Lipase catalysis: an environmentally friendly production for polyol esters (biolubricant) from microalgae oil.

ABSTRACTThis study aims to investigate the optimized lipase-catalysed esterification reaction from novel feedstock microalgae (Chlorella protothecoides) oil-free fatty acids (FFAs) for biolubricant (Trimethylolpropane (TMP)-triesters) synthesis. FFAs were obtained from microalgae oil by enzymatic hydrolysis. Response surface methodology (RSM) with the central composite design was performed to investigate the effect of experimental factors (lipase amount, TMP/FFAs molar ratio, reaction temperature) on the FFAs conversion and also investigated to resolve the optimum design points. After the experimental studies, the highest FFAs conversion of 93% with 92% triester and 8% mono, di esters contents were found when the lipase amount was 5.5%. TMP/FFAs molar ratio was 0.33 and the reaction temperature value was 60°C. The model fitted with the experimental values with R2 = 0.97. It was also supported by gas chromatography and FTIR analyses that the product obtained was a lubricant.

Modeling the oxygen inhibition in microalgae: An experimental approach based on photorespirometry.

Microalgae cultivation has been the object of relevant interest for many industrial applications. Where high purity of the biomass/product is required, closed photobioreactors (PBRs) appear to be the best technological solution. However, as well as cost, the major drawback of closed systems is oxygen accumulation, which is well known to be responsible for growth inhibition. Only a few quantitative approaches have attempted to describe and model oxygen inhibition, which is the result of different biological mechanisms. Here, we have applied a photorespirometric protocol to assess and quantify the effect of high oxygen concentration on photosynthetic production rate. In particular, the effects of light intensity and biomass concentration were assessed, resulting in different maximum inhibitory oxygen concentrations. Literature models available were found not to fully represent experimental data as a function of concentration and light. Accordingly, a new formulation was proposed and validated to describe the photosynthetic rate as a function of external oxygen concentration.

The growth and lutein accumulation in heterotrophic Chlorella protothecoides provoked by waste Monascus fermentation broth feeding.

Although the potential of heterotrophic microalgae served as a sustainable source for lutein, it was still crucial to formulate a suitable medium to offset the cost involved in algal biomass cultivation while improve inherent lutein productivity. The objective of this study was to investigate the feasibilities of waste Monascus fermentation broth medium (MFBM) toward heterotrophic Chlorella protothecoides-enriched lutein. The results indicated that C. protothecoides subjected to MFBM batch feeding achieved 7.1 g/L biomass and 7.27 mg/g lutein. The resulting lutein productivity (7.34 mg/L/day) represented 1.54-fold more than that of frequently used Basal medium. Concurrently, the effective metabolism and absorption of carbon, nitrogen, and phosphorus in MFBM by C. subellipsoidea cultivation make it easily complied with the permissible dischargeable limits for fermentation broth. When response to fed-batch culture mode, the biomass and lutein productivity peaked 20.4 g/L and 9.11 mg/L/day with concentrated MFBM feeding. Transcriptomics data hinted that MFBM feeding manipulated lutein biosynthesis key checkpoints (e.g., lycopene ß-cyclase and lycopene ε-cyclase) while accelerated energy pathways (e.g., glycolysis and TCA cycle) to contribute such high lutein productivity in C. protothecoides. These encouraging findings not only provided indications in applying nutrient-rich fermentation broth for affordable microalgae cultivation but also presented possibilities in linking algal high value-added products like lutein with high-efficient biological nutrition removal from industrial fermentation processing.

A kinetic metabolic study of lipid production in Chlorella protothecoides under heterotrophic condition.

BACKGROUND: Microalgae have been proposed as potential platform to produce lipid-derived products, such as biofuels. Knowledge on the intracellular carbon flow distribution may identify key metabolic processes during lipid synthesis thus refining culture/genetic strategies to maximize cell lipid productivity. A kinetic metabolic model simulating cell metabolic behavior and lipid production was first applied in the microalgae platform Chlorella protothecoides under heterotrophic condition. It combines both physiology and flux information in a kinetic approach. Cell nutrition, growth, lipid production and almost 30 metabolic intermediates covering central carbon metabolism were included and simulated. RESULTS: Model simulations were shown to adequately agree with experimental data, which is suggesting that the proposed model copes with Chlorella protothecoides cells' biology. The dynamic metabolic flux analysis using the model showed a reversible starch flux from accumulation to decomposing when glucose reached depletion, while net lipid flux shows a quasi-constant rate. The sensitive flux parameters on starch and lipid metabolism suggested that starch synthesis is the major competing pathway that affects lipid accumulation in C. protothecoides. Flux analysis also demonstrated that high lipid yield under heterotrophic condition is accompanied with high lipid flux and low TCA activity. Meanwhile, the dynamic flux distribution also suggests a relatively constant ratio of glucose distributed to biomass, lipid, starch, nucleotides as well as pentose phosphate pathway. CONCLUSION: The model described not only experimental data, but also unraveled intracellular carbon flow distribution and identify key metabolic processes during lipid synthesis. Most of the metabolic kinetics also showed statistical significance for metabolic mechanism. Therefore, this study unravels the mechanisms of the glucose impact on the dynamic carbon flux distribution, thus improving our understanding of the links between carbon fluxes and lipid metabolism in C. protothecoides.

Cultivation of Chlorella protothecoides under different growth modes and its utilisation in oil/water emulsions.

Microalgae can be incorporated in different bio-based products; however, the green colour is a barrier for a successful integration. This study aims to overcome this barrier by growing microalgae in different cultivation modes. Mixotrophic cultivation of Chlorella protothecoides resulted in the highest biomass production after 5 days (5.56 ±â€¯0.09 g/L), followed by heterotrophic and photoautotrophic cultivation (4.33 ±â€¯0.15 and 1.80 ±â€¯0.05 g/L, respectively). Mixotrophically and heterotrophically produced biomass presented a reduced greenish colouration compared to photoautotrophically produced biomass. Chlorophyll content resulted in 1.46 ±â€¯0.21 and 0.95 ±â€¯0.28 mg/g dry weight (DW) in mixotrophic and heterotrophic cultures, respectively, and 25.98 ±â€¯1.28 mg/g DW in photoautotrophic cultures. In contrast, the fraction of carotenoids in the total pigments was much higher. With the whole microalgae fractions after cell disruption as ingredients, stable emulsions containing 50% oil could be produced. No syneresis with serum separation was observed 24 h after preparation.

Effects of sugarcane bagasse hydrolysate (SCBH) on cell growth and fatty acid accumulation of heterotrophic Chlorella protothecoides.

Microalgal lipid production by Chlorella protothecoides using sugarcane bagasse hydrolysate was investigated in this study. First, maximum glucose and reducing sugar concentrations of 15.2 and 27.0 g/L were obtained in sugarcane bagasse hydrolysate (SCBH), and the effects of different percentages of glucose and xylose on algal cultivation were investigated. Afterwards, SCBH was used as a carbon source for the cultivation of C. protothecoides and higher biomass concentration of 10.7 g/L was achieved. Additionally, a large amount of fatty acids, accounting up to 16.8% of dry weight, were accumulated in C. protothecoides in the nitrogen-limited (0.1-1 mmol/L) culture. Although SCBH inhibited fatty acid accumulation to a certain degree and the inhibition was aggravated by nitrogen starvation, SCBH favored microalgal cell growth and fatty acid production. The present study is of significance for the integration of cost-effective feedstocks production for biodiesel with low-cost SCBH as well as environmentally friendly disposal of lignocellulosic wastes.

Respirometry as a tool to quantify kinetic parameters of microalgal mixotrophic growth.

Modeling microalgal mixotrophy is challenging, as the regulation of algal metabolism is affected by many environmental factors. A reliable tool to simulate microalgal behavior in complex systems, such as wastewaters, may help in setting the proper values of operative variables, provided that model parameters have been properly evaluated. In this work, a new respirometric protocol is proposed to quickly obtain the half-saturation constant values for several nutrients. The protocol was first verified for autotrophic exploitation of ammonium and phosphorus (Monod kinetics), as well as of light intensity (Haldane model), further elaborated on specific light supply basis. It was then applied to measure the kinetic parameters of heterotrophic growth. The half-saturation constants for nitrogen and phosphorus resulted comparable with autotrophic ones. The dependence on acetate and dissolved oxygen concentration was assessed. Mixotrophy was modeled as the combination of autotrophic/heterotrophic reactions, implemented in AQUASIM, and validated on batch curves with/without bubbling, under nutrient limitation, and different light intensities. It was shown that the reliability of the proposed respirometric protocol is useful to measure kinetic parameters for nutrients, and therefore to perform bioprocess simulation.

Microalgae-bacteria gas exchange in wastewater: how mixotrophy may reduce the oxygen supply for bacteria.

Microalgae-bacteria consortia application to wastewater treatment is considered as a potential and cheap strategy towards a self-sustaining oxygen-carbon dioxide gas exchange. However, microalgae can also carry out mixotrophy, thus reducing the net oxygen production, due to consumption of organic substrates. In this work, respirometric tests were used to quantify the oxygen reduction in the presence of biodegradable COD (chemical oxygen demand), which resulted up to 70%, depending on the biodegradability of the carbon substrate. The implication of mixotrophic metabolism on nutrient removal in urban wastewater was also measured by co-cultivating C. protothecoides with bacteria from activated sludge. To better understand the contribution of different populations, ad hoc experiments under controlled conditions were designed to quantify the nutrient consumption of bacteria and microalgae. Microalgae and bacteria were cultivated together and separately, with and without external bubbling, so to better ascertain the specific role of gas production and nutrient removal. Results showed that microalgae can remove up to 100 and 85% of P and N respectively, but the contribution on COD consumption may affect the net O2 supply to heterotrophic bacteria. However, a mutual COD consumption by microalgae and bacteria was proved by both experimental growth curves and mass balance application, based on stoichiometry experimentally adjusted.

[Effect of sodium chloride on growth and lipid accumulation of Chlorella protothecoides CS-41].

With basal medium, we studied the growth status, lipid droplet distribution, total lipid content of Chlorella protothecoides CS-41 treated with different concentrations of sodium chloride (0, 150, 300 and 600 mmol/L) by optical microscopy, electron microscopy, confocal laser focusing and Nile red staining. Results show that the addition of NaCl affected the growth of Chlorella protothecoides CS-41. With the increase of NaCl concentration, the growth rate of Chlorella was inhibited. Chlorella cell wall became thicker, and lipid droplets increased. At the early stage, the amount of lipid droplets in the 600 mmol/L NaCl culture was the highest, but at the late-log stage, the amount of lipid droplets increased with the increase of the biomass of culture in 150 and 300 mmol/L NaCl culture. At the stable stage, biomass (dry weight) in 300 mmol/L NaCl culture was 73.55% of that in the control, but the total lipid content was 2.22 times higher than that in the control. A certain concentration of sodium chloride treatment can significantly increase the lipid content of Chlorella protothecoides CS-41.

Current lipid extraction methods are significantly enhanced adding a water treatment step in Chlorella protothecoides.

BACKGROUND: Microalgae have the potential to rapidly accumulate lipids of high interest for the food, cosmetics, pharmaceutical and energy (e.g. biodiesel) industries. However, current lipid extraction methods show efficiency limitation and until now, extraction protocols have not been fully optimized for specific lipid compounds. The present study thus presents a novel lipid extraction method, consisting in the addition of a water treatment of biomass between the two-stage solvent extraction steps of current extraction methods. The resulting modified method not only enhances lipid extraction efficiency, but also yields a higher triacylglycerols (TAG) ratio, which is highly desirable for biodiesel production. RESULTS: Modification of four existing methods using acetone, chloroform/methanol (Chl/Met), chloroform/methanol/H2O (Chl/Met/H2O) and dichloromethane/methanol (Dic/Met) showed respective lipid extraction yield enhancement of 72.3, 35.8, 60.3 and 60.9%. The modified acetone method resulted in the highest extraction yield, with 68.9 ± 0.2% DW total lipids. Extraction of TAG was particularly improved with the water treatment, especially for the Chl/Met/H2O and Dic/Met methods. The acetone method with the water treatment led to the highest extraction level of TAG with 73.7 ± 7.3 µg/mg DW, which is 130.8 ± 10.6% higher than the maximum value obtained for the four classical methods (31.9 ± 4.6 µg/mg DW). Interestingly, the water treatment preferentially improved the extraction of intracellular fractions, i.e. TAG, sterols, and free fatty acids, compared to the lipid fractions of the cell membranes, which are constituted of phospholipids (PL), acetone mobile polar lipids and hydrocarbons. Finally, from the 32 fatty acids analyzed for both neutral lipids (NL) and polar lipids (PL) fractions, it is clear that the water treatment greatly improves NL-to-PL ratio for the four standard methods assessed. CONCLUSION: Water treatment of biomass after the first solvent extraction step helps the subsequent release of intracellular lipids in the second extraction step, thus improving the global lipids extraction yield. In addition, the water treatment positively modifies the intracellular lipid class ratios of the final extract, in which TAG ratio is significantly increased without changes in the fatty acids composition. The novel method thus provides an efficient way to improve lipid extraction yield of existing methods, as well as selectively favoring TAG, a lipid of the upmost interest for biodiesel production.

CO2 utilization in the production of biomass and biocompounds by three different microalgae.

The atmospheric CO2 increase is considered the main cause of global warming. Microalgae are photosynthetic microorganisms that can help in CO2 mitigation and at the same time produce value-added compounds. In this study, Scenedesmus obliquus, Chlorella vulgaris, and Chlorella protothecoides were cultivated under 0.035 (air), 5 and 10% (v/v) of CO2 concentrations in air to evaluate the performance of the microalgae in terms of kinetic growth parameters, theoretical CO2 biofixation rate, and biomass composition. Among the microalgae studied, S. obliquus presented the highest values of specific growth rate (µ = 1.28 d-1), maximum productivities (P max = 0.28 g L-1d-1), and theoretical CO2 biofixation rates (0.56 g L-1d-1) at 10% CO2. The highest oil content was found at 5% CO2, and the fatty acid profile was not influenced by the concentration of CO2 in the inflow gas mixture and was in compliance with EN 14214, being suitable for biodiesel purposes. The impact of the CO2 on S. obliquus cells' viability/cell membrane integrity evaluated by the in-line flow cytometry is quite innovative and fast, and revealed that 86.4% of the cells were damaged/permeabilized in cultures without the addition of CO2.

Effect of sodium chloride on growth and lipid accumulation of Chlorella protothecoides CS-41 / 生物工程学报

With basal medium, we studied the growth status, lipid droplet distribution, total lipid content of Chlorella protothecoides CS-41 treated with different concentrations of sodium chloride (0, 150, 300 and 600 mmol/L) by optical microscopy, electron microscopy, confocal laser focusing and Nile red staining. Results show that the addition of NaCl affected the growth of Chlorella protothecoides CS-41. With the increase of NaCl concentration, the growth rate of Chlorella was inhibited. Chlorella cell wall became thicker, and lipid droplets increased. At the early stage, the amount of lipid droplets in the 600 mmol/L NaCl culture was the highest, but at the late-log stage, the amount of lipid droplets increased with the increase of the biomass of culture in 150 and 300 mmol/L NaCl culture. At the stable stage, biomass (dry weight) in 300 mmol/L NaCl culture was 73.55% of that in the control, but the total lipid content was 2.22 times higher than that in the control. A certain concentration of sodium chloride treatment can significantly increase the lipid content of Chlorella protothecoides CS-41.

Salt stress induced lipid accumulation in heterotrophic culture cells of Chlorella protothecoides: Mechanisms based on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration.

Salt stress as an effective stress factor that could improve the lipid content and lipid yield of glucose in the heterotrophic culture cells of Chlorella protothecoides was demonstrated in this study. The highest lipid content of 41.2% and lipid yield of 185.8mg/g were obtained when C. protothecoides was stressed under 30g/L NaCl condition at its late logarithmic growth phase. Moreover, the effects of salt and osmotic stress on lipid accumulation were comparatively analyzed, and it was found that the effects of NaCl and KCl stress had no significant differences at the same osmolarity level of 1150mOsm/kg with lipid contents of 41.7 and 40.8% as well as lipid yields of 192.9 and 186.8mg/g, respectively, whereas these results were obviously higher than those obtained under the iso-osmotic glycerol and sorbitol stresses. Furthermore, basing on the multi-level analysis of oxidative response, key enzyme activity and biochemical alteration, the superior performance of salt stress driving lipid over-synthesis was probably ascribed to the more ROS production as a result of additional ion effect besides the osmotic effect, subsequently mediating the alteration from carbohydrate storage to lipid accumulation in signal transduction process of C. protothecoides.

Biodiesel Production from Chlorella protothecoides Oil by Microwave-Assisted Transesterification.

In this study, biodiesel production from microalgal oil by microwave-assisted transesterification was carried out to investigate its efficiency. Transesterification reactions were performed by using Chlorella protothecoides oil as feedstock, methanol, and potassium hydroxide as the catalyst. Methanol:oil ratio, reaction time and catalyst:oil ratio were investigated as process parameters affected methyl ester yield. 9:1 methanol/oil molar ratio, 1.5% KOH catalyst/oil ratio and 10 min were optimum values for the highest fatty acid methyl ester yield.

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides.

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope-assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate-limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS-GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate-free and less ammonium-affinity GDH pathway, or alternatively the energy-consuming GS-GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.

A rapid and accurate quantification method for real-time dynamic analysis of cellular lipids during microalgal fermentation processes in Chlorella protothecoides with low field nuclear magnetic resonance.

The rapid and real-time lipid determination can provide valuable information on process regulation and optimization in the algal lipid mass production. In this study, a rapid, accurate and precise quantification method of in vivo cellular lipids of Chlorella protothecoides using low field nuclear magnetic resonance (LF-NMR) was newly developed. LF-NMR was extremely sensitive to the algal lipids with the limits of the detection (LOD) of 0.0026g and 0.32g/L in dry lipid samples and algal broth, respectively, as well as limits of quantification (LOQ) of 0.0093g and 1.18g/L. Moreover, the LF-NMR signal was specifically proportional to the cellular lipids of C. protothecoides, thus the superior regression curves existing in a wide detection range from 0.02 to 0.42g for dry lipids and from 1.12 to 8.97gL(-1) of lipid concentration for in vivo lipid quantification were obtained with all R(2) higher than 0.99, irrespective of the lipid content and fatty acids profile variations. The accuracy of this novel method was further verified to be reliable by comparing lipid quantification results to those obtained by GC-MS. And the relative standard deviation (RSD) of LF-NMR results were smaller than 2%, suggesting the precision of this method. Finally, this method was successfully used in the on-line lipid monitoring during the algal lipid fermentation processes, making it possible for better understanding of the lipid accumulation mechanism and dynamic bioprocess control.

Isolation and Analysis of the Cppsy Gene and Promoter from Chlorella protothecoides CS-41.

Phytoene synthase (PSY) catalyzes the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, the first colorless carotene in the carotenoid biosynthesis pathway. So it is regarded as the crucial enzyme for carotenoid production, and has unsurprisingly been involved in genetic engineering studies of carotenoid production. In this study, the psy gene from Chlorella protothecoides CS-41, designated Cppsy, was cloned using rapid amplification of cDNA ends. The full-length DNA was 2488 bp, and the corresponding cDNA was 1143 bp, which encoded 380 amino acids. Computational analysis suggested that this protein belongs to the Isoprenoid_Biosyn_C1 superfamily. It contained the consensus sequence, including three predicted substrate-Mg(2+) binding sites. The Cppsy gene promoter was also cloned and characterized. Analysis revealed several candidate motifs for the promoter, which exhibited light- and methyl jasmonate (MeJA)-responsive characteristics, as well as some typical domains universally discovered in promoter sequences, such as the TATA-box and CAAT-box. Light- and MeJA treatment showed that the Cppsy expression level was significantly enhanced by light and MeJA. These results provide a basis for genetically modifying the carotenoid biosynthesis pathway in C. protothecoides.

Alterations of the lipid content and fatty acid profile of Chlorella protothecoides under different light intensities.

Chlorella protothecoides is a valuable source of lipids that may be used for biodiesel production. The present work shows analysis of the potential of photoheterotrophic cultivation of C. protothecoides under various light intensities aiming to identify the conditions with maximal biomass and lipid content. An increase in light intensity was associated with an increased specific growth rate and a shortened doubling time. Also, the relative total lipid content increased from 24.8% to 37.5% with increase of light intensity. The composition of fatty acid methyl esters was affected by light intensity with the C16-18 fatty acids increased from 76.97% to 90.24% of total fatty acids. However, the content of linolenic acids decreased with the increase of the culture irradiance. These studies indicate that cultures irradiated with high light intensities achieve the minimal specifications for biodiesel quality on linolenic acids and thus are suitable for biodiesel production.

High yields of fatty acid and neutral lipid production from cassava bagasse hydrolysate (CBH) by heterotrophic Chlorella protothecoides.

The fermentation process for high yields of fatty acid and neutral lipid production from cassava bagasse hydrolysate (CBH) was developed by heterotrophic Chlorella protothecoides. An efficient single-step enzymatic hydrolysis of cassava bagasse (CB) by cellulase was firstly developed to produce >30 g/L of reducing sugars. The concentrated CBH was subsequently applied in a batch culture, producing 7.9 g/L of dry biomass with yield of 0.44 g/g reducing sugar and 34.3 wt% of fatty acids and 48.6 wt% of neutral lipids. Furthermore, fed-batch fermentation using CBH achieved higher yields of fatty acids (41.0 wt% and a titer of 5.83 g/L) and neutral lipids (58.4 wt% and yield of 0.22 g/g reducing sugar). Additionally, the fatty acid profile analysis showed that the intercellular lipid was suitable to prepare high-quality biodiesel. This study demonstrated the feasibility of using CBH as low-cost feedstock to produce crude algal oil for sustainable biodiesel production.

Enhanced biomass and oil production from sugarcane bagasse hydrolysate (SBH) by heterotrophic oleaginous microalga Chlorella protothecoides.

The potential use of sugarcane bagasse hydrolysate (SBH) for microalgal oil in a heterotrophic mode and the oil accumulation mechanisms by SBH-induced Chlorella protothecoides cells were investigated in this study. Results demonstrated that SBH performed better than glucose for cell growth and lipid accumulation under the same reducing sugar concentration. The lipid productivity of 0.69g/L/d was accomplished at 40g/L of reducing sugar by batch culture. Under the fed-batch culture condition, the maximum biomass and lipid productivity were 24.01g/L and 1.19g/L/d, respectively. Metabolic pathway analysis results indicated that xylose and arabinose involved in pentose phosphate pathway might be predominant over sole glucose involved in glycolysis for lipid accumulation in cells. Three metabolic checkpoints in the proposed metabolic network, including xylulose kinase, acyl-CoA dehydrogenase, and dihydrolipoyl dehydrogenase reveal new possibilities in developing genetic and metabolic engineering microalgae for desirable lipid productivity.