Semi-continuous Culture Research Articles

Creation of consortia of hydrocarbon-oxidizing microorganisms effectively degrading xylene isomers

Due to the serious danger to the environment and human health, aromatic hydrocarbons have recently become the subject of extensive research. They can easily spread in soils and groundwater as aresult of the oil production process, leaking tanks and problems encountered during transportation overlong distances. Therefore, their removal is one of the most important environmental problems. Biological methods of purification of the natural environment are the safest, most effective and less expensivein comparison with the methods of chemical and physical purification. Currently, most efforts are aimedat finding bacterial communities capable of mineralizing pollutants into less harmful, non-hazardouscompounds. Moreover, the use of consortia can further increase the efficiency of decomposition of hydrocarbons, since they have multiple metabolic capabilities. Earlier, we isolated hydrocarbon-oxidizingmicroorganisms capable of oxidizing o-xylene from oil-contaminated soils of Western Kazakhstan. Theaim of this work was the creation of bacterial consortia, and study the efficiency of degradation of xyleneisomer mixture. By the method of semi-continuous cultivation, variants of strains with increased oxidative potential were obtained. Their ability to grow on m- and p-xylene was tested. Based on selected active strains of hydrocarbon-oxidizing bacteria there have been created consortia which effectivelydegrade xylene isomers. After 24 h of cultivation of xylene oxidizing consortia, the residual content ofo-xylene in the medium was 6.1-12.7%, m-xylene – 4.5-10.2%, p-xylene – 10.2-20.2%. The highestactivity was shown by the consortium 8 (B. megaterium 1/1 an, B. thuringiensis 2/4 fl, B. tropicus 2/6 fl,B. megaterium P1-fl1-5), which utilized 89.8-95.5% test substrates.Key words: biodegradation, hydrocarbon-oxidizing bacteria, consortium, xylene isomers.

Enriching Rotifers with "Premium" Microalgae: Rhodomonas lens.

The nutritional value of the marine cryptophyte Rhodomonas lens for the filter feeder Brachionus plicatilis as well as its biotechnological potential as a source of phycoerythrin (PE) and polyunsaturated fatty acids (PUFA) were evaluated in semi-continuous cultures maintained with different daily renewal rates (RR), from 10% (R10) to 50% (R50) of the total volume. Steady-state cell density decreased from 22 to 7 × 106cellsmL-1 with increasing RR, with the maximum cell productivity, nearly 0.4gL-1day-1, observed with R40. PE cell content attained the highest values with the highest RR (circa 9pgcell-1). All treatments of R. lens maintained under nitrate-saturated conditions (R20-R50) showed a similar high content of PUFAs, > 60% of total fatty acids (FA), with linolenic acid (18:3n-3) and 18:4n-3, representing 12 and 29% of total FA respectively. The PUFA level in the nitrogen-limited R10 cultures was significantly lower (37%). R. lens promoted higher weight gain in the rotifer B. plicatilis than Tisochrysis lutea (T-ISO), a species commonly used for rotifer culture and enrichment. Significant differences were found in the protein content and in the ratio n-3/n-6 fatty acids among rotifers fed with R. lens from different RRs, with higher values being found in those fed with R. lens from higher RRs. The enrichment of the rotifers for short periods of 3h was sufficient to modify the biochemical composition of the rotifers, but it was evidenced as too short for the accumulation of PUFAs, when compared to long-term (24h) enrichment. The rotifers reflected the higher protein and PUFA content of R. lens cultivated with nutrient sufficient microalgae (R40) after only 3h of enrichment. These results demonstrate that semi-continuous culture of R. lens under appropriate conditions can strongly enhance the nutritional value of this species, being reflected in the growth and biochemical composition of the filter feeder, even in short exposure periods.

Rapid development of clone-specific, high-performing perfusion media from established feed supplements.

Perfusion cultivation of recombinant CHO cells is of substantial interest to the biopharmaceutical industry. This is due to increased space–time‐yields (STYs) and a short residence time of the recombinant protein in the bioreactor. Economic processes rely on cultivation media supporting rapid growth in the exponential phase and high protein production in the stationary phase at minimal media consumption rates. To develop clone‐specific, high‐performing perfusion media we present a straightforward and rapid two‐step approach combining commercially available basal media and feed supplements using design‐of‐experiment. First, the best performing feed supplements are selected in batch cultures. Then, the mixing ratio of selected feed supplements is optimized in small‐scale semicontinuous perfusion cultures. The final media formulation is supported by statistical response surface modeling of a set of cultivation experiments with blended media formulations. Two best performing novel media blends were finally applied to perfusion bioreactor verification runs to reach 200 × 106 c/ml within 2 weeks at minimum cell‐specific perfusion rates as low as 10–30 pL/c/d. Obtained STYs of 0.4–1.2 g/L/d represent a 10‐fold increase compared to batch cultures. This general workflow is universally applicable to any perfusion platform combining a specific cell line, basal medium, and established feed solutions.

Continuous influenza virus production in a tubular bioreactor system provides stable titers and avoids the "von Magnus effect".

Continuous cell culture-based influenza vaccine production could significantly reduce footprint and manufacturing costs compared to current batch processing. However, yields of influenza virus in continuous mode can be affected by oscillations in virus titers caused by periodic accumulation of defective interfering particles. The generation of such particles has also been observed previously in cascades of continuous stirred tank reactors (CSTRs) and is known as the “von Magnus effect”. To improve virus yields and to avoid these oscillations, we have developed a novel continuous tubular bioreactor system for influenza A virus production. It was built using a 500 mL CSTR for cell growth linked to a 105 m long tubular plug-flow bioreactor (PFBR). Virus propagation took place only in the PFBR with a nominal residence time of 20 h and a production capacity of 0.2 mL/min. The bioreactor was first tested with suspension MDCK cells at different multiplicities of infection (MOI), and then with suspension avian AGE1.CR.pIX cells at a fixed nominal MOI of 0.02. Maximum hemagglutinin (HA) titers of 2.4 and 1.6 log10(HA units/100 μL) for suspension MDCK cells and AGE1.CR.pIX cells, respectively, were obtained. Flow cytometric analysis demonstrated that 100% infected cells with batch-like HA titers can be obtained at a MOI of at least 0.1. Stable HA and TCID50 titers over 18 days of production were confirmed using the AGE1.CR.pIX cell line, and PCR analysis demonstrated stable production of full-length genome. The contamination level of segments with deletions (potentially defective interfering particles), already present in the virus seed, was low and did not increase. Control experiments using batch and semi-continuous cultures confirmed these findings. A comparison showed that influenza virus production can be achieved with the tubular bioreactor system in about half the time with a space-time-yield up to two times higher than for typical batch cultures. In summary, a novel continuous tubular bioreactor system for cell culture-based influenza virus production was developed. One main advantage, an essentially single-passage amplification of viruses, should enable efficient production of vaccines as well as vectors for gene and cancer therapy.

A feasibility study of utilizing hydrothermal liquefaction derived aqueous phase as nutrients for semi-continuous cultivation of Tetraselmis sp.

The feasibility of substituting 50% nutrients by aqueous phase liquid (APL), derived from hydrothermal liquefaction of Tetraselmis sp. biomass, in a semi-continuous cultivation of Tetraselmis sp. was studied. Growth experiments were conducted in indoor photobioreactor and outdoor raceway tank for three consecutive cycles. At the end of exponential growth pahse, 75% of the culture was harvested, and the supernatant was returned to the cultivation system. For control cultures, fresh nutrients were added; however, for the experimental cultures, an appropriate volume of APL was added to replace half of the nutrients. Either indoor or outdoor, the growth rate and biomass yield in APL-added cultures were either equal or slightly better compared to control culture; although APL had little to no effect on the metabolite content of Tetraselmis sp., metabolites profile of Tetraselmis sp. varied between APL-added and control cultures. Nevertheless, 50% nutrients requirements for microalgae cultivation could be replaced by APL.

Enhanced and Balanced Microalgal Wastewater Treatment (COD, N, and P) by Interval Inoculation of Activated Sludge.

Although chemical oxygen demand (COD) is an important issue for wastewater treatment, COD reduction with microalgae has been less studied compared to nitrogen or phosphorus removal. COD removal is not efficient in conventional wastewater treatment using microalgae, because the algae release organic compounds, thereby finally increasing the COD level. This study focused on enhancing COD removal and meeting the effluent standard for discharge by optimizing sludge inoculation timing, which was an important factor in forming a desirable algae/bacteria consortium for more efficient COD removal and higher biomass productivity. Activated sludge has been added to reduce COD in many studies, but its inoculation was done at the start of cultivation. However, when the sludge was added after 3 days of cultivation, at which point the COD concentration started to increase again, the algal growth and biomass productivity were higher than those of the initial sludge inoculation and control (without sludge). Algal and bacterial cell numbers measured by qPCR were also higher with sludge inoculation at 3 days later. In a semi-continuous cultivation system, a hydraulic retention time of 5 days with sludge inoculation resulted in the highest biomass productivity and N/P removal. This study achieved a further improved COD removal than the conventional microalgal wastewater treatment, by introducing bacteria in activated sludge at optimized timing.

In-pond strain selection of euryhaline Tetraselmis sp. strains for reliable long-term outdoor culture as potential sources of biofuel and other products

Studies on microalgae species and strain selection have demonstrated the potential of a broad range of algal species suitable for large-scale mass production, but provided little data on the reliability and productivity of the cultures under outdoor conditions over a long period. Long-term studies as part of the development of large-scale microalgae mass production are essential at small scale to minimise expenditure in scaling-up and large-scale operations. The current study describes the long-term semi-continuous culture of 4 wild-type isolates of euryhaline Tetraselmis sp. grown in 1 m2 raceway ponds outdoors in 7% (w/w NaCl) saline water enriched with N and P without any additional carbon source under the environmental conditions in Perth, Western Australia. The long-term investigation indicated that Tetraselmis MUR-231 was not able to grow outdoors, while Tetraselmis strains MUR-167, MUR-230 and MUR-233 grew outdoors over long periods. Tetraselmis MUR-167 was cultured for over 16 months outdoors before terminated due to cell clump formation and heavy biofilm development on the pond walls, whereas Tetraselmis strains MUR-230 and MUR-233 were cultured in semi-continuous culture over 20 months. The latter two strains grew at high solar irradiance with daily maximum values of 58890 W m−2 and temperatures ranging from about 40 to 0 °C. Both strains had a similar average specific growth rate of 0.19 day−1 over the experimental period. Maximum biomass productivities were 9.11 g m−2 day−1 and 9.99 g m−2 day−1 for Tetraselmis MUR-230 and MUR-233, respectively. The maximum total lipid productivity of 4.30 g m−2 day−1 of Tetraselmis MUR-230 was slightly higher than Tetraselmis MUR-233 (4.05 g m−2 day−1). These results clearly indicate the potential of these two Tetraselmis strains for commercial application, such as a high lipid yielding feedstock under minimal culture management conditions in arid and semi-arid coastal regions such as northern Western Australia.

Definition of toxicity detergent and disinfectant Sanimol L using infusoria Tetrachymena pyriformis

The results of research of toxicity cleaning and disinfecting products for milking and dairy equipment using as bioindicator infusoria Tetrachymena piriformis were presented. The study was developed by us on the product Sanimol L (active ingredients: cation SAS — 4.0%, alkali — 7.0%, sodium metasilicate — 3.5%, Trilon B — 0.6%) and conducted compared with the available products on the market, in particular, CircoSuper AF (sodium hypochlorite — 4.0%, alkali — 10.0%) and Sulfohlorantin (anion SAS — 35.0%, chlororganic compounds — 36.5% and phosphates — 10.0%). The infusoria were kept in an organic medium by the method of semi-continuous cultivation at a temperature of 22±1°C with weekly replacement part of the medium. To infusoria that located in a nutrient medium on Petri dishes added aqueous solutions of detergents and disinfectants. It was determined that all the research products (Sanimol L, CircoSuper AF and Sulfohlorantin) in 1.0% concentration results in less the number of infusoria and their activity during the first minute. At this concentration in one hour Sulfohlorantin product causes a lethal effect. With influence of CircoSuper AF and Sanimol L on infusoria, their mortality occurred after 3 hours and 24 hours, respectively. At 0.1% concentration solutions of Sanimol L product survival rate of infusoria during 1 hour exposure was similar as in control. Products CircoSuper AF and Sulfohlorantin with that same concentration and exposure caused a decrease in the survival rate of infusoria by an average of 7.0%. After 24 hours, the number of infusoria under the influence of all studied products decreased by 68.0%. With an increase exposure up to 36 hours, product Sulfohlorantin showed a lethal effect and products Sanimol L and CircoSuper AF caused 73.0% infusoria mortality. After 48 hours, the infusoria mortality under the influence of these products was on average 90.0%. Within 56 hours, the products Sanimol L and CircoSuper AF showed a lethal effect. The toxicity product Sanimol L at exposure 24 hours was lower by5.0% and 35.0% compared with the products Circo Super AF and Sulfohlorantin. Toxicological studies on infusoria indicate that Sanimol L is a prospective ecologically safe product and it can be used in dairy cattle breeding for improvement ecological situation in areas of intensive production.

A novel process for the mixotrophic production of lutein with Chlorella sorokiniana MB-1-M12 using aquaculture wastewater

In this study, microalgal cultivation was applied as a feasible strategy for treating shrimp culture wastewater (SCW) from a shrimp farm in southern Tainan. Chlorella sorokiniana MB-1-M12 was first grown on BG-11 medium with 0.5% salinity, obtaining a biomass concentration and productivity of 4.35 g/L and 1.56 g/L/d, respectively. When 80% of BG-11 nutrients were added to 75% strength SCW, lutein content and productivity increased to 5.19 mg/g and 5.55 mg/L/d, respectively. A novel operation strategy involving periodic exchange of freshwater and SCW was designed for semi-continuous cultivation of MB-1-M12 strain for optimal biomass and lutein production. The average biomass concentration, productivity, lutein content, and productivity were 3.5 g/L, 1.3 g/L/d, 3.89 mg/g and 5.0 mg/L/d, respectively. Although microalgae have been considered as an alternative natural source of lutein, this work is among the earliest reports describing lutein production from microalgae cultivated with wastewater via a circular economy concept.

Solid state fermentation pilot-scaled plug flow bioreactor, using partial least square regression to predict the residence time in a semicontinuous process

Plug flow bioreactor (PFB) used in solid state fermentation gives the possibility to have semicontinuous culture. However, it is complicated to follow a single particle during its residence inside the bioreactor and to therefore study precisely the culture process all along the device. In this study, semicontinuous production of fungal compounds was successfully obtained by cultivating Aspergillus niger with a PFB prototype. Kinetic productions of the same metabolites along the bioreactor were obtained at laboratory scale to predict the residence time of the particles inside the PFB from Partial Least Square (PLS). This original methodology allowed (1) the comparison between the production kinetics at the two production scales (pilot and laboratory) and (2) the observation of eventual changes following the scale-up of the process offering a good overall insight of the PFB performance.

A scale-up evaluation of a semicontinuous culture of Scenedesmus sp. in a raceway under greenhouse conditions using a commercial fertilizer as culture medium

In the present work, Scenedesmus sp. was grown under a semicontinuous regime in fertilizer (Bayfolan® Forte, 1 mL L−1) medium, and Bold’s Basal Medium (BBM) as control, in raceways of 200 and 2000 L under greenhouse conditions. The biomass, lipid, protein, carbohydrate and total pigment production was determined. The culture under the semicontinuous regime allowed an increase in the productivities in each consecutive cycle, reaching a lipid productivity of 20.00 ± 6.29 mg L−1 d−1 in Bayfolan medium. The biomass productivity in the medium supplemented with fertilizer was significantly higher than in the BBM. The protein productivity was significantly higher in Bayfolan (1.93 ± 0.28 L−1 d−1). The average carbohydrate productivity of the cycles was 10.10 ± 2.01 mg L−1 d−1. The fertilizer allowed an increase of up to 3-fold in total pigment productivity compared to the BBM. The fatty acids obtained were mainly composed of C16:0, C18:3n3 (α-linolenic) and C18:0. Palmitic acid (C16:0) was the main fatty acids methyl ester accumulated along with stearic (C18:0) and myristic (C14:0) acids. It was shown that the utilization of the liquid fertilizer Bayfolan is a potential alternative to replace conventional media.

Screening of Isochrysis strains for simultaneous production of docosahexaenoic acid and fucoxanthin

Microalgae are capable of synthesizing a wide range of value-added compounds such as docosahexaenoic acid (DHA) and fucoxanthin. In the present study, the potential for simultaneous production of DHA and fucoxanthin by the marine alga Isochrysis was investigated via strain screening and culture optimization. A comparative analysis of sixteen Isochrysis strains revealed that CCMP1324 was superior with regard to growth and production of DHA and fucoxanthin. In response to various nutritional and environmental cues, CCMP1324 accumulated DHA and fucoxanthin in a coordinated and growth-dependent manner, which reached ~17.1 and 14.1 mg g−1, respectively. A semi-continuous culture strategy leading to high productivities of DHA (9.05 mg L−1 day−1) and fucoxanthin (7.96 mg L−1 day−1) was developed. Taken together, our study represents a pioneering work studying the accumulation coordination between DHA and fucoxanthin in Isochrysis. These findings provide valuable insights into future exploration for the integrated production of high-value compounds.

Effect of ocean acidification on the nutritional quality of marine phytoplankton for copepod reproduction.

Phytoplankton are the oceans’ principal source of polyunsaturated fatty acids that support the growth and reproduction of consumers such as copepods. Previous studies have demonstrated ocean acidification (OA) can change the availability of polyunsaturated fatty acids to consumer diets which may affect consumer reproduction. Two laboratory experiments were conducted to examine the effects of feeding high-pCO2-reared phytoplankton on copepod egg production, hatching success, and naupliar survival. Marine phytoplankton Rhodomonas salina, Skeletonema marinoi, Prorocentrum micans, and Isochrysis galbana were exponentially grown in semi-continuous cultures at present (control) (400 ppm CO2, pH~8.1) and future (1,000 ppm CO2, pH~7.8) conditions and provided to Acartia tonsa copepods over 4 consecutive days as either nitrogen-limited (Exp. I) or nitrogen-depleted (Exp. II) mixed assemblage of phytoplankton. The composition of FAs in the phytoplankton diet was affected by pCO2 concentration and nitrogen deficiency; the ratio of essential fatty acids to total polyunsaturated fatty acids decreased in phytoplankton grown under high pCO2 and the mass of total fatty acids increased under nitrogen depletion. Additionally, total concentrations of essential fatty acids and polyunsaturated fatty acids in the diet mixtures were less under the high-pCO2 compared to the control-pCO2 treatments. Median egg production, hatching success, and naupliar survival were 48–52%, 4–87%, and 9–100% lower, respectively, in females fed high-pCO2 than females fed low-pCO2 phytoplankton, but this decrease in reproductive success was less severe when fed N-depleted, but fatty acid-rich cells. This study demonstrates that the effects of OA on the nutritional quality of phytoplankton (i.e., their cellular fatty acid composition and quota) were modified by the level of nitrogen deficiency and the resulting negative reproductive response of marine primary consumers.

Enhancement of C-phycocyanin productivity by Arthrospira platensis when growing on palm oil mill effluent in a two-stage semi-continuous cultivation mode

Palm oil mill effluent (POME) is well known as agricultural wastewater that has a high potential as a medium for microalgal growth due to its high macro- and micronutrient content. The cyanobacterium Arthrospira platensis is considered as a species with a high C-phycocyanin (C-PC) content which is important for fine chemical and pharmaceutical applications. However, cultivation of A. platensis on POME to produce economically feasible amounts of C-PC has not been well explored. For this, environmental, nutritional, and cultivation modes (batch, semi-continuous) were varied to optimize C-PC productivity when cultivated at various POME concentrations. Arthrospira platensis was found to grow well on POME. Highest biomass and C-PC concentrations were found on 30–100% POME. Central composite rotatable design (CCRD) response surface methodology demonstrated that C-PC productivity was influenced by urea addition at the optimum salinity. The highest C-PC productivity was found on 100% POME during semi-continuous cultivation, while the addition of phosphorus and urea did not significantly improve C-PC productivity. By applying semi-continuous cultivation with 50% POME at the first stage and 100% POME at the second stage, a similarly high C-PC productivity (4.08 ± 1.3 mg L−1 day−1) was achieved as compared with (artificial) Zarrouk medium during batch cultivation. We conclude that, when using a two-stage semi-continuous cultivation process, A. platensis can produce economically feasible amounts of C-PC when cultivated on 100% POME.

Long-term reliable culture of a halophilic diatom, Amphora sp. MUR258, in outdoor raceway ponds

The microalgal industry as a source of high-value products (i.e. β-carotene, astaxanthin) was established over 50 years ago. However, only a very small number of species have been commercialised. There is a need for new species and new products to expand this industry. The objective of this study was to examine the reliability and productivities of long-term outdoor culture of a newly isolated halophilic diatom, Amphora sp. MUR258 (Bacillariophyceae), in raceway ponds in Perth, Western Australia. The Amphora sp. was grown in outdoor raceway ponds as a semi-continuous culture for about 13 months at a culture salinity between 8.6 and 14.9% (w/v) NaCl. The highest cell density (167 × 104 cells mL−1), specific growth rate (0.29 day−1) and biomass and lipid productivities (24 and 6.8 g m−2 day−1, respectively) were achieved in summer. The annual average of biomass (ash-free dry weight) and lipid productivities was 7 and 2.2 g AFDW m−2 day−1, respectively. Minor contamination by a Navicula sp. was seen during winter, but was not a significant problem. No major protozoan contamination was seen. These results indicate the potential of reliable large-scale cultivation of Amphora sp. MUR258 as a potential source of diatom lipid and/or fucoxanthin.

A natural algal polyculture outperforms an assembled polyculture in wastewater-based open pond biofuel production

Use of diverse algal polycultures could enhance the viability of mass algal cultivation by increasing the productivity and stability of production. However, there are multiple approaches for selecting productive polycultures, ranging from a synthetic ecology approach using carefully selected laboratory strains, to collection and cultivation of naturally occurring polycultures. In this study, we compared the performance of a systematically assembled and a naturally occurring polyculture under two conditions: 1) pilot-scale, semi-continuous culture conditions and 2) grazing and resource stress conditions. The naturally occurring polyculture had 25% higher mean biomass productivity and 19% higher N-removal efficiency than the assembled polyculture under semi-continuous culture. Differences became even more pronounced under grazing and resource stress conditions; mean biomass productivity was 60% higher in the naturally occurring polyculture when exposed to stress. Lipid and protein content (% dry mass) were significantly higher in the assembled polyculture, while carbohydrate content was higher in the naturally occurring polyculture. The naturally occurring polyculture maintained higher diversity than the assembled polyculture throughout the experiment, suggesting a greater degree of niche complementarity as a potential mechanism for differences in biomass yields and nutrient uptake. Our results show that systematically engineered algal communities may not always lead to greater productivity than algal communities found in nature per se. We suggest that future studies using polycultures assembled from lab strains should test them against those occurring naturally; collectively, this would both serve as a benchmark for progress in synthetic ecology and would test whether there are generalities in the performance of natural vs. assembled polycultures.

Nutritional value and production performance of the rotifer Brachionus plicatilis Müller, 1786 cultured with different feeds at commercial scale

The rotifer Brachionus plicatilis is the first live feed in larviculture of marine fish species. Rotifer diets differ in their biochemical composition, physical properties, and production technology while feeding protocols largely vary among facilities. The objective of the present study was to determine the effects of two different forms of Nannochloropsis oculata and commonly used commercial diets on growth performance and biochemical composition of rotifers produced under commercial conditions. Rotifers were fed one of five different types of feed: Algome® (dried Schizochytrium sp.), Protein Plus® (PP), Inactive Baker’s Yeast® (INBY), spray-dried Nannochloropsis oculata (SDN), or freshly cultured Nannochloropsis oculata (FN). Rotifers fed SDN diet resulted in significantly higher rotifer biomass during 16 days of semi-continuous culture, with an increasing biomass trend that lasted 11 days, high egg production, and egg-carrying female numbers, whereas rotifers fed PP showed highest ∑n-3, arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid contents. Amino acid profiles of rotifers were enhanced by utilization of both INBY and SDN diets. Overall, the results indicated that SDN is optimal for long-term biomass production of rotifers. However, their nutritional profile needs to be enriched by feeding PP (EFA source) and INBY (EAA source) once desired biomass production is obtained.

Influence of Nitrogen Limitation on Lipid Accumulation and EPA and DHA Content in Four Marine Microalgae for Possible Use in Aquafeed

Microalgae are regarded as a promising alternative that can replace fishmeal and fish oil in aquaculture. Under N-limitation, many microalgae species change their carbon storage patterns in favor of neutral lipids (NLs) mainly in the form of triacylglycerol (TAG), but fatty acids in polar lipids (PL) are nutritionally more available for fish than those esterified into NLs. In the present study, the effect of N-limitation on the lipid content and fatty acid profiles in different lipid class of Phaeodactylum tricornutum, Isochrysis aff. galbana clone T-Iso, Rhodomonas baltica and Nannochloropsis oceanica were investigated. The microalgae cells were cultivated by two different methods, batch and semi-continuous culture, to create strong and moderate N-limitation, and this in turn will significantly affect the biomass and lipid productivity. All four species accumulated lipids mainly in the form of TAG in response to strong nitrogen limitation. N. oceanica, however accumulated 51% of the dry weight as lipid in moderate nitrogen limitation and up to 87% of the fatty acid was in TAG. Isochrysis aff. galbana clone T-Iso was the only species where the fraction of polyunsaturated fatty acid (PUFA), especially the fraction of docosahexaenoic acid (DHA) increased with increasing nitrogen limitation. Total lipid productivity showed no increase in batch culture although stronger nitrogen limitation led to lipid accumulation. P. tricornutum had the highest eicosapentaenoic acid (EPA) content, while N. oceanica showed the highest EPA productivity due to the high content of lipid. The highest DHA productivity was found in Isochrysis aff. galbana clone T-Iso from moderate N-limitation mainly due to the high biomass productivity. Based on the results from the current study, N. oceanica and T-Iso are two promising microalgae strains as long-term sustainable source of n-3 long chain -PUFAs under moderate N-limitation. As shown in the present study, increased lipid content in microalgal cells due to strong N-limitation induction may not increase the lipid productivity because biomass production usually is reduced. Therefore, a combination of approaches such as metabolic engineering, conditioning and selection may be needed to further increase the n-3 LC-PUFA productivity without substantial loss of biomass.

Growth and Nutrient Utilization of Green Algae in Batch and Semicontinuous Autotrophic Cultivation Under High CO2 Concentration.

The growth performance of Chlorella protothecoides, Chlorella pyrenoidosa, and Chlorella sp. in autotrophic cultivation with 10% carbon dioxide (CO2) was evaluated. The biomass production of C. protothecoides, along with its carbon, nitrogen (N), and phosphorus (P) utilization, in batch and semicontinuous autotrophic cultivation with 20% CO2 was also determined. Among the three algae species, C. protothecoides obtained the highest biomass yield (1.08g/L) and P assimilation (99.4%). Compared with the CO2 flow rate and inoculation ratio in batch cultivation, light intensity considerably improved biomass yield, N and P assimilation, and CO2 utilization. In the semicontinuous cultivation of C. protothecoides, a hydraulic retention time (HRT) of 8days kept the system at a stable running state, thereby demonstrating that an HRT of 8days was better than an HRT of 5days. Among the three N/P ratios for C. protothecoides in semicontinuous cultivation with 20% CO2, 2:1 provided the highest biomass productivity (0.19g/L/day) and CO2 fixation rate (0.37g/L/day). Therefore, this lower N/P ratio is more suitable than 10:1 and 50:1 for the growth of C. protothecoides with 20% CO2. Compared with the batch cultivation of C. protothecoides, semicontinuous cultivation improved the CO2 fixation rate (by 1.5-2 times) and CO2 utilization efficiency (by 3-6 times) of C. protothecoides.

Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies

A lutein-enriched mutant, Chlorella sorokiniana MB-1-M12 was grown mixotrophically for lutein production. The lutein production efficiency of the strain was enhanced via optimizing the operating strategies. The results show that using semi-continuous cultivation with a medium replacement ratio of 75% resulted in a higher lutein productivity and lutein concentration of 6.24 mg/L/d and 50.6 mg/L, respectively, which were markedly higher than those obtained from batch and fed-batch cultivation. Cultivation under simulated outdoor cultivation conditions (i.e., temperature of 35 °C/25 °C for a 12 h/12 h light/dark cycle) could achieve the highest lutein productivity and lutein concentration of 3.34 mg/L/d and 30.8 mg/L, respectively. Lutein production via outdoor cultivation of MB-1-M12 strain with a 60-L tubular photobioreactor was performed using semi-continuous operation. With a medium replacement ratio of 75%, a good lutein productivity (4.46 mg/L/d) and concentration (27.4 mg/L) was obtained, indicating the feasibility of producing lutein under outdoor cultivation of the microalgal strain.