Effect of Biochemical Stimulants on Biomass Productivity and Metabolite Content of the Microalga, Chlorella sorokiniana

The influence of 12 biochemical stimulants, namely 2-phenylacetic acid (PAA; 30 ppm), indole-3 butyric acid (IBA; 10 ppm), 1-naphthaleneacetic acid (NAA; 2.5, 5 and 10 ppm ), gibberellic acid (GA3, 10 ppm), zeatin (ZT; 0.002 ppm), thidiazuron (0.22 ppm), humic acid (20 ppm), kelp extract (250 ppm), methanol (500 ppm), ferric chloride (3.2 ppm ), putrescine (0.09 ppm), spermidine (1.5 ppm) were prescreened for their impact on growth and chlorophyll for the green alga—Chlorella sorokiniana. C. sorokiniana responded best to phytohormones in the auxin family, particularly NAA. Thereafter, two studies were conducted on combinations of phytohormones to compare blends from within the auxin family as well as against other families. These treatments were NAA_{5 ppm}+PAA_{30 ppm}, NAA_{2.5 ppm}+PAA_15 ppm, NAA_5 ppm+IBA_10 ppm, NAA_5 ppm+GA3_10 ppm, NAA_5 ppm+ZT_1 ppm, and NAA_5 ppm+GA3_10 ppm+ZT_1 ppm. Combinations of NAA with other auxins did not have synergistic or antagonistic effects on the growth. However, combinations of compounds from different phytohormone families, such as NAA_5 ppm+GA3_10 ppm+ZT_1 ppm, dramatically increased the biomass productivity by 170% over the control followed by the treatments: NAA _5 ppm+GA3_10 ppm (138%), NAA _5 ppm+ZT_1 ppm (136%), and NAA _5 ppm ( 133%). The effect of biochemical stimulants were also measured on metabolites such as chlorophyll, protein, and lipids in C. sorokiniana. Renewed interest in microalgae for biotechnology and biofuel applications may warrant the use of biochemical stimulants for cost reduction in large-scale cultivation through increased biomass productivity.     

Analysis on the Chlorophyll Content of Commercial Green Leafy Vegetables

The objectives of the present study were to evaluate the chlorophyll content of green leafy vegetables found commercially and carry out a comparative investigation between in vivo and in vitro data. The chlorophyll of green leafy vegetable can be used as visible parameters of the quality of vegetables during storage, since it will be degraded gradually along with post-harvest senescence. Therefore, the development of reliable in vivo chlorophyll measurement should be advantageous rather than visual observation for the purpose of quality control and product sortation. Here, the existence of chlorophylls in ten green leafy vegetables were reported as SPAD values of a handheld SPAD-502 chlorophyll meter and % N of an Agriexpert CCN-6000 nitrogen meter (in vivo data), as well as total peak area data of HPLC measurement for chlorophyll a and b after exhaustive extraction using methanol (in vitro data). Both in vivo and in vitro measurement gave comparable grouping of vegetables with high and low content of chlorophyll. Moreover, correlation plots between SPAD values and total peak area of HPLC showed adequate linear correlation (R² > 0.7), revealing the potency of in vivo observation for the prediction of actual chlorophyll content in commercial leafy vegetables. SPAD values and % N presented strong linear relationship (R² > 0.9), in which SPAD-meter performed better detection at very low values. The calibration curve for each species of vegetable should be substantial to overcome the limiting factors of in vivo observation, such as leaf size, tissue thickness, and variation of chloroplast distribution.     

镧和铈对大豆开花期叶绿素含量的影响

以大豆东农42和47为试验材料,采用盆栽方式,使用乙醇分光光度计法研究在大豆开花期喷施不同浓度稀土元素La(Ⅲ)和Ce(Ⅲ)(RE1/60 mg·L-1,RE2/90 mg·L-1,RE3/120 mg·L-1和RE4/150 mg·L-1)对叶片叶绿素含量(Chl)的影响。结果表明:与对照组(CK)相比,La对两种大豆Chl含量呈现"低促高抑"的现象:在RE1浓度下,Chl含量高于CK值,并且达到最大值,随着浓度增大,促进作用降低,抑制作用明显,RE2～RE4浓度下Chl含量降低,并且低于CK值;Ce对两种大豆Chl含量呈现下降趋势,在RE1浓度下下降幅度最为明显,RE2～RE4浓度下叶绿素含量有所上升并低于CK值。     

镧对青菜生物量、植株氮磷钾含量及土壤酶活性的影响

利用盆栽试验,研究了镧对青菜生物量、植株氮磷钾含量及土壤酶活性的影响.结果表明,施用镧显著增加了青菜生物量,与对照相比地上部干重提高了5.49%～19.78%,根干重提高了13.51%～51.35%.在低浓度下,随镧浓度的升高,青菜茎叶和根的氮、磷、钾含量逐渐增加,到一定浓度后,随着镧浓度的继续升高,含量反而逐渐降低.镧施入土壤后对脲酶、酸性磷酸酶、转化酶活性表现出刺激作用并随浓度的升高而增强,而对过氧化氢酶活性有轻微的抑制作用,对脱氢酶活性有强烈的抑制作用.     

Thidiazuron-Induced Changes in Biomass Parameters,Total Phenolic Content,and Antioxidant Activity in Callus Cultures of Artemisia absinthium L.

Callus culture of Artemisia absinthium L. was established for enhanced production of phenolics and higher antioxidant activity. Callus was induced from seed-derived leaf explants, incubated on to MS media supplemented with thidiazuron (TDZ; 0.5–5.0 mg/l) either alone or in combination with α-naphthalene acetic acid (NAA; 1.0 mg/l). These callus cultures were investigated for their growth kinetics, total phenolic content, and antioxidant activity on weekly basis for a period of 49 days. Maximum dry biomass accumulation of 8.73 g/l was observed on day 42 in response to 1.0 mg/l TDZ and 1.0 mg/l NAA. Furthermore, maximum level of total phenolic content of 8.53 mg GAE/g DW and highest 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of 72.6 % were observed in calli formed in response to 1.0 mg/l TDZ on day 42. The results showed a positive correlation of total phenolic content and DPPH radical scavenging activity in most of the callus cultures of A. absinthium L.     

The Utilization of Algae and Seaweed Biomass for Bioremediation of Heavy Metal-Contaminated Wastewater

The presence of heavy metals in water bodies is linked to the increasing number of industries and populations. This has serious consequences for the quality of human health and the environment. In accordance with this issue, water and wastewater treatment technologies including ion exchange, chemical extraction, and hydrolysis should be conducted as a first water purification stage. However, the sequestration of these toxic substances tends to be expensive, especially for large scale treatment methods that require tedious control and have limited efficiency. Therefore, adsorption methods using adsorbents derived from biomass represent a promising alternative due to their great efficiency and abundance. Algal and seaweed biomass has appeared as a sustainable solution for environmentally friendly adsorbent production. This review further discusses recent developments in the use of algal and seaweed biomass as potential sorbent for heavy metal bioremediation. In addition, relevant aspects like metal toxicity, adsorption mechanism, and parameters affecting the completion of adsorption process are also highlighted. Overall, the critical conclusion drawn is that algae and seaweed biomass can be used to sustainably eliminate heavy metals from wastewater.     

Effects of Phytohormones and Jasmonic Acid on Glucosinolate Content in Hairy Root Cultures of Sinapis alba and Brassica rapa

Although some study have established hairy root cultures from brassicaceous plants with glucosinolates (GS) as characteristic secondary metabolite, studies are missing which compare hairy roots with the corresponding mother plants. Therefore, two different plant species—Sinapis alba and Brassica rapa subsp. rapa pygmeae teltoviensis—were transformed with the Agrobacterium rhizogenes strain A4. Aliphatic and indolyl GS were present in B. rapa, exhibiting larger quantities in leaves than in roots. Aromatic p-hydroxybenzyl GS were found particularly in the leaves of S. alba. However, the proportion of indolyl GS increased suddenly in transformed hairy roots of S. alba and B. rapa. Cultivation with the phytohormone kinetin (0.5 mg?L^?1) enhanced GS accumulation in B. rapa hairy roots, however not in S. alba, but 2,4-D (0.4 mg?L^?1) induced de-differentiation of roots in both species and reduced GS levels. GS levels especially of 1-methoxyindol-3ylmethyl GS increased in hairy roots in response to JA, but root growth was inhibited. While 2 weeks of cultivation in 100 to 200 μM JA were determined at optimum for maximum GS yield in S. alba hairy root cultures, 4 weeks of cultivation in 50 to 100 μM JA was the optimum for B. rapa.     

Effect of Water Hardness on Catechin and Caffeine Content in Green Tea Infusions

The health benefits of green tea are associated with its high catechin content. In scientific studies, green tea is often prepared with deionized water. However, casual consumers will simply use their local tap water, which differs in alkalinity and mineral content depending on the region. To assess the effect of water hardness on catechin and caffeine content, green tea infusions were prepared with synthetic freshwater in five different hardness levels, a sodium bicarbonate solution, a mineral salt solution, and deionized water. HPLC analysis was performed with a superficially porous pentafluorophenyl column. As water hardness increased, total catechin yield decreased. This was mostly due to the autoxidation of epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Epicatechin (EC), epicatechin gallate (ECG), and caffeine showed greater chemical stability. Autoxidation was promoted by alkaline conditions and resulted in the browning of the green tea infusions. High levels of alkaline sodium bicarbonate found in hard water can render some tap waters unsuitable for green tea preparation.     

Low-Cost Production of Green Microalga Botryococcus braunii Biomass with High Lipid Content Through Mixotrophic and Photoautotrophic Cultivation

Botryococcus braunii is a microalga that is regarded as a potential source of renewable fuel because of its ability to produce large amounts of lipid that can be converted into biodiesel. Agro-industrial by-products and wastes are of great interest as cultivation medium for microorganisms because of their low cost, renewable nature, and abundance. In this study, two strategies for low-cost production of B. braunii biomass with high lipid content were performed: (i) the mixotrophic cultivation using molasses, a cheap by-product from the sugar cane plant as a carbon source, and (ii) the photoautotrophic cultivation using nitrate-rich wastewater supplemented with CO₂ as a carbon source. The mixotrophic cultivation added with 15 g L^?1 molasses produced a high amount of biomass of 3.05 g L^?1 with a high lipid content of 36.9 %. The photoautotrophic cultivation in nitrate-rich wastewater supplemented with 2.0 % CO₂ produced a biomass of 2.26 g L^?1 and a lipid content of 30.3 %. The benefits of this photoautotrophic cultivation are that this cultivation would help to reduce accumulation of atmospheric carbon dioxide and more than 90 % of the nitrate could be removed from the wastewater. When this cultivation was scaled up in a stirred tank photobioreactor and run with semi-continuous cultivation regime, the highest microalgal biomass of 5.16 g L^?1 with a comparable lipid content of 32.2 % was achieved. These two strategies could be promising ways for producing cheap lipid-rich microalgal biomass that can be used as biofuel feedstocks and animal feeds.     

The Influence of Lactic Acid Fermentation on Selected Properties of Pickled Red,Yellow, and Green Bell Peppers

Red, yellow, and green peppers are vegetables rich in natural pigments. However, they belong to seasonal vegetables and need to be treated to prolong their shelf life. One new approach to processing vegetables is to pickle them using lactic acid bacteria. The use of such a process creates a new product with high health value, thanks to the active ingredients and lactic acid bacteria. Therefore, this study aimed to evaluate the effect of the applied strain of lactic acid bacteria (LAB) on the chemical properties, including the content of active compounds (pigments) and the physical properties of the peppers. Levilactobacillus brevis, Limosilactobacillus fermentum, and Lactoplantibacillus plantarum were used for fermentation and spontaneous fermentation. The pigments, polyphenols content, and antioxidant properties were determined in the pickled peppers, as well as sugar content, color, dry matter, texture properties, and the count of lactic acid bacteria. In all samples, similar growth of LAB was observed. Significant degradation of chlorophylls into pheophytins was observed after the fermentation process. No significant differences were observed in the parameters tested, depending on the addition of dedicated LAB strains. After the fermentation process, the vitamin C and total polyphenols content is what influenced the antioxidant activity of the samples. It can be stated that the fermentation process changed the red bell pepper samples in the smallest way and the green ones in the highest way.     

The Effect of Drying Temperature on the Composition of Biomass

The compositional quality of different lignocellulosic feedstocks influences their performance and potential demand at a biorefinery. Many analytical protocols for determining the composition or performance characteristics of biomass involve a drying step, where the drying temperature can vary depending on the specific protocol. To get reliable data, it is important to determine the correct drying temperature to vaporize the water without negatively impacting the compositional quality of the biomass. A comparison of drying temperatures between 45 °C and 100 °C was performed using wheat straw and corn stover. Near-infrared (NIR) spectra were taken of the dried samples and compared using principal component analysis (PCA). Carbohydrates were analyzed using quantitative saccharification to determine sugar degradation. Analysis of variance was used to determine if there was a significant difference between drying at different temperatures. PCA showed an obvious separation in samples dried at different temperatures due to sample water content. However, quantitative saccharification data shows, within a 95% confidence interval, that there is no significant difference in sugar content for drying temperatures up to 100 °C for wheat straw and corn stover.     

Resveratrol Content and Expression of Phenylalanine Ammonia-lyase and Stilbene Synthase Genes in Cell Cultures of Vitis amurensis Treated with Coumaric Acid

The present study examines the effect of p-coumaric acid (CA), a precursor of stilbenes and isoflavonoids, on biosynthesis of resveratrol in cell cultures of Vitis amurensis. Earlier, we transformed V. amurensis with the rolB gene of Agrobacterium rhizogenes and showed increased level of resveratrol production in the rolB transgenic cell culture. We used control and the rolB-transgenic cell culture of V. amurensis as a model system in this study. CA was capable of increasing resveratrol production in the control and the rolB-transgenic cell cultures in 10.3 and 1.5 times, respectively. The CA-treated control and rolB transgenic calli produced up to 0.06% and 1.1% DW of resveratrol. Using quantitative real-time RT-PCR, we characterized the expression of phenylalanine ammonia-lyase (PAL) and stilbene synthase (STS) genes in the CA-treated control and rolB transgenic cell cultures. The expression of PAL genes remained essentially unchanged under 0.1 mM of CA, while expression of VaPAL1, VaPAL2, VaPAL3, and VaPAL5 genes was considerably decreased under 0.5 and 2 mM CA compared with the untreated cells. In the CA-treated calli, expression of VaSTS2 and VaSTS3 was increased, while expression of VaSTS5, VaSTS8, VaSTS9, and VaSTS10 was significantly decreased. These results indicate that CA increased resveratrol accumulation in V. amurensis calli via selective enhancement of expression of individual STS genes.     

Effect of Irrigation on the Oil Content and Fatty Acid Composition of Some Sunflower Seeds

The effect of irrigation on the seed yield, oil yield, and oil composition of sunflower in populations of Ekiz1 and VNIIMK 8931, a mixture of N₂, N₃, and N₄ lines, and a synthetic variety obtained from these lines has been studied. The major fatty acid was found to be oleic acid, in addition to linoleic, palmitic, and stearic acids. Irrigation increased seed yield, oil yield, and oil content of all samples. In two of them, the synthetic variety and VNIIMK 8931, the increase was found to be statistically significant. In addition, irrigation of the samples did not increase significantly the amount of oleic and linoleic acids. On the contrary, the amount of linoleic acid in the mixture of N₂, N₃, and N₄ lines and oleic acid in VNIIMK 8931 decreased statistically significantly.     

Production of Verbascoside,Isoverbascoside and Phenolic Acids in Callus,Suspension, and Bioreactor Cultures of Verbena officinalis and Biological Properties of Biomass Extracts

Callus, suspension and bioreactor cultures of Verbena officinalis were established, and optimized for biomass growth and production of phenylpropanoid glycosides, phenolic acids, flavonoids and iridoids. All types of cultures were maintained on/in the Murashige and Skoog (MS) media with 1 mg/L BAP and 1 mg/L NAA. The inoculum sizes were optimized in callus and suspension cultures. Moreover, the growth of the culture in two different types of bioreactors—a balloon bioreactor (BB) and a stirred-tank bioreactor (STB) was tested. In methanolic extracts from biomass of all types of in vitro cultures the presence of the same metabolites—verbascoside, isoverbascoside, and six phenolic acids: protocatechuic, chlorogenic, vanillic, caffeic, ferulic and rosmarinic acids was confirmed and quantified by the HPLC-DAD method. In the extracts from lyophilized culture media, no metabolites were found. The main metabolites in biomass extracts were verbascoside and isoverbascoside. Their maximum amounts in g/100 g DW (dry weight) in the tested types of cultures were as follow: 7.25 and 0.61 (callus), 7.06 and 0.48 (suspension), 7.69 and 0.31 (BB), 9.18 and 0.34 (STB). The amounts of phenolic acids were many times lower, max. total content reached of 26.90, 50.72, 19.88, and 36.78 mg/100 g DW, respectively. The highest content of verbascoside and also a high content of isoverbascoside obtained in STB (stirred-tank bioreactor) were 5.3 and 7.8 times higher than in extracts from overground parts of the parent plant. In the extracts from parent plant two iridoids—verbenalin and hastatoside, were also abundant. All investigated biomass extracts and the extracts from parent plant showed the antiproliferative, antioxidant and antibacterial activities. The strongest activities were documented for the cultures maintained in STB. We propose extracts from in vitro cultured biomass of vervain, especially from STB, as a rich source of bioactive metabolites with antiproliferative, antioxidant and antibacterial properties.     

Chemical Elicitors-Induced Variation in Cellular Biomass,Biosynthesis of Secondary Cell Products,and Antioxidant System in Callus Cultures of Fagonia indica

Fagonia indica is a rich source of pharmacologically active compounds. The variation in the metabolites of interest is one of the major issues in wild plants due to different environmental factors. The addition of chemical elicitors is one of the effective strategies to trigger the biosynthetic pathways for the release of a higher quantity of bioactive compounds. Therefore, this study was designed to investigate the effects of chemical elicitors, aluminum chloride (AlCl₃) and cadmium chloride (CdCl₂), on the biosynthesis of secondary metabolites, biomass, and the antioxidant system in callus cultures of F. indica. Among various treatments applied, AlCl₃ (0.1 mM concentration) improved the highest in biomass accumulation (fresh weight (FW): 404.72 g/L) as compared to the control (FW: 269.85 g/L). The exposure of cultures to AlCl₃ (0.01 mM) enhanced the accumulation of secondary metabolites, and the total phenolic contents (TPCs: 7.74 mg/g DW) and total flavonoid contents (TFCs: 1.07 mg/g DW) were higher than those of cultures exposed to CdCl₂ (0.01 mM) with content levels (TPC: 5.60 and TFC: 0.97 mg/g) as compared to the control (TPC: 4.16 and TFC: 0.42 mg/g DW). Likewise, AlCl₃ and CdCl₂ also promoted the free radical scavenging activity (FRSA; 89.4% and 90%, respectively) at a concentration of 0.01 mM, as compared to the control (65.48%). For instance, the quantification of metabolites via high-performance liquid chromatography (HPLC) revealed an optimum production of myricetin (1.20 mg/g), apigenin (0.83 mg/g), isorhamnetin (0.70 mg/g), and kaempferol (0.64 mg/g). Cultures grown in the presence of AlCl₃ triggered higher quantities of secondary metabolites than those grown in the presence of CdCl₂ (0.79, 0.74, 0.57, and 0.67 mg/g). Moreover, AlCl₃ at 0.1 mM enhanced the biosynthesis of superoxide dismutase (SOD: 0.08 nM/min/mg-FW) and peroxidase enzymes (POD: 2.37 nM/min/mg-FW), while CdCl₂ resulted in an SOD activity up to 0.06 nM/min/mg-FW and POD: 2.72 nM/min/mg-FW. From these results, it is clear that AlCl₃ is a better elicitor in terms of a higher and uniform productivity of biomass, secondary cell products, and antioxidant enzymes compared to CdCl₂ and the control. It is possible to scale the current strategy to a bioreactor for a higher productivity of metabolites of interest for various pharmaceutical industries.     

A Synthetic Chlorophyll Dimer Appending Fullerene: Effect of Chlorophyll Pairing on (Photo)redox Properties

Accurately mimicking structure and function of natural chlorophyll (Chl) assemblies is very challenging. Herein, we report the synthesis of a fullerene-appended Chl dimer being capable of intramolecular photoinduced charge separation (CS) with a unique structure reminiscent of reaction centers (RCs) in phototrophs. Structural analyses revealed that the Chl dimer adopts a bird-like structure in which two Chl components overlapped partially with one of the four pyrrole rings in a Chl ring similar to in a Chl pair in the natural RC complexes. A comparative study including voltammetry and spectrometric analyses using the Chl dimer and its corresponding monomer with and without a fullerene moiety was performed to gain insight into the effect of Chl pairing on (photo)redox properties. Our results suggest that the present dimer motif that closely resemble the Chl pair in natural RCs lead to more facile oxidation and lower energy of the CS state of the Chl dimer than those of the Chl monomer, resulting in its photoredox behavior different from that of the monomer Chl.     

Methyl Jasmonate Effect on Betulinic Acid Content and Biological Properties of Extract from Senna obtusifolia Transgenic Hairy Roots

It is known that Senna obtusifolia has been used in medicine since ancient times due to the content of many valuable compounds with a pro-health effect. One of them is betulinic acid, which is a pentacyclic triterpene with antimalarial, antiviral, anti-inflammatory and anticancer properties. In this work, a continuation of our previous research, an attempt was made to increase the level of betulinic acid accumulation by the cultivation of transgenic hairy roots that overexpress the squalene synthase gene in a 10 L sprinkle bioreactor with methyl jasmonate elicitation. We present that the applied strategy allowed us to increase the content of betulinic acid in hairy root cultures to the level of 48 mg/g dry weight. The obtained plant extracts showed a stronger cytotoxic effect on the U87MG glioblastoma cell line than the roots grown without elicitors. Additionally, the induction of apoptosis, reduction of mitochondrial membrane potential, chromosomal DNA fragmentation and activation of caspase cascades are demonstrated. Moreover, the tested extract showed inhibition of topoisomerase I activity.     

镉胁迫对紫花苜蓿生长及植株镉含量的影响

采用盆栽实验,以多叶苜蓿和准格尔苜蓿2个品种为材料,研究了Cd胁迫(0、5、10、20、50mg·kg-1)对紫花苜蓿生长和植株Cd含量的影响。结果显示,Cd处理质量分数为5mg·kg-1时,2个品种的株高、主根长、干质量较之对照组均有所提高,但差异不显著;随Cd处理质量分数增加,上述指标呈显著降低趋势,尤其是当处理质量分数达到50mg·kg-1时,其下降幅度则多叶苜蓿明显小于准格尔苜蓿;在重度Cd胁迫下,2个品种地上部和根部的Cd含量都增大,准格尔苜蓿的Cd含量高于多叶苜蓿。试验表明,在Cd质量分数超过10mg·kg-1的土壤中不宜种植紫花苜蓿。     

Effect of External Acid Site Content in PdHZSM-5 Catalyst on Its Activity and Selectivity in Hydroisomerization of n-Hexane

We have studied the relationship between the external acidity and the catalytic properties of zeolite PdHZSM-5 (Si/Al = 21) in hydroconversion of n-hexane. We have established that decreasing the external H-site content by 5-9 mol/g at 548 K leads to an increase in conversion by 18-19% and a decrease in the selectivity of the catalyst with respect to the isomerization products by 47-70%. We suggest an explanation for this substantial effect of the external H-sites on the activity and selectivity of the catalyst.     

Cultivation of Chlorella protothecoides with Urban Wastewater in Continuous Photobioreactor: Biomass Productivity and Nutrient Removal

The capability to grow microalgae in nonsterilized wastewater is essential for an application of this technology in an actual industrial process. Batch experiments were carried out with the species in nonsterilized urban wastewater from local treatment plants to measure both the algal growth and the nutrient consumption. Chlorella protothecoides showed a high specific growth rate (about 1 day^?1), and no effects of bacterial contamination were observed. Then, this microalgae was grown in a continuous photobioreactor with CO₂–air aeration in order to verify the feasibility of an integrated process of the removal of nutrient from real wastewaters. Different residence times were tested, and biomass productivity and nutrients removal were measured. A maximum of microalgae productivity was found at around 0.8 day of residence time in agreement with theoretical expectation in the case of light-limited cultures. In addition, N-NH₄ and P-PO₄ removal rates were determined in order to model the kinetic of nutrients uptake. Results from batch and continuous experiments were used to propose an integrated process scheme of wastewater treatment at industrial scale including a section with C. protothecoides.