERK pathway via C6 ceramide treatment increased ERK activation and lipid production simultaneously, while PD98059 mediated inhibition of the pathway yielded opposite results. Interestingly, suppression of the JNK pathway with SP600125 resulted in a substantial decrease in cell viability under osmotic stress. These results suggest that ERK and JNK MAP kinases have important roles in microalgal lipid accumulation and cell growth under osmotic stress, respectively.

Isolation, phenotypic characterization and genome wide analysis of a Chlamydomonas reinhardtii strain naturally modified under laboratory conditions: towards enhanced microalgal biomass and lipid production for biofuels.

BACKGROUND: Microalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing. RESULTS: In this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation. CONCLUSION: Our phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production.

Development of direct conversion method for microalgal biodiesel production using wet biomass of Nannochloropsis salina.

In this work, the effects of several factors, such as temperature, reaction time, and solvent and acid quantity on in situ transesterification yield of wet Nannochloropsis salina were investigated. Under equivalent total solvent volume to biomass ratio, pure alcohol showed higher yield compared to alcohol-chloroform solvent. For esterifying 200 mg of wet cells, 2 ml of methanol and 1 ml of ethanol was sufficient to complete in situ transesterification. Under temperatures of 105 °C or higher, 2.5% and 5% concentrations of sulfuric acid was able to successfully convert more than 90% of lipid within 30 min when methanol and ethanol was used as solvents respectively. Also, it was verified that the optimal condition found in small-scale experiments is applicable to larger scale using 2 L scale reactor as well.

Water use and its recycling in microalgae cultivation for biofuel application.

Microalgal biofuels are not yet economically viable due to high material and energy costs associated with production process. Microalgae cultivation is a water-intensive process compared to other downstream processes for biodiesel production. Various studies found that the production of 1 L of microalgal biodiesel requires approximately 3000 L of water. Water recycling in microalgae cultivation is desirable not only to reduce the water demand, but it also improves the economic feasibility of algal biofuels as due to nutrients and energy savings. This review highlights recently published studies on microalgae water demand and water recycling in microalgae cultivation. Strategies to reduce water footprint for microalgal cultivation, advantages and disadvantages of water recycling, and approaches to mitigate the negative effects of water reuse within the context of water and energy saving are also discussed.

Utilization of lipid extracted algal biomass and sugar factory wastewater for algal growth and lipid enhancement of Ettlia sp.

The present study assessed the use of hydrolysate of lipid extracted algal biomass (LEA) combined with the sugar factory wastewater (SFW) as a low cost nutrient and a carbon source, respectively for microalgal cultivation. Microalgal strain Ettlia sp. was both mixotrophically and heterotrophically cultivated using various amounts of hydrolysate and SFW. The culture which was grown in medium containing 50% LEA hydrolysate showed highest growth, achieving 5.26 ± 0.14 gL(-1) after 12 days of cultivation. The addition of SFW increased the lipid productivity substantially from 5.8 to 95.5 mg L(-1)d(-1) when the culture medium was fortified with 20% SFW. Gas chromatography analysis indicated a noticeable increase of 20% in C16 and C18 fraction in FAME distribution under above condition. Therefore, it can be concluded that the combination of LEA hydrolysate and sugar factory waste water can be a powerful growth medium for economical algal cultivation.

Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method.

Identification of novel microalgal strains with high lipid productivity is one of the most important research topics in renewable biofuel research. However, the major bottleneck in the strain screening process is that currently known methods for the estimation of microalgal lipid are laborious and time-consuming. The present study successfully employed sulpho-phospho-vanillin (SPV) colorimetric method for direct quantitative measurement of lipids within liquid microalgal culture. The SPV reacts with lipids to produce a distinct pink color, and its intensity can be quantified using spectrophotometric methods by measuring absorbance at 530nm. This method was employed for a rapid quantification of intracellular lipid contents within Chlorella sp., Monoraphidium sp., Ettlia sp. and Nannochloropsis sp., all of which were found to have lipid contents ranging in between 10% and 30%. Subsequent analysis of the biomass using gas chromatography confirmed that our protocol is highly accurate (R(2)=0.99).

T-cell dysfunction and inhibitory receptors in hepatitis C virus infection.

Dysfunction of the virus-specific T cells is a cardinal feature in chronic persistent viral infections such as one caused by hepatitis C virus (HCV). In chronic HCV infection, virus-specific dysfunctional CD8 T cells often overexpress various inhibitory receptors. Programmed cell death 1 (PD-1) was the first among these inhibitory receptors that were identified to be overexpressed in functionally impaired T cells. The roles of other inhibitory receptors such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) have also been demonstrated in T-cell dysfunctions that occur in chronic HCV patients. Blocking these inhibitory receptors in vitro restores the functions of HCV-specific CD8 T cells and allows enhanced proliferation, cytolytic activity and cytokine production. Therefore, the blockade of the inhibitory receptors is considered as a novel strategy for the treatment of chronic HCV infection.