Two-stage Culture Research Articles

Environmental Regulation of PndbA600, an Auto-Inducible Promoter for Two-Stage Industrial Biotechnology in Cyanobacteria.

Cyanobacteria are photosynthetic prokaryotes being developed as sustainable platforms that use renewable resources (light, water, and air) for diverse applications in energy, food, environment, and medicine. Despite the attractive promise that cyanobacteria offer to industrial biotechnology, slow growth rates pose a major challenge in processes which typically require large amounts of biomass and are often toxic to the cells. Two-stage cultivation strategies are an attractive solution to prevent any undesired growth inhibition by de-coupling biomass accumulation (stage I) and the industrial process (stage II). In cyanobacteria, two-stage strategies involve costly transfer methods between stages I and II, and little work has been focussed on using the distinct growth and stationary phases of batch cultures to autoregulate stage transition. In the present study, we identified and characterised a growth phase-specific promoter, which can serve as an auto-inducible switch to regulate two-stage bioprocesses in cyanobacteria. First, growth phase-specific genes were identified from a new RNAseq dataset comparing two growth phases and six nutrient conditions in Synechocystis sp. PCC 6803, including two new transcriptomes for low Mg and low K. A type II NADH dehydrogenase (ndbA) showed robust induction when the cultures transitioned from exponential to stationary phase growth. Behaviour of a 600-bp promoter sequence (PndbA600) was then characterised in detail following the expression of PndbA600:GFP in Synechococcus sp. PCC 7002. Culture density and growth media analyses showed that PndbA600 activation was not dependent on increases in culture density per se but on N availability and on another activating factor present in the spent media of stationary phase cultures (Factor X). PndbA600 deactivation was dependent on the changes in culture density and in either N availability or Factor X. Electron transport inhibition studies revealed a photosynthesis-specific enhancement of active PndbA600 levels. Our findings are summarised in a model describing the environmental regulation of PndbA600, which can now inform the rational design of two-stage industrial processes in cyanobacteria.

Novel, automated, semi-industrial modular photobioreactor system for cultivation of demanding microalgae that produce fine chemicals—The next story of H. pluvialis and astaxanthin

Recently, there has been increased interest in the use of microorganisms in the production of pharmaceuticals, nutraceuticals and energy supply products, which is due to their rapid growth rate and ability to biosynthesize fine chemicals or biotransform specific xenobiotics. To achieve the desired scale of production and optimization of microbial cultures, it is necessary to design bioreactors that enable process automation, control of working parameters, reduction of microbial and chemical contaminations, and culture independence of climate conditions. In response to this need, an original, modular airlift-type photobioreactor system was designed and manufactured. This novel semitechnical system, with a total volume of 1000 dm3, was operated via computer control, which enabled the creation of time profiles of red, blue and white LED illumination and of carbon dioxide and air dosing. The quality and usefulness of the developed system was demonstrated via the case study, namely two-stage cultivation of the microalgae Haematococcus pluvialis—a species commonly used in the production of natural astaxanthin. The experimentally developed procedure ensures a repeatable and efficient biomass multiplication process and the maintenance of a light- and chemical-mediated effective stress mechanism that allows the production of up to 3.2% natural astaxanthin in terms of dry biomass.

Two-stage cultivation of Chlorella vulgaris using light and salt stress conditions for simultaneous production of lipid, carotenoids, and antioxidants

The effects of light and salt stress (NaCl and MgCl2) on the two-stage cultivation of Chlorella vulgaris, with or without a medium replacement, for the simultaneous production of lipid, carotenoids, and antioxidant compounds were investigated. The highest lipid productivity (15.59 ± 0.10 mg L−1 day−1) was obtained at 5 g L−1 MgCl2 and 140 μmol photons m−2 s−1 light intensity. The saturated fatty acids (SFA) ranged from 52.35–81.64%, monounsaturated fatty acids (MUFA) from 7.38–34.26%, and polyunsaturated fatty acids (PUFA) from 7.25–25.10%, with palmitic (C16:0), stearic (C18:0), and oleic (C18:1) acids as predominant fatty acids. Under high light intensity and nitrogen limitation in the two-stage cultivation, supplementation of 10 g L−1 NaCl with a medium replacement caused a marked increase in the total carotenoids (4.37 ± 0.33 μg mL−1). Cultivation of C. vulgaris in a medium containing 5 g L−1 NaCl or Mg Cl2, with or without a medium replacement step and with exposure to 140 μmol photons m−2 s−1 light intensity, led to enhanced antioxidant activities (65–79%). The different levels of antioxidant activities of the C. vulgaris extracts suggested the variation in the phytochemical compounds, a result of the stressed conditions.

Construction and optimization of engineered Bacillus subtilis for surfactin production

Surfactin has great potential applications in enhancing oil recovery, agriculture, pharmaceuticals, foods and beverages, and cosmetics due to its extraordinary surface activity, biodegradability, anti-bacterial activity and biocompatibility. Enhancing surfactin production by engineering surfactin-producer and optimizing culture conditions is the key of its industrial production and subsequent applications. In this study, the effect of fatty acid synthesis pathway on surfactin synthesis was investigated, and Bacillus subtilis THBS-2 and THBS-8 with high surfactin titer were constructed by overexpressing key genes involved in the fatty acid synthesis pathway. To optimize culture condition, the amount and adding time of isopropyl-beta-D-thiogalactopyranoside (IPTG) and amino acids were studied, and a two-stage culture method was obtained: IPTG (final concentration: 1.25 mmol/L) and leucine (final concentration: 5 g/L) were added at 3 h, leucine (final concentration 5 g/L) and condensed culture medium (5 mL) were added at 24 h. Applying this strategy, the surfactin titer of B. subtilis THBS-2 reached to 24 g/L in shake flask at 48 h and up to 34 g/L after 68 h fermentation in a 30-L fermentor. The results provide basis for large-scale production and broad application of surfactin.

Triton X-100 improves co-production of β-1,3-D-glucan and pullulan by Aureobasidium pullulans.

The effects of several surfactants on the biosynthesis of β-1,3-D-glucan (β-glucan) and pullulan by Aureobasidium pullulans CCTCC M 2012259 were investigated, and Triton X-100 was found to decrease biomass formation but increase β-glucan and pullulan production. The addition of 5g/L Triton X-100 to the fermentation medium and bioconversion broth significantly increased β-glucan production by 76.6% and 69.9%, respectively, when compared to the control without surfactant addition. To reveal the physiological mechanism underlying the effect of Triton X-100 on polysaccharides production, the cell morphology and viability, membrane permeability, key enzyme activities, and intracellular levels of UDPG, NADH, and ATP were determined. The results indicated that Triton X-100 increased the activities of key enzymes involved in β-glucan and pullulan biosynthesis, improved intracellular UDPG and energy supply, and accelerated the transportation rate of precursors across the cell membrane, all of which contributed to the enhanced production of β-glucan and pullulan. Moreover, a two-stage culture strategy with combined processes of batch fermentation and bioconversion was applied, and co-production of β-glucan and pullulan in the presence of 5g/L Triton X-100 additions was further improved. The present study not only provides insights into the effect of surfactant on β-glucan and pullulan production but also presents a feasible approach for efficient production of analogue exopolysaccharides. KEY POINTS: • Triton X-100 increased β-glucan and pullulan production under either batch fermentation or bioconversion. • Triton X-100 increased the permeability of cell membrane and accelerated the transportation rate of precursors across cell membrane. • Activities of key enzymes involved in β-glucan and pullulan biosynthesis were increased in the presence of Triton X-100. • Intracellular UDPG levels and energy supply were improved by Triton X-100 addition.

Production of 3-hydroxypropionic acid from acetate using metabolically-engineered and glucose-grown Escherichia coli

Acetate can be used as carbon feedstock for the production of 3-hydroxypropionic acid (3-HP), but the production level was low due to inefficient cell growth on acetate. To better utilize acetate, a two-stage strategy, whereby glucose is used for cell growth and acetate for 3-HP formation, was attempted. Dissected malonyl-CoA reductase of Chloroflexus aurantiacus, alone or along with acetyl-CoA carboxylase and/or transhydrogenase, was overexpressed, and by-products formation pathway, glyoxylate shunt (GS) and gluconeogenesis were modified. When GS or gluconeogenesis was disrupted, cell growth on glucose was not hampered, while on acetate it was completely abolished. Consequently, 3-HP production, at production stage, were low, though 3-HP yield on acetate was increased, especially in the case of aceA deletion. In two-stage bioreactor, strain with upregulated GS produced 7.3 g/L 3-HP with yield of 0.26 mol/mol acetate. This study suggests that two-stage cultivation is a good strategy for 3-HP production from acetate.

Production of TiO2-deposited Diatoms and Their Applications for Photo-catalytic Degradation of Aqueous Pollutants

Biogenic TiO2 diatoms can be prepared by metabolic incorporation of TiO2 on the frustule surface of the diatoms. To utilize the photo-catalytic activity of biogenic TiO2 for practical applications, high production of TiO2-deposited diatoms is required. Here, we evaluated the growth rate and biogenic TiO2 production of several domestic diatom species. Among the tested diatoms, Caloneis schroederi (CS) showed high biomass production (4.01 g/L) in a two-stage cultivation system and proper exoskeletal rigidity for efficient production of biogenic TiO2. The dry weight of the TiO2-deposited CS frustules (biogenic CS-TiO2) was 4.52 g/L. Biogenic CS-TiO2 showed significant photo-catalytic degradation of Congo Red in water with an efficiency of 27.8%, which is higher than that of standard TiO2 (17.4%). In addition, chloroform could be removed from water through the photo-catalytic activity of biogenic CS-TiO2 (91%). Therefore, the biogenic CS-TiO2 developed in this study can be employed for the treatment of pollutant-contaminated wastewater.

Enhanced polyhydroxybutyrate production through incorporation of a hydrogen fuel cell and electro-fermentation system

The integration of a hydrogen fuel cell with an electro-fermentation system represents a novel approach for improving polyhydroxybutyrate (PHB) accumulation in Ralstonia eutropha H16, using a sustainable energy source. In this study, electro-fermentation noticeably affected cell growth, biomass production, substrate consumption, and PHB accumulation. Final residual biomass concentrations and maximum specific growth rates were enhanced by supplying a 10-mA electric current. Furthermore, a remarkable enhancement in PHB content (30% higher than control) was achieved by redox-mediated electro-fermentation with a 10 mA electric current, upon the addition of a redox mediator. Two-stage cultivation limited the growth suppression caused by redox-mediated electro-fermentation, and also increased the maximum PHB productivity of the system. The additional electrons supplied upon supplementation of the redox mediator accelerated the glycolytic pathway and redox cycling of NADH/NAD+, led to a spontaneous boost for adenosine triphosphate (ATP) generation, and further facilitated the biosynthesis of PHB.

Optimization of the production of lipids and carotenoids in the microalga Golenkinia aff. brevispicula

Microalgae are a promising platform to produce natural pigments and lipids when compared to traditional sources, since they present faster growth and biomass productivity and there is a wide diversity that has not been studied yet. The green microalgae belonging to the genus Golenkinia presents few reports of its biomass production and applications. In this work, the productivity of lipids and carotenoids was studied in the strain Golenkinia aff. brevispicula FAUBA-3 using a two-stage cultivation system: a first step for biomass production under semicontinuous cultures, and a second step for the accumulation of lipids and carotenoids under batch salinity stress (35 g L−1). The highest biomass productivity (0.92 g L−1 day−1) was obtained under a dilution rate of 0.4 day−1. The growth rate (μ) was a hyperbolic function of average irradiance inside the culture (Eav). The optimum Eav was 88.4 μmol photons m−2 s−1 and the maximum specific growth rate (μmax) was 0.85 day−1. Photoinhibition was observed above 570 μmol photons m−2 s−1 of Eav. The best photosynthetic performance was obtained at 0.2 day−1 dilution rate but with lower biomass productivity due to nutrients and light limitation. High content of protein (30% DW) and polyunsaturated fatty acids (50% of total PUFAs) and low content of carbohydrates (19% DW) were observed at high dilution rate, and the opposite response at low dilution rate. In the second culture stage, an increase of salinity induced lipid and carotenoid accumulation which improved the productivity to 89 mg L−1 day−1 and 1.3 mg L−1 day−1 of total lipids and carotenoids respectively compared to one-stage culture. Lutein, β-carotene and astaxanthin were the main carotenoid identified in a decreasing order. Those results confirm that two-stage culture is an adequate strategy to optimize the carotenoid and lipid productivity in the promising strain Golenkinia FAUBA-3.

Effect of plant growth regulators on growth and lipid accumulation of microalgal Haematococcus pluvialis Flotow in two-stage culture

Haematococcus pluvialis cells were cultured in aerated liquid Bold’s Basal medium in two-stage (initial stage during in 7 weeks for increased biomass growth and second stage during in 3 weeks for increased lipid accumulation) with different volumes 250 mL, 10 L, and 1,000 L. With a volume of 250 mL, the medium was supplied with benzyl adenine (BA), indole-3-acetic acid (IAA) or gibberellic acid (GA3) at concentration from 0.1 - 0.2 mg/L in initial stage and IAA or GA3 at concentration from 0.1 - 0.2 mg/L in second stage. After 10 weeks of culture, results showed that supplement of 0.1 mg/L BA in initial stage and 0.125 mg/L IAA in second stage increased cell density, and microalgal cells had green color with a spherical shape. On the contrary, supplement of 0.15 mg/L IAA in initial stage and 0.175 mg/L GA3 in second stage increased lipid accumulation, and microalgal cells had red color with a spherical shape. With a volume of 10 L, the medium was supplied with 0.1 mg/L BA in initial stage, and treated with separation or combination from 2 - 3 of these factors (nitrogen starvation, 0.5% NaCl, 4.98 mg/L FeSO4) were applied in second stage. The result showed that the cultures was treated with nitrogen starvation increased dry biomass and biofuel, but treated with 4.98 mg/L FeSO4 only increased biofuel. With a volume of 1,000 L, microalgal cells were cultured in BB liquid medium in initial stage, and treated with 4.98 mg/L FeSO4 increased fresh 78.67 mg/mL and dry biomass 2.05 mg/L and total lipid content 28.24 %/ DW.

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.

Nitrogen Deficiency-Dependent Abiotic Stress Enhances Carotenoid Production in Indigenous Green Microalga Scenedesmus rubescens KNUA042, for Use as a Potential Resource of High Value Products

The microalgal strain Scenedesmus rubescens KNUA042 was identified in freshwater in Korea and characterized by evaluating its stress responses in an effort to increase lipid and carotenoid production. Under a two-stage cultivation process, the algal strain that generally exhibits optimal growth at a nitrate (source of nitrogen) concentration of 0.25 g L−1 was challenged to different exogenous stimuli—salinity (S), light intensity (L), combined L and S (LS), and nitrogen deficiency (C)—for 14 days. Lipid production and carotenoid concentration increased in a time-dependent manner under these physicochemical conditions during the culture periods. Lipid accumulation was confirmed by thin layer chromatography, BODIPY staining, and fatty acid composition analysis, which showed no differences in the algal cells tested under all four (C, S, L, and LS) conditions. The quality of biodiesel produced from the biomass of the algal cells met the American Society for Testing and Materials and the European standards. Total carotenoid content was increased in the LS-treated algal cells (6.94 mg L−1) compared with that in the C-, S-, and L-treated algal cells 1.75, 4.15, and 1.32 mg L−1, respectively). Accordingly, the concentration of canthaxanthin and astaxanthin was also maximized in the LS-treated algal cells at 1.73 and 1.11 mg g−1, respectively, whereas lutein showed no differences in the cells analyzed. Conversely, chlorophyll a level was similar among the C-, S-, and LS-treated algal cells, except for the L-treated algal cells. Thus, our results suggested that S. rubescens KNUA042 was capable of producing carotenoid molecules, which led to the maximum values of canthaxanthin and astaxanthin concentrations when exposed to the combined LS condition compared with that observed when exposed to the salinity condition alone. This indicates that the algal strain could be used for the production of high-value products as well as biofuel. Furthermore, this article provides the first evidence of carotenoid production in S. rubescens KNUA042.

Optimization of growth and EPS production in two Porphyridum strains

Due to the relatively low yield of exopolysaccharides from microalgae compared to other microorganisms, their production is more challenging and needs to be optimized. Porphyridium sordidum and Porphyridium purpureum growth and exopolysaccharide production were optimized by testing influence of light at different wavelengths and varying nutrients as well as abiotic factors. White light was identified as the most suitable for both strains concerning growth and exopolysaccharide production. CaCl2 was identified to have the greatest influence on growth and exopolysaccharide production for P. sordidum; NaCl and KH2PO4 revealed to be most beneficial for P. purpureum. The cultivation medium was optimized to enhance biomass production using the Taguchi design. Additionally, the most prominent abiotic factors for exopolysaccharide induction were determined. Finally, the results were combined in an optimized two-stage cultivation and increased EPS production by 3.4 times for P. sordidum and by 1.2 times for P. purpureum compared to the initial conditions.

Economical lipid production from crude glycerol using Rhodosporidiobolus fluvialis DMKU-RK253 in a two-stage cultivation under non-sterile conditions

To enhance the microbial lipid production through Rhodosporidiobolus fluvialis DMKU-RK253, a two-stage cultivation process with a focus on cost reduction was developed. Low-cost raw materials, which are biodiesel-derived crude glycerol and monosodium glutamate were utilized to produce microbial lipids. A two-stage cultivation process was performed under non-sterile cultivation, and a response surface methodology was used to determine the effects of the interaction among factors in the first and second stages of cultivation while using shaking flask cultivation. In the first stage of cultivation, cells were cultivated in a nitrogen-rich medium for cell mass production. The optimal crude glycerol (30 g/L) and monosodium glutamate (5 g/L) concentrations, and the inoculum age (96 h), were found to improve cell mass production. In the second stage, a non-sterile crude glycerol solution was used to enhance lipid production without any aseptic techniques. An optimal crude glycerol concentration of 57 g/L with an initial pH of 7 and an initial cell concentration of optical density at 600 nm of 20 resulted in a high lipid production of 27.81 ± 1.86 g/L and maximum cell mass of 39.14 ± 0.08 g/L. These high lipid and cell mass concentrations consequent to achieve a high lipid content of 71.42% of the dry cell mass, a lipid yield of 0.34 g/g glycerol and a cell mass yield of 0.48 g/g glycerol. The low-cost raw materials were estimated to be US$0.80/kg for microbial lipid production. Therefore, a potential condition for the cultivation process of yeast that produces a high amount of inexpensive lipids has been demonstrated.

An unconventional two-stage cultivation strategy to increase the lipid content and enhance the fatty acid profile on Chlorella minutissima biomass cultivated in a novel internal light integrated photobioreactor aiming at biodiesel production

Biodiesel quality is dependent upon the fatty acids distribution of the raw material used in the trans/esterification reactions. While microalgae cells are regarded as potential oil suppliers for the biodiesel production chain, a common criticism is related to the low lipid productivities and often to a poor fatty acid distribution. In the present work we propose to submit microalgal cultivation to a second stage with nutritional stress by a simple addition of a 70% nitrate source depleted medium at the beginning of stationary phase, without harvesting cells, saving time and water. Chlorella minutissima was cultivated in a novel unconventional photobioreactor, named Internally Illuminated Integrated Photobioreactor (ILI-PBR), to verify the efficiency of the proposed strategy. Biomass concentration, lipid content and volumetric lipids productivity increased 24.9%, 120.7% and 55.6%, respectively, from Stage 1 to Stage 2, when ILI-PBR was illuminated by constant blue LED and 0.5 vvm of aeration. The fatty acid profiles, evaluated in terms of LED illumination (white, red and blue), showed a predominance of palmitic acid in all cases (59.49%, 57.28% and 46.51%, respectively), which allowed to enhance some biodiesel properties such as cetane number, cold filter plugging point, density and kinematic viscosity, according to international specifications.

Lipid Production from Amino Acid Wastes by the Oleaginous Yeast Rhodosporidium toruloides

Microbial lipids have been considered as promising resources for the production of renewable biofuels and oleochemicals. Various feedstocks, including sugars, crude glycerol, and volatile fatty acids, have been used as substrates for microbial lipid production, yet amino acid (AA) wastes remain to be evaluated. Here, we describe the potential to use AA wastes for lipid production with a two-stage culture mode by an oleaginous yeast strain Rhodosporidium toruloides CGMCC 2.1389. Each of the 20 proteinogenic AAs was evaluated individually as sole carbon source, with 8 showing capability to facilitate cellular lipid contents of more than 20%. It was found that L-proline was the most favored AA, with which cells accumulated lipids to a cellular lipid content of 37.3%. When blends with AA profiles corresponding to those of meat industry by-products and sheep viscera were used, the cellular lipid contents reached 27.0% and 28.7%, respectively. The fatty acid compositional analysis of these lipid products revealed similar profiles to those of vegetable oils. These results, thus, demonstrate a potential route to convert AA wastes into lipids, which is of great importance for waste management and biofuel production.

Lipid Production from Sugarcane Top Hydrolysate and Crude Glycerol with Rhodosporidiobolus fluvialis using a Two-Stage Batch-Cultivation Strategy with Separate Optimization of Each Stage.

Lipids from oleaginous microorganisms, including oleaginous yeasts, are recognized as feedstock for biodiesel production. A production process development of these organisms is necessary to bring lipid feedstock production up to the industrial scale. This study aimed to enhance lipid production of low-cost substrates, namely sugarcane top and biodiesel-derived crude glycerol, by using a two-stage cultivation process with Rhodosporidiobolus fluvialis DMKU-SP314. In the first stage, sugarcane top hydrolysate was used for cell propagation, and in the second stage, cells were suspended in a crude glycerol solution for lipid production. Optimization for high cell mass production in the first stage, and for high lipid production in the second stage, were performed separately using a one-factor-at-a-time methodology together with response surface methodology. Under optimum conditions in the first stage (sugarcane top hydrolysate broth containing; 43.18 g/L total reducing sugars, 2.58 g/L soy bean powder, 0.94 g/L (NH4)2SO4, 0.39 g/L KH2PO4 and 2.5 g/L MgSO4 7H2O, pH 6, 200 rpm, 28 °C and 48 h) and second stage (81.54 g/L crude glycerol, pH 5, 180 rpm, 27 °C and 196 h), a high lipid concentration of 15.85 g/L, a high cell mass of 21.07 g/L and a high lipid content of 73.04% dry cell mass were obtained.

Bacteriophage amplification – A comparison of selected methods

The bactericidal properties of bacteriophages have been used almost since the moment of the discovery of bacterial viruses. In the light of the rapidly growing number of antibiotic-resistant bacteria, phage therapy is considered one of the most promising alternatives to classical treatment. Phage amplification is one of the most common procedures of working with phages, and high-titer preparations are beneficial at the experimental stage of studies as well as in practice. The objective of this study was to compare five commonly applied methods of phage amplification: (i) pooled plaques method, (ii) the plate wash method, (iii) the agar culture method, (iv) the two-stage culture method, and (v) in liquid culture. All methods were tested for fifteen different phages. The results described herein indicate that there is no optimal, universal method for phage amplification, and the most effective method has to be established individually for each phage.

Enhanced production of anthraquinones and phenolic compounds using chitosan from the adventitious roots of Morinda coreia Buck. and Ham.

This report establishes the production and characterization of anthraquinones and phenolic compounds from the adventitious root cultures of Morinda coreia, induced from the leaves on half strength Murashige and Skoog’s (MS) medium fortified with 1.0 mg l−1 of Indole-3 butyric acid (IBA). The in vitro regenerated adventitious roots (AR) were treated with various concentrations of IBA (0.5–5.0 mg l-1) containing half-strength of liquid MS medium with sucrose (1.5 %). The maximum amount of anthraquinones (149.80 mg g−1 dry weight of AR) and phenolic compounds (71.6 mg g−1 dry weight of AR) were detected on 1.0 mg l−1 and 4.0 mg l−1 of IBA respectively. These proliferated AR on IBA contained 2.10 times more anthraquinones and 1.89 times more phenolics as compared with the yield of in vivo grown roots of M. coreia. Other parameters, such as growth ratio (5.082), fresh weight (1.568 g) and dry weight (0.163 g) of AR were recorded maximum with the concentration of 1.0 mg l−1 IBA. These AR were introduced into the two-stage culture system with various concentrations of chitosan (0.2, 0.4 and 0.8 mg ml-1) for elicitation. Among these, 0.4 mg ml-1 chitosan was observed as the best concentration and further used for the production of secondary metabolites (anthraquinones and phenolics) at different time intervals (2–8 days). Growth ratio and biomass (fresh and dry weight) were decreased from second to eighth day of elicitation but the amount of anthraquinones (292.038 mg g-1 dry weight) and phenolics (86.8 mg g-1 dry weight) were increased till 4th day of the elicitation. The methods developed in this study could be used for further proliferation of AR and large scale production of anthraquinones and phenolic compounds using in vitro raised leaves of M. coreia.

Enhanced lipid productivity of Chlamydomonas reinhardtii with combination of NaCl and CaCl2 stresses.

Salinity (NaCl) stress treatment is a strategy to induce lipid accumulation in microalgae. This study aimed to investigate the effect of a combination of two salts (NaCl/CaCl2) on lipid productivity of Chlamydomonas reinhardtii. C. reinhardtii was cultured in a two-stage culture comprising 9-day active growth in C medium followed by 3-day salt stress in C medium with various concentrations of NaCl (50‒200mM)/CaCl2 (100mM). In salt stress stage, NaCl (200mM), CaCl2 (100mM), and the NaCl/CaCl2 mixture inhibited growth but increased the lipid content in C. reinhardtii in comparison with NaCl (0, 50, and 100mM) conditions. Combinatorial treatment with 100mM NaCl/100mM CaCl2 resulted in the highest lipid content (73.4%) and lipid productivity (10.9mg/L/days), being 3.5- and 2.1-fold, respectively, in salt-free control conditions, and 1.8- and 1.5-folds, respectively, with 200mM NaCl. Furthermore, 100mM NaCl/100mM CaCl2 treatment markedly upregulated glycerol-3-phosphate dehydrogenase (GPDH), lysophosphatidic acid acyltransferase (LPAAT), and diacylglycerol acyltransferase (DAGAT), which are involved in lipid accumulation in C. reinhardtii. The upregulation of these genes with 100mM NaCl/100mM CaCl2 resulted in the highest lipid content in C. reinhardtii. Therefore, stress treatment using two salts, 100mM NaCl/100mM CaCl2, is a potentially promising strategy to enhance lipid productivity in microalgae.