Development of a Saccharomyces cerevisiae strain for increasing the accumulation of triacylglycerol as a microbial oil feedstock for biodiesel production using glycerol as a substrate

Triacylglycerol (TAG) is a microbial oil feedstock for biodiesel production that uses an inexpensive substrate, such as glycerol. Here, we demonstrated the overproduction of TAG from glycerol in engineered Saccharomyces cerevisiae via the glycerol‐3‐phosphate (G3P) pathway by overexpressing the major TAG synthesis. The G3P accumulation was increased 2.4‐fold with the increased glycerol utilization gained by the overexpression of glycerol kinase (GUT1). By overexpressing diacylglycerol acyltransferase (DGA1) and phospholipid diacylglycerol acyltransferase (LRO1), the engineered YPH499 (pGutDgaLro1) strain produced 23.0 mg/L lipids, whereas the YPH499 (pESC‐TRP) strain produced 6.2 mg/L total lipids and showed a lipid content that was increased 1.4‐fold compared with 3.6% for the wild‐type strain after 96 h of cultivation. After 96 h of cultivation using glycerol, the overall content of TAG in the engineered strain, YPH499 (pGutDgaLro1), yielded 8.2% TAG, representing a 2.3‐fold improvement, compared with 3.6% for the wild‐type strain. The results should allow a reduction of costs and a more sustainable production of biodiesel. Biotechnol. Bioeng. 2013; 110: 343–347. © 2012 Wiley Periodicals, Inc.     

Transgenic parthenocarpic eggplants: superior germplasm for increased winter production

Winter production of three eggplant hybrids transgenic for the parthenocarpic gene DefH9-iaaM was compared, in an unheated greenhouse, to the performance of two untransformed control hybrids and the commercial parthenocarpic cultivar Talina. Each hybrid was either treated or untreated with a commercial formulation of phytohormones to induce fruit set and growth. The productivity of the transgenic parthenocarpic hybrids was not influenced by the hormonal treatment. On the contrary, the productivity of untransformed hybrids was significantly improved by hormonal treatment of the flower buds. The yield of the transgenic hybrids was significantly higher than that obtained in the corresponding untransformed hybrids, even when the latter were treated with phytohormones. The yield increment due to the parthenocarpic trait was particularly evident when compared to the yield of the two corresponding hybrid combinations, which are identical except for the presence of the DefH9-iaaM gene. The transgenic hybrids allowed an increase in productivity of ca. 25%. This increment coincided with a 10% reduction in cultivation cost, mainly due to the labour needed for the hormonal sprays, and to the production of fruits of better quality. Thus, the DefH9-iaaM gene is a biotechnological tool superior to both agronomic and traditional genetic parthenocarpic mutants.     

Effect of green and red light in lipid accumulation and transcriptional profile of genes implicated in lipid biosynthesis in Chlamydomonas reinhardtii

Microalgae have the potential to accumulate triacylglycerols under different light spectra. In this work, Chlamydomonas reinhardtii was grown under white (400–700 nm), red (650 nm), and green (550 nm) lights. According to our results, red light (650 nm) has a positive effect in the microalgae growth and chlorophyll concentration. About the lipid content, the control culture (white light‐illuminated) reached a 4.4% of dry cell weight (dcw), whereas the culture grown at 550 nm showed an increase of 1.35‐fold in the lipids accumulation (5.96% dcw). Interestingly, the most significant accumulation was found in the culture grown at 650 nm (14.78% dcw) which means 3.36‐fold higher with respect to the white light‐illuminated culture. The most abundant fatty acids found in lipid extracts obtained from the cultures under different light wavelength were palmitic (C16: 0), oleic (C18: 1n9), stearidonic (C18: 4), and linoleic (C18: 2), which are useful in the biodiesel production. Changes in gene expression in response to different wavelength illuminations were assessed; however, an in‐depth analysis of a larger number of genes involved in lipid biosynthesis is necessary to fully explain the highest accumulation of lipids in the culture grown under red light. This approach will be useful to find a sustainable source of lipids for biodiesel production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1404–1411, 2016     

Parasitism by Cuscuta pentagona sequentially induces JA and SA defence pathways in tomato

While plant responses to herbivores and pathogens are well characterized, responses to attack by other plants remain largely unexplored. We measured phytohormones and C₁₈ fatty acids in tomato attacked by the parasitic plant Cuscuta pentagona, and used transgenic and mutant plants to explore the roles of the defence‐related phytohormones salicylic acid (SA) and jasmonic acid (JA). Parasite attachment to 10‐day‐old tomato plants elicited few biochemical changes, but a second attachment 10 d later elicited a 60‐fold increase in JA, a 30‐fold increase in SA and a hypersensitive‐like response (HLR). Host age also influenced the response: neither Cuscuta seedlings nor established vines elicited a HLR in 10‐day‐old hosts, but both did in 20‐day‐old hosts. Parasites grew larger on hosts deficient in SA (NahG) or insensitive to JA [jasmonic acid‐insensitive1 (jai1) ], suggesting that both phytohormones mediate effective defences. Moreover, amounts of JA peaked 12 h before SA, indicating that defences may be coordinated via sequential induction of these hormones. Parasitism also induced increases in free linolenic and linoleic acids and abscisic acid. These findings provide the first documentation of plant hormonal signalling induced by a parasitic plant and show that tomato responses to C. pentagona display characteristics similar to both herbivore‐ and pathogen‐induced responses.     

Effect of nitrogenous resource on growth,biochemical composition and ultrastructure of Isochrysis galbana (Isochrysidales,Haptophyta)

The nitrogenous resource used to promote algal growth has cost implications for mass culture processes. The present study therefore aimed to determine the effect of different nitrogenous resources (nitrate, ammonium and urea) on various performance parameters (growth, final cell yield, pigmentation, lipid yield and cellular and sub‐cellular characteristics) in Isochrysis galbana. Growth rate was unaffected by nitrogenous resource, but the final cellular yield in the nitrate and urea treatments far exceeded that evident in the ammonium treatments. The reduced cell yield in ammonium treatments and the earlier onset of the stationary phase was brought about by nitrogen‐starvation due to an increase in pH and resultant ammonia volatilization. This starvation initiated an early onset of lipid accumulation, chlorophyll depletion and an increase in the carotenoid to chlorophyll ratio relative to the other nitrogen (N) source treatments. Hence, in spite of being potentially the preferred source of N by algae (due to its reduced state), ammonium‐nitrogen is undesirable for mass culture. The performance parameters of Isochrysis grown in urea (an organic N source) and nitrate (an inorganic N source) were similar, but lipid accrued earlier in cells grown in medium supplemented with urea. This is advantageous for lipid acquisition for the production of biodiesel since it would reduce the duration of photobioreactor runs. Urea is easily available and considerably cheaper than all the other N sources tested and is thus recommended as the nitrogenous resource for large‐scale culture of I. galbana for biodiesel production.     

Growth promotion of Chlorella ellipsoidea by co-inoculation with Brevundimonas sp. isolated from the microalga

Eight bacterial strains identified as P1, P2, Y1, Y2, W1, W2, G, and R were isolated from a long-term laboratory culture of the green alga Chlorella ellipsoidea. Although it is unknown how these bacterial strains have been maintained with the C. ellipsoidea culture, all appeared to promote the growth of C. ellipsoidea. Co-inoculation of each bacterial strain with C. ellipsoidea resulted in 0.5–3 times greater algal growth than that of C. ellipsoidea alone. The most effective bacterium (i.e., strain P1) was selected and further characterized. Biochemical analysis and transmission electron microscopy revealed that strain P1 is closely related to the genus Brevundimonas. Sequence analysis of the 16S rRNA of strain P1 showed 99.9 and 99.4% nucleotide sequence identity to that of B. nasdae and B. vesicularis, respectively. In addition to the growth promotion of C. ellipsoidea by strain P1, the growth of strain P1 was also significantly enhanced by co-culturing with C. ellipsoidea, indicating a symbiotic relationship between the bacterium and alga. Scanning electron microscopy showed the direct adhesion of strain P1 cells to the surface of C. ellipsoidea cells, as well as the development of abundant crinkles on the surface of co-cultured C. ellipsoidea cells. Handling editor: J. Padisak     

Characterization of the cellulolytic secretome of Trichoderma harzianum during growth on sugarcane bagasse and analysis of the activity boosting effects of swollenin

This study demonstrates the production of an active enzyme cocktail produced by growing Trichoderma harzianum on sugarcane bagasse. The component enzymes were identified by LCMS‐MS. Glycosyl hydrolases were the most abundant class of proteins, representing 67% of total secreted protein. Other carbohydrate active enzymes involved in cell wall deconstruction included lytic polysaccharide mono‐oxygenases (AA9), carbohydrate‐binding modules, carbohydrate esterases and swollenin, all present at levels of 1%. In total, proteases and lipases represented 5 and 1% of the total secretome, respectively, with the rest of the secretome being made up of proteins of unknown or putative function. This enzyme cocktail was efficient in catalysing the hydrolysis of sugarcane bagasse cellulolignin to fermentable sugars for potential use in ethanol production. Apart from mapping the secretome of T. harzianum, which is a very important tool to understand the catalytic performance of enzyme cocktails, the gene coding for T. harzianum swollenin was expressed in Aspergillus niger. This novel aspect in this work, allowed increasing the swollenin concentration by 95 fold. This is the first report about the heterologous expression of swollenin from T. harzianum, and the findings are of interest in enriching enzyme cocktail with this important accessory protein which takes part in the cellulose amorphogenesis. Despite lacking detectable glycoside activity, the addition of swollenin of T. harzianum increased by two‐fold the hydrolysis efficiency of a commercial cellulase cocktail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:327–336, 2016     

Efficacy of Chlorella pyrenoidosa and Scenedesmus abundans for Nutrient Removal in Rice Mill Effluent (Paddy Soaked Water)

Microalgae are product of sustainable development owing to its ability to treat variety of wastewater effluents and thus produced biomass can serve as value added product for various commercial applications. This paper deals with the cultivation of microalgae species namely Chlorella pyrenoidosa and Scenedesmus abundans in rice mill effluent (i.e., paddy soaked water) for nutrient removal. In order to investigate the nutrient removal capability, microalgae are subjected to cultivation in both raw and autoclaved samples. The maximum phosphate removal by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 98.3% and 97.6%, respectively, whereas, the removal of ammoniacal nitrogen by Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 92% and 90.3%, respectively. The growth (measured in terms of chlorophyll content) of Scenedesmus abundans and Chlorella pyrenoidosa in raw sample was 3.88 mg/l and 5.55 mg/l, respectively. The results indicate the suitability of microalgae cultivation in rice mill effluent treatment for nutrient removal.     

Simultaneous heat shock and in situ adsorption enhance plumbagin production in Plumbago indica root cultures

Plumbago indica L. is an important source of plumbagin, a commercially valuable bioactive compound. However, the uses of plumbagin are limited due to its low supply as well as low yields and slow growth of the plant sources. This study evaluated the use of a simple, easy, and low‐cost approach using heat shock (HS) and ultrasound (US), and an in situ adsorption using a nonpolar copolymer adsorbent styrene‐divynilbenzene resin (Diaion^® HP‐20) to enhance plumbagin production in Plumbago indica root cultures. Treatment with HS (60°C) for 10 min significantly increased the production of plumbagin (5.51 mg/g DW) by up to five‐fold, compared to the level in untreated root cultures (1.14 mg/g DW). In contrast, treatments with US alone or with HS treatment produced no satisfactory increase of plumbagin production. However, combined treatment of a 20‐day‐old root culture with HS (60°C, for 10 min) in the presence of Diaion^® HP‐20 (10 g/L) markedly increased the production up to 20.28 mg/g DW of plumbagin that was almost 14‐fold higher, compared to the level in an untreated root culture. Such an increase would be sufficient for commercial applications of this method to produce plumbagin.     

Economic analysis of pilot‐scale production of B‐phycoerythrin

β‐Phycoerythrin is a color protein with several applications, from food coloring to molecular labeling. Depending on the application, different purity is required, affecting production cost and price. Different production and purification strategies for B‐phycoerythrin have been developed, the most studied are based on the production using Porphyridium cruentum and purified using chromatographic techniques or aqueous two‐phase systems. The use of the latter can result in a less expensive and intensive recovery of the protein, but there is lack of a proper economic analysis to study the effect of using aqueous two‐phase systems in a scaled‐up process. This study analyzed the production of B‐Phycoerythrin using real data obtained during the scale‐up of a bioprocess using specialized software (BioSolve, Biopharm Services, UK). First, a sensitivity analysis was performed to identify critical parameters for the production cost, then a Monte Carlo analysis to emulate real processes by adding uncertainty to the identified parameters. Next, the bioprocess was analyzed to determine its financial attractiveness and possible optimization strategies were tested and discussed. Results show that aqueous two‐phase systems retain their advantages of low cost and intensive recovery (54.56%); the costs of production per gram calculated (before titer optimization: US$15,709 and after optimization: US$2,374) allowed to obtain profit (in the range of US$millions in a 10‐year period) for a potential company taking this production method by comparing the production cost against commercial prices. The bioprocess analyzed is a promising and profitable method for the generation of a highly purified B‐phycoerythrin. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1472–1479, 2016     

Serum replacement with albumin‐associated lipids prevents excess aggregation and enhances growth of induced pluripotent stem cells in suspension culture

Suspension culture systems are currently under investigation for the mass production of pluripotent stem (PS) cells for tissue engineering; however, the control of cell aggregation in suspension culture remains challenging. Existing methods to control aggregation such as microwell culture are difficult to scale up. To address this issue, in this study a novel method that incorporates the addition of KnockOut Serum Replacement (KSR) to the PS cell culture medium was described. The method regulated cellular aggregation and significantly improved cell growth (a 2‐ to 10‐fold increase) without any influence on pluripotency. In addition, albumin‐associated lipids as the major working ingredient of KSR responsible for this inhibition of aggregation were identified. This is one of the simplest methods described to date to control aggregation and requires only chemically synthesizable reagents. Thus, this method has the potential to simplify the mass production process of PS cells and thus lower their cost. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1009–1016, 2016     

Use of biodiesel‐derived crude glycerol for vancomycin production by Amycolatopsis orientalis XMU‐VS01

Crude glycerol is a primary by‐product in the biodiesel industry. Microbial fermentation on crude glycerol for producing value‐added products provides opportunities to utilize a large quantity of this by‐product. This study investigates the potential of using the crude glycerol to produce vancomycin (glycopeptide antibiotics) through fermentation of Amycolatopsis orientalis XMU‐VS01. The results show that crude glycerol was the most effective carbon source for mycelium growth and vancomycin production, with 40–60 g/L glycerol concentration as optimal range. Among other culture medium components, potato protein (nitrogen source) and the phosphate concentration had significant effects (p<0.05) for vancomycin production. A Box‐Behnken design and response surface methodology were employed to formulate the optimal medium. Their optimal values were determined as 52.73 g/L of glycerol, 17.36 g/L of potato protein, and 0.1 g/L of dipotassium phosphate. A highest vancomycin yield of 7.61 g/L with biomass concentration of 15.8 g/L was obtained after 120 h flask fermentation. The yield of vancomycin was 3.5 times higher than with basic medium. The results suggest that biodiesel‐derived crude glycerol is a promising feedstock for production of vancomycin from A. orientalis culture.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Purine salvage in Methanocaldococcus jannaschii: Elucidating the role of a conserved cysteine in adenine deaminase

Adenine deaminases (Ade) and hypoxanthine/guanine phosphoribosyltransferases (Hpt) are widely distributed enzymes involved in purine salvage. Characterization of the previously uncharacterized Ade (MJ1459 gene product) and Hpt (MJ1655 gene product) are discussed here and provide insight into purine salvage in Methanocaldococcus jannaschii. Ade was demonstrated to use either Fe(II) and/or Mn(II) as the catalytic metal. Hpt demonstrated no detectable activity with adenine, but was equally specific for hypoxanthine and guanine with a k_cat/K_M of 3.2 × 10⁷ and 3.0 × 10⁷ s^{? 1}M^{? 1}, respectively. These results demonstrate that hypoxanthine and IMP are the central metabolites in purine salvage in M. jannaschii for AMP and GMP production. A conserved cysteine (C127, M. jannaschii numbering) was examined due to its high conservation in bacterial and archaeal homologues. To assess the role of this highly conserved cysteine in M. jannaschii Ade, site‐directed mutagenesis was performed. It was determined that mutation to serine (C127S) completely abolished Ade activity and mutation to alanine (C127A) exhibited 10‐fold decrease in k_cat over the wild type Ade. To further investigate the role of C127, detailed molecular docking and dynamics studies were performed and revealed adenine was unable to properly orient in the active site in the C127A and C127S Ade model structures due to distinct differences in active site conformation and rotation of D261. Together this work illuminates purine salvage in M. jannaschii and the critical role of a cysteine residue in maintaining active site conformation of Ade. Proteins 2016; 84:828–840. © 2016 Wiley Periodicals, Inc.     

Use of ceramic-based cell immobilization to produce 1,3-propanediol from biodiesel-derived waste glycerol with Klebsiella pneumoniae

Aims: The feasibility of the continuous production of a valuable bioplastic raw material, namely 1,3‐propanediol (1,3‐PDO) from biodiesel by‐product glycerol, using immobilized cells was investigated. In addition, the effect of hydraulic retention time (HRT) was also analysed. Methods and Results: Ceramic balls and ceramic rings were used for the immobilization of a locally isolated strain; Klebsiella pneumoniae (GenBank no. 27F HM063413 ). HRT of 1 h is the best one in terms of volumetric production rate (g 1,3‐PDO l^?1 h^?1). The results indicated that ceramic‐based cell immobilization achieved a 2‐fold higher production rate (10 g 1,3‐PDO l^?1 h^?1) in comparison with suspended cell system (4·9 g 1,3‐PDO l^?1 h^?1). Conclusions: Continuous cultures with immobilized cells revealed that 1,3‐PDO production was more effective and more stable than suspended culture systems. Furthermore, cell immobilization had also obvious benefits especially for resistance of the production for extreme conditions (high organic loading rates, cell washouts). The results were important for understanding the significance of continuous immobilization process among other well‐known 1,3‐PDO fermentation processes. Significance and Impact of the Study: This work is a promising process for further studies, as the immobilized micro‐organism was able to reach high volumetric production rates at short HRT, it has an important role in tolerating and converting glycerol during fermentation. Therefore, HRT is a very significant operational parameter (P value <0·05) directly affecting the bioreactor performance and production rate.     

Effects of outdoor cultures on the growth and lipid production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> using closed photobioreactors

One of the principal challenges for large scale production of microalgae is the high costs of biomass production. Aiming for minimize this problem, microalgal biodiesel production should focus on outdoors cultures, using available solar light and allowing lower energy cost process. Testing species that proved to be common and easy to culture may be a good approach in this process. The present work reports indoor-outdoor cultures of Phaeodactylum tricornutum using different bioreactors types, using cell growth, biochemical composition, and the profiles of the fatty acids produced as the parameters to test the optimization processes. The results show that the use of outdoor cultures is a good choice to obtain P. tricornutum biomass with a good potential for biodiesel production. The microalgae produced reached better growth efficiency, major lipid content and showed an increment in the percentage of saturated fatty acids (required on the biodiesel production) respect indoor cultures. These results are important to show the relevance of using outdoor cultures as a way to improve the efficiency and the energetic balance of the biodiesel production with P. tricornutum algae.     

The expression of the Cuphea palustris thioesterase CpFatB2 in Yarrowia lipolytica triggers oleic acid accumulation

The conversion of industrial by‐products into high‐value added compounds is a challenging issue. Crude glycerol, a by‐product of the biodiesel production chain, could represent an alternative carbon source for the cultivation of oleaginous yeasts. Here, we developed five minimal synthetic glycerol‐based media, with different C/N ratios, and we analyzed the production of biomass and fatty acids by Yarrowia lipolytica Po1g strain. We identified two media at the expense of which Y. lipolytica was able to accumulate ～5 g L^?1 of biomass and 0.8 g L^?1 of fatty acids (0.16 g of fatty acids per g of dry weight). These optimized media contained 0.5 g L^?1 of urea or ammonium sulfate and 20 g L^?1 of glycerol, and were devoid of yeast extract. Moreover, Y. lipolytica was engineered by inserting the FatB2 gene, coding for the CpFatB2 thioesterase from Cuphea palustris, in order to modify the fatty acid composition towards the accumulation of medium‐chain fatty acids. Contrary to the expected, the expression of the heterologous gene increased the production of oleic acid, and concomitantly decreased the level of saturated fatty acids. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:26–35, 2016     

The limit of resistance to salinity in the freshwater cyanobacterium Microcystis aeruginosa is modulated by the rate of salinity increase

The overall mean levels of different environmental variables are changing rapidly in the present Anthropocene, in some cases creating lethal conditions for organisms. Under this new scenario, it is crucial to know whether the adaptive potential of organisms allows their survival under different rates of environmental change. Here, we used an eco‐evolutionary approach, based on a ratchet protocol, to investigate the effect of environmental change rate on the limit of resistance to salinity of three strains of the toxic cyanobacterium Microcystis aeruginosa. Specifically, we performed two ratchet experiments in order to simulate two scenarios of environmental change. In the first scenario, the salinity increase rate was slow (1.5‐fold increase), while in the second scenario, the rate was faster (threefold increase). Salinity concentrations ranging 7–10 gL^‐1 NaCl (depending on the strain) inhibited growth completely. However, when performing the ratchet experiment, an increase in salinity resistance (9.1–13.6 gL^‐1 NaCl) was observed in certain populations. The results showed that the limit of resistance to salinity that M. aeruginosa strains were able to reach depended on the strain and on the rate of environmental change. In particular, a higher number of populations were able to grow under their initial lethal salinity levels when the rate of salinity increment was slow. In future scenarios of increased salinity in natural freshwater bodies, this could have toxicological implications due to the production of microcystin by this species.     

How Do Different Watering Regimes Affect the Growth,Chlorophyll Fluorescence,Phytohormone, and Phenolic Acid Content of Greenhouse-Grown Ceratotheca triloba?

We evaluated the effect of different watering regimes on the growth, chlorophyll fluorescence, phytohormones, and phenolic acids in Ceratotheca triloba (Bernh.) Hook.f., a commonly consumed African indigenous leafy vegetable. The study was conducted in the greenhouse under different watering regimes [seven (daily); three (thrice); two (twice); one (once) day(s) per week] for a period of 2 and 4-months. In each pot (7.5 cm diameter; 150 ml volume), 50 ml of water was applied per treatment. At the end of the experiment, plant growth, chlorophyll fluorescence, phytohormones, and phenolic acids were determined. A decrease in water availability resulted in a consistent decline in plant growth after a 4-month growth period. The severity of reduced water availability was more noticeable in plants watered once a week with a 1.4-fold reduction in growth and quantum efficiency of PSII (Fv/Fm) value of 0.80. The significant decline in growth and chlorophyll fluorescence was probably due to the increased production of abscisic acid (ABA) and cytokinin (CK) content together with the detected phytohormones in plants with restricted water supply. Furthermore, plants watered once a week had a trade-off between growth and phenolic acid production, with significantly higher (threefolds) concentrations of vanillic, ferulic, caffeic, and 4-coumaric acids in 4-month-old plants. Even though C. triloba grew best in well-watered soil, the plant had the potential to adapt and survive in soils with limited water supply for longer periods of growth. These findings suggest that regulation of phytohormones and phenolic acids played an important role in improving the growth of C. triloba under limited water conditions.