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Xylose is the second most abundant sugar in lignocellulosic biomass and on the planet. In the second-generation bioethanol production process, it is essential to metabolize all sugars obtained in the hydrolysis of lignocellulose to increase ethanol titers and conversion yields of biomass to biofuel and decrease production costs. Wild-type Saccharomyces cerevisiae strains, the workhorse microorganisms in ethanol production, are not able to ferment xylose. In this review, a comparison in bioethanol production from xylose was carried out between non-conventional yeasts and recombinant strains of Saccharomyces, highlighting the advantages and disadvantages according to the most important factors that influence sugar metabolism to ethanol, such as the assimilation pathway used, co-assimilation with glucose, oxygen levels, tolerance to fermentation inhibitors, and process temperature, among others. The review begins reviewing the metabolic pathways of xylose assimilation, followed by the associated cofactor imbalance, heterologous expression of xylose isomerase (XI), regulation of xylose reductase (XR) and xylitol dehydrogenase (XDH), and the relevance of genes involved in the pentose phosphate pathway (PPP). Likewise, the importance of other yeasts, such as Scheffersomyces stipitis, Candida shehatae, and Kluyveromyces marxianus, as non-conventional yeasts to produce bioethanol is highlighted, due to their ability to ferment a wide range of sugars, including xylose, compared to S. cerevisiae.

Ibervillea sonorae (S. Watson) Greene, (huereque) is an endemic shrub whose roots are used in traditional medicine to treat diabetes. Here, we establish cell cultures of I. sonorae to evaluate the presence of antidiabetic metabolites. Stem and roots sections were incubated on media with different concentrations of growth regulators to induce calli. Using B5 medium containing 25 g L−1 sucrose, 150 mg L−1ascorbic acid, 6 g L−1 agar–agar, supplemented with 0.5 mg L−1 α-naphthalene-acetic acid, 0.5 mg L−1 benzyladenine and 1 mg L−1 indole-3-acetic acid, 95% calli induction was attained. Flavonoids and phenols in I. sonorae cultured cell suspension were investigated. The methanol extracts exhibited antioxidant activity that was attributed to phenolic compounds. These extracts stimulated glucose uptake more efficaciously than I. sonorae plant roots. This study demonstrates that in vitro cultured cells stand as a good option for production of hypoglycemic metabolites.

The objectives of this study were to investigate whether elicitors induce the production of taxoids in Taxus globosa by testing the hypothesis that the cells induce a greater accumulation of taxoids depending on the type and concentration of the elicitor treatment tested. Cell cultures were initiated from Taxus globosa friable calli for producing taxoids using the Gamborg medium supplemented with different initial combinations of growth regulators as follows: naphthaleneacetic acid, benzylaminopurine, picloram, and polyvinylpyrrolidone. The cell suspension cultures were then used for evaluating taxoid production through different elicitor treatments, such as methyl jasmonate, ethanol, buthionine sulphoximine, and hydrogen peroxide. The cell suspension cultures showing the best growth characteristics were found in the medium supplemented with 10.74 µM of naphthaleneacetic acid and 3.33 µM of picloram. The highest biomass for the cell cultures was obtained in the EtOH-2 treatment (0.12 ± 0.005 mg·g−1 of dry weight) after 8 days, while the biomass in the control treatment at that time was 0.095 ± 0.2 mg·g−1 of dry weight. The exogenous application of a combination of elicitors buthionine and hydrogen peroxide in the cell suspension cultures significantly increased the concentration of the 10-deacetylbaccatin (1662 µg·g−1 of dry weight), cephalomannine (334.32 µg·g−1 of dry weight), and the production of taxol (157.0 µg·g−1 of dry weight).

Lignocellulose hydrolysates are rich in fermentable sugars such as xylose, cellobiose and glucose, with high potential in the biotechnology industry to obtain bioproducts of higher economic value. Thus, it is important to search for and study new yeast strains that co-consume these sugars to achieve better yields and productivity in the processes. The yeast Clavispora lusitaniae CDBB-L-2031, a native strain isolated from mezcal must, was studied under various culture conditions to potentially produce ethanol and xylitol due to its ability to assimilate xylose, cellobiose and glucose. This yeast produced ethanol under microaerobic conditions with yields of 0.451 g

Spodoptera frugiperda J.E. Smith (Lepidoptera: Noctuidae) is a polyphagous insect pest native to America. Due to its capacity for adaptation and migration, it is currently located in Africa, Asia, and Oceania, where it threatens agricultural crops. The ability of S. frugiperda to develop resistance to insecticides is one of the reasons for the continuous search for more effective, low-cost, and environmentally friendly control products. In the present work, the insecticidal activity of ethanolic and hexane extracts obtained from fresh and dehydrated leaves of Piper auritum Kunth (Piperales: Piperaceae), Piper umbellatum L. (Piperales: Piperaceae), and Cedrela odorata L. (Sapindales: Meliaceae) was studied against first instar larvae of S. frugiperda. The ethanolic extracts of the dehydrated leaves of C. odorata and P. auritum presented insecticidal activity as high (100% mortality at a concentration of 92 mg/cm2) as that obtained with the positive control, Melia azedarach L. (Sapindales: Meliaceae). The GC-MS analysis of the extracts revealed the presence of phytochemicals classified mainly into the groups of monoterpenes, sesquiterpenes, diterpenes, phenylpropanoids, alcohols, and fatty acids. P. auritum grows and propagates rapidly. In addition, due to its low toxicity in mammals and non-target insects, it is a plant with the potential to be used as a botanical insecticide. The exposure of S. frugiperda larvae to low concentrations of ethanolic extract of P. auritum allowed us to observe their biological activity in the development of this insect. The LC50 was 22.1 mg/cm2. At sublethal concentrations (LC21 and LC35) the low fertility of the emerging adults was noticeable.

The enzymatic hydrolysis of sugar cane bagasse (SCB) releases simple fermentable sugars however, phenolic compounds (PCs) are also released from the lignin present in the structure of the lignocellulosic biomass limiting the production of sugars and therefore the fermentative process to convert these sugars to bioethanol. During the saccharification of SCB with holocellulases from Cellulomonas flavigena PR-22, 46 μg mL −1 of PCs were produced, which inhibited 87 and 74% carboxymethyl cellulase (CMCase) and xylanases respectively resulting in a low concentration of reducing sugars (5.5 g L −1 ) at the end of the process. Polymers such as polyethylene glycol 4000 (PEG) and polyvinylpyrrolidone (PVP) can prevent this inhibition due to their ability to chemically adsorb PCs. PEG and PVP were added to C. flavigena PR-22 enzyme extracts at 0.4% (w/v). PEG 4000 prevented CMCase inhibition, maintaining the original activity after the saccharification process, whereas xylanase production was increased by 47% when PVP was added.

Specialized or secondary metabolism of plants from two genera, Uncaria and Hamelia, has been widely studied generating a huge investment in research and development of patents. This review highlights the most recent patents granted so far, with some exceptions, covering different areas of knowledge including biotechnological aspects of plant specialized/secondary metabolism. Several patent databases were consulted in order to collect patent information. There are patents for specialized/secondary metabolite isolation methods, synthesis, production, genes, development of medicaments, drug delivery systems, equipment, machines, industrial processes, plant culture techniques, and new plant varieties, among others. This review of the state-of-the art about patents related to Uncaria and Hamelia, as well as their metabolites, can be a valuable source of organized information for those researchers developing projects in many aspects related to these two genera. Uncaria and Hamelia have an enormous potential to become important economically crops.

Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding β-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active β-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L−1 and 222 IU g−1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L−1) as the sole carbon source for 48 h. Ethanol production was 5 g L−1 after 96 h of culture, which represented a yield of 0.41 g g−1 of substrate consumed (12 g L−1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the β-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

Specialized or secondary metabolism of plants from the genus Cannabis has been widely studied, generating a huge investment in research and development of patents. This review highlights the most recent patents granted so far, with some exceptions, covering different areas of knowledge including biotechnological aspects of plant specialized/secondary metabolism. Several patent databases were consulted in order to collect patent information. There are patents for specialized/secondary metabolite isolation methods, synthesis, production, genes, development of medicaments, drug delivery systems, equipment, machines, industrial processes, plant culture techniques, improved plant varieties, among others. Furthermore, important aspects such as nutrition and fiber production having unlimited applications in several areas are discussed. Endocannabinoid system is a special topic that has been included in this review due to interconnections among cannabinoids, endocannabinoids and its medical effects in human beings. This review of the state-of-the-art about Cannabis patents has the potential to be a valuable source of organized information for those researchers developing projects in many aspects related to this important natural resource.

The organic fraction of municipal solid waste (OFMSW), mainly composed of lignocellulosic polymers, is extremely complex. Therefore, it is necessary to apply pretreatments to remove the lignin content and decrease the cellulose crystallinity in order to use the OFMSW for gas biofuels production in the context of biorefineries from waste. This work focused on critically reviewing the conventional pretreatments applied to OFMSW, with the goal of improving the H2 production, as well as other biofuels in modern biorefineries. There are a wide variety of pretreatments that have successfully been used, mainly alkaline, milling and dilute acid. In addition, some research has focused on the recovery and reutilization of the alkali, acid or solvents after the pretreatment, to be incorporated into new cycles of production, minimizing the environmental impacts. Moreover, it would be necessary to incorporate analytical tools, in order to determine the sustainability of the biorefinery project. It is concluded that waste pretreatments could significantly contribute to increased yields of biogas fuels in organic waste-based biorefineries. Therefore, establishing preliminary stages for conditioning biomass or wastes is essential to improve the degradation of wastes and bio-product generation. © 2016 Society of Chemical Industry

Occurrence of calcium oxalate (CaC2O4 – CaOX) crystals has been observed in more than 215 plant families. However, very little is known about the effects of calcium oxalate on biomass pretreatment and saccharification. Agave bagasse (AGB) was used as a model material due to its natural high levels of CaOX. To understand the physicochemical changes in function of biomass pretreatment, both raw AGB and CaOX-extracted agave bagasse (EAB) were subjected to ionic liquid (IL) with 1-Butyl-3-methylimidazolium chloride [C4C1Im][Cl] and alkaline hydrogen peroxide (AHP) pretreatments. Physicochemical changes were monitored by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and wet chemistry methods. Results show that free CaOX crystals affected negatively (by ca 39%) the saccharification of AHP-pretreated EAB compared to AGB. On the other hand, IL pretreatment achieved higher sugar yield (7.8 g dm−3) and lower crystallinity (14%) with EAB than for AHP (5.4 g dm−3 and 29%, respectively).

Jatropha curcas has considerable potential for production of biodiesel and secondary metabolites with medicinal uses. Herein, J. curcas cell suspension cultures were established to study the effect of jasmonic acid (JA) elicitation on triterpene production and oxidative responses. Cell cultures grown in dark conditions reached maximum biomass accumulation at the 12th day of culture (14.3 ± 0.45 g DW L−1) with a specific growth rate μ = 0.131 d−1. Elicitation with JA (200 or 400 μM) on 4-days-old cell cultures caused reduction in biomass and triterpene contents. In contrast, application of 200 μM JA at the 7th day of culture triggered triterpene accumulation by three times (1180 ± 12.3 μg g−1 DW, at day 2) with respect to control, without significant changes in biomass and viability. After 2 days of elicitation, betulin increased up to 7.3-fold (from 110.6 ± 20.7 to 808.7 ± 55.4 μg g−1 DW), while betulinic acid reached the maximum amount at day 6 after elicitation (245.6 ± 3.7 to 835 ± 41.5 μg g−1 DW). Lupeol presented a moderate increase (167.9 ± 51.0–288.8 ± 7.3 μg g−1 DW) along 8 days after elicitation. In correlation with triterpene production, JA application induced oxidative responses evaluated by an increase in the H2O2 levels up to three times and of malondialdehyde by 59 %. At day 4 after elicitation, catalase showed higher increase (122 %) than peroxidases (63 %) and ascorbate peroxidase (26 %). Incorporation of radioactive labels from (R,S)-[2-14C]mevalonic acid in triterpenes and sterols confirmed its role as metabolic precursor.

Cellulase (CMCase) and xylanase enzyme production and saccharification of sugar cane bagasse were coupled into two stages and named enzyme production and sugar cane bagasse saccharification. The performance of Cellulomonas flavigena (Cf) PR-22 cultured in a bubble column reactor (BCR) was compared to that in a stirred tank reactor (STR). Cells cultured in the BCR presented higher yields and productivity of both CMCase and xylanase activities than those grown in the STR configuration. A continuous culture with Cf PR-22 was run in the BCR using 1% alkali-pretreated sugar cane bagasse and mineral media, at dilution rates ranging from 0.04 to 0.22 1/h. The highest enzymatic productivity values were found at 0.08 1/h with 1846.4 ± 126.4 and 101.6 ± 5.6 U/L·h for xylanase and CMCase, respectively. Effluent from the BCR in steady state was transferred to an enzymatic reactor operated in fed-batch mode with an initial load of 75 g of pretreated sugar cane bagasse; saccharification was then performed in an STR at 55°C and 300 rpm for 90 h. The constant addition of fresh enzyme as well as the increase in time of contact with the substrate increased the total soluble sugar concentration 83% compared to the value obtained in a batch enzymatic reactor. This advantageous strategy may be used for industrial enzyme pretreatment and saccharification of lignocellulosic wastes to be used in bioethanol and chemicals production from lignocellulose. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:321-326, 2016.

The primary carotenoid synthesized by Xanthophyllomyces dendrorhous is astaxanthin, which is used as a feed additive in aquaculture. Cell growth kinetics and carotenoid production were correlated with the mRNA levels of the idi, crtE, crtYB, crtI, crtS and crtR genes, and the changes in gene sequence between the wild-type and a carotenoid overproducer XR4 mutant strain were identified. At the late stationary phase, the total carotenoid content in XR4 was fivefold higher than that of the wild-type strain. Additionally, the mRNA levels of crtE and crtS increased during the XR4 growth and were three times higher than the wild-type strain in the late stationary phase. Moreover, the nucleotide sequences of crtYB, crtI and crtR exhibited differences between the strains. Both the higher crtE and crtS transcript levels and the crtYB, crtI and crtR mutations can, at least in part, act to up-regulate the carotenoid biosynthesis pathway in the XR4 strain.

Jatropha curcas has significant potential for production of biodiesel and secondary metabolites with medical applications. The effect of jasmonic acid (JA) on flavonoid production and antioxidant responses in two Mexican J. curcas plants (accessions I-64 and I-52), growing under controlled environmental conditions, was studied. Foliar application of JA (0.25, 0.5, and 1.0 mM) increased flavonoid content by 3.3-fold (445.6 ± 62.2 µg QE g−1 DW) at 1.0 mM in the I-64 accession after 120 h. At this time, individual flavonoid levels of kaempferol, apigenin, and vitexin were 6.8 (106.8 ± 18.6 µg g−1 DW), 4.4 (15.5 ± 1.5 µg g−1 DW), and 8.1 (6.27 ± 1.3 µg g−1 DW) times higher than in the controls, while for the I-52 specimens, the highest increase of flavonoids occurred at 24 h. In treated I-52 plants, enhancement of 58% (74.7 ± 9.2 µg g−1 DW) in anthocyanins occurred after 120 h, while in I-64 plants, they remained similar to controls. Phenylalanine ammonia lyase showed a peak of activity after 120 h in treated I-64 plants, while the I-52 accession showed peaks at 24 and 120 h. Elicited I-64 plants presented H2O2 levels similar to controls with a 30.6% increase in catalase (CAT) and 3.3-fold in ascorbate peroxidase (APX) activities. In elicited I-52 plants, a 2.4-fold increase in H2O2 concentration was related to a 65.6% decrease of CAT and a 1.8-fold increase of APX activities. Therefore, under JA elicitation, J. curcas plants increased antioxidant responses including flavonoid and anthocyanin production to maintain cell redox balance.

The catalytic fraction of the Cellulomonas flavigena PN-120 oligomeric β-glucosidase (BGLA) was expressed both intra- and extracellularly in a recombinant diploid of Saccharomyces cerevisiae, under limited nutrient conditions. The recombinant enzyme (BGLA¹⁵) expressed in the supernatant of a rich medium showed 582 IU/L and 99.4 IU/g dry cell, with p-nitrophenyl-β-D-glucopyranoside as substrate. BGLA¹⁵ displayed activity against cello-oligosaccharides with 2-5 glucose monomers, demonstrating that the protein is not specific for cellobiose and that the oligomeric structure is not essential for β-D-1,4-bond hydrolysis. Native β-glucosidase is inhibited almost completely at 160 mM glucose, thus limiting cellobiose hydrolysis. At 200 mM glucose concentration, BGLA¹⁵ retained more than 50 % of its maximal activity, and even at 500 mM glucose concentration, more than 30 % of its activity was preserved. Due to these characteristics of BGLA¹⁵ activity, recombinant S. cerevisiae is able to utilize cellulosic materials (cello-oligosaccharides) to produce bioethanol.

The aim of this work was to compare the H2 production in a lab scale anaerobic fluidized bed bioreactors (AFBRs) at two levels of operational temperature: ambient temperature (A) and 35 °C (M) and two organic volumetric loading rates Bv: 5 and 8 g sucrose/L.day, with a constant hydraulic residence time of 1 day. The increase of Bv from 5 to 8 g sucrose/L. day had a positive effect on the performance of our anaerobic fluidized bed reactors. In conclusion, at a Bv of 8 g sucrose/L.day the average performance of AFBR-A was superior to that of AFBR-M: 1.8 times for the H2 concentration in the biogas (54% and 31% respectively) and 2.2 times for the H2 productivity (920 and 427 NmLH2/Lbed .day, respectively). The H2 production in an AFBR operated at ambient temperature was superior to that of the mesophilic units and showed encouraging results for H2 production in submerged fermentation of moderate concentration of sucrose.

Hamelia patens is a perennial shrub native from Mexico which is used in traditional medicine to treat chronic wounds and tumors. Bioactive monoterpenoid indole alkaloids (MIAs) and monoterpenoid oxindole alkaloids (MOAs) have been isolated from this species, associated with the ethnomedical use. In order to study the biosynthesis and production of these alkaloids, H. patens plants were treated with the stress hormone jasmonic acid. Nuclear magnetic resonance-based metabolic profiles and multivariate data analysis were applied to explore changes or variability in elicited and control plants. According to the partial least square-discriminant analysis from MeOH/H2O fractions, sucrose, tryptophan, glutamic acid, glutamine, strictosidine, p-coumaric acid, chlorogenic acid, and the MOAs isopteropodine, palmirine and pteropodine were predominant in the treated plants, while glucose, aspartic acid, acetic acid, and loganic acid concentrations were higher in control plants. HPLC analysis showed that accumulation of pteropodine, isopteropodine, speciophylline, rumberine, hameline and palmirine in elicited plants was preceded by an increase in the transcription level of 1-deoxy-d-xylulose-5-phosphate synthase (DXS, EC 2.2.1.7) and strictosidine synthase (STR, EC 4.3.3.2), and STR activity during a time course. However, no inductions of transcription and enzyme activity of strictosidine β-glucosidase (SGD, EC 3.2.1.105) were observed in elicited plants. Correlations between primary and secondary metabolism biosynthetic pathways, such as the alkaloid and chlorogenic acid biosynthesis were found. Also, their implications in the plant fitness to stress are proposed. Confirmation of tryptophan as a precursor of MIA and MOA, via shikimate pathway, was done by L-[3′-14C]-tryptophan feeding.

Derepressed mutant PR-22 was obtained by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg(-1) and cellulolytic activity from 1.9 to 4 U mg(-1); this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20-32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l(-1) of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H 2 O 2 , 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H 2 O 2 (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ = 0.045 days -1 and maximum biomass of 12.08 ± 1.24 g DW/L (at day 30). Roots accumulated 3α-dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H 2 O 2 had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H 2 O 2 , MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H 2 O 2 , reduced by 30-40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H 2 O 2 with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H 2 O 2 in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6- and 2-times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

This work describes the accumulation and dis- tribution of the herbicide atrazine in soil, water, and roots from three wetland model systems using the monocots Typha domingensis, Sagittaria lancifolia, and Echinochloa pyra- midalis. Results were analyzed from a 3 3 full factorial experimental design, in order to describe the effect of accumulation of atrazine and times of exposure in the species evaluated. We found that accumulation depends on the species and the herbicide concentration; about 30% was accumulated in soil, 40% in roots, and 10-20% in water. By the end of the experiment, E. pyramidalis accumulated 8.47 mg/l of atrazine and 14.39 mg/l T. domingensis; in all cases, adsorption accounted for 1.4%, fitting a Langmuir model with a kd of 14.47.

A number of species of the genus Erythrina are rich in secondary metabolites, particularly phenolics and alkaloids that exhibit interesting anti-inflammatory, anti-plasmodial, bactericidal, curariform and fungicidal activities. Unfortunately, the isolation of these compounds through the extraction of organs and seeds of whole plants is becoming more difficult since the natural growing areas of many of the species, and particularly of Erythrina americana, are being urbanised. Plant tissue culture not only constitutes a viable method through which to preserve the species, but may also represent a constant and stable source of target alkaloids. Currently, however, in vitro systems, and especially cell suspension cultures, only accumulate low levels of the desired products. A number of strategies for increasing production have been proposed, the most successful of which involves elicitation of suspension cells with plant growth regulators. In this context, excellent results have been reported following elicitation of cell cultures derived from different species and genera with jasmonic acid and methyl jasmonate. The present paper provides a brief review of the novel approaches to the use of Erythrina alkaloids that have recently been described, and of the advances that have been made in the formation of tissue cultures of E. americana and their subsequent elicitation in attempts to augment alkaloid accumulation.

To obtain micro propagated Uncaria tomentosa plantlets with enhanced secondary metabolites production, long-term responses to salicylic acid (SA) pre-treatments at 1 and 100 µM were evaluated after propagation of the plantlets in a SA-free medium.SA pre-treatments of single node cuttings OF U. tomentosa produced long-term responses in microplants grown for 75 days in a SA-free medium. Reduction in survival rate, root formation, and stem elongation were observed only with 100 µM SA pre-treatments with respect to the control (0 + DMSO).Both pre-treatments enhanced H2O2 and inhibited superoxide dismutase and catalase activities, while guaiacol peroxidase was increased only with 1 µM SA. Also, both pre-treatments increased total monoterpenoid oxindole alkaloids by ca. 55 % (16.5 mg g(-1) DW), including isopteropodine, speciophylline, mitraphylline, isomitraphylline, rhynchopylline, and isorhynchopylline; and flavonoids by ca. 21 % (914 μg g(-1) DW), whereas phenolic compounds were increased 80 % (599 μg g(-1) DW) at 1 µM and 8.2 % (359 μg g(-1) DW) at 100 µM SA.Pre-treatment with 1 µM SA of U.tomentosa microplants preserved the survival rate and increased oxindole alkaloids, flavonoids, and phenolic compounds in correlation with H2O2 and peroxidase activity enhancements, offering biotechnological advantages over non-treated microplants.

Polycyclic aromatic hydrocarbons (PAHs) such as phenanthrene, anthracene and Benzo[a]pyrene (BaP) are toxic for the environment. Removing these components from soil is difficult as they are resistant to degradation and more so in soils with high pH and large salt concentrations as in soil of the former lake Texcoco, but stimulating soil micro-organisms growth by adding nutrients might accelerate soil restoration. Soil of Texcoco and an agricultural Acolman soil, which served as a control, were spiked with phenanthrene, anthracene and BaP, added with or without biosolid or inorganic fertilizer (N, P), and dynamics of PAHs, N and P were monitored in a 112-day incubation. Concentrations of phenanthrene did not change significantly in sterilized Acolman soil, but decreased 2-times in unsterilized soil and >25-times in soil amended with biosolid and NP. The concentration of phenanthrene in unsterilized soil of Texcoco was 1.3-times lower compared to the sterilized soil, 1.7-times in soil amended with NP and 2.9-times in soil amended with biosolid. In unsterilized Acolman soil, degradation of BaP was faster in soil amended with biosolid than in unamended soil and soil amended with NP. In unsterilized soil of Texcoco, degradation of BaP was similar in soil amended with biosolid and NP but faster than in the unamended soil. It was found that application of biosolid and NP increased degradation of phenanthrene, anthracene and BaP, but to a different degree in alkaline-saline soil of Texcoco compared to an agricultural Acolman soil.

An in vitro presymbiotic system between mesquite [Prosopis laevigata (Willd.) M.C. Johnst], a semi-arid leguminous plant, and pregerminated spores of Gigaspora rosea Nicol. & Schenck was established. After characteristic hyphal branching, high performance liquid chromatographic analyses of methanol extracts from P. laevigata roots revealed a concentration change in one ultraviolet-detectable product. This product was identified by nuclear magnetic resonance and mass spectrometry as trigonelline, a pyridine alkaloid. The concentration of trigonelline was constant in the aerial parts of the plant with or without G. rosea, but its concentration in the roots increased 1.8-fold when G. rosea was present. Trigonelline may be a regulatory factor during early signal events in the establishment of the arbuscular mycorrhizal symbiosis in P. laevigata.

The production of erythroidines and other alkaloids was studied in cotyledons, callus and cell suspension cultures of Erythrina americana Miller. The cell suspension cultures, grown in Murashige & Skoog medium with naphthaleneacetic acid (3 mg l−1) and kinetin (2 mg l−1), produced 89 and 17 μg α- and β-erythroidines respectively per g dry wt.

Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

The aim of this work was to evaluate the effect of buffer addition and process temperature (ambient and 35°C) on H2 production in batch fermentation of cheese whey (CW). When the H2 production reached a plateau, the headspace of the reactors were flushed with N2 and reactors were re-incubated. Afterwards, only the reactors with phosphate buffer showed a second cycle of H2 production and 48% more H2 was obtained. The absence of a second cycle in non-buffered reactors could be related to a lower final pH than in the buffered reactors; the low pH could drive the fermentation to solvents production. Indeed a high solvent production was observed in non-buffered bioreactors as given by low ρ ratios (defined as the ratio between sum of organic acid production and sum of solvents production). Regarding the process temperatures, no significant difference between the H2 production of reactors incubated at ambient temperature and at 35°C was described. After flushing the headspace of bioreactors with N2 at the end of the second cycle, the H2 production did not resume (in all reactors).

This manuscript evaluates the phytotoxicity and biotransformation of n-hexadecane as well as peroxidase activity and cytochrome P450 concentration in microsomes for cell suspension cultures of Cinchona robusta and Dioscorea composita. Phytotoxicity was evaluated based on viability and growth. Cell cultures were exposed to a 2 and 4% (v/v) dose of n-hexadecane. The biotransformation of n-hexadecane was determined based on labeled recovery in polar, nonpolar, and cell residue fractions after cell culture extraction during exponential cell growth phase and stationary phase. Differences were observed in accumulation of label during cell growth phase and stationary phase for the cells of the two plants. Differences also were observed between phases for label in polar and nonpolar fractions. Thin-layer chromatography determined labeled intermediates and some were identified. The activity of peroxidase and concentration of cytochrome P450 was lower in C. robusta than in controls and greater in D. composita than in controls. In vitro biotransformation was not successful.

A suspension culture of Cinchona robusta which under normal culture conditions does not produce anthraquinones, produces a range of these anthraquinones after elicitation. Eight new anthraquinones were identified, robustaquinones A–H, in addition to two known anthraquinones, 1,3,8-trihydroxy-2-methoxyanthraquinone and copareolatin 6-methyl ether. Their oxygenation pattern, characterized by a 6,7-disubstitution in the A-ring, is rare and raises questions about their biogenetic origin. They might be derived from phenylpropanoids and not from o -succinylbenzoic acid.

Dioscorea deltoidea cell suspension cultures were established in modified Murashige and Skoog medium. The diosgenin production increased from 0.10 g−1 to 3.98 g−1 dry cell weight when cells were cultivated in the light and in a growth medium limited in phosphate and sucrose. The addition of 1.3 g of autoclaved fungal mycelium of Alternaria tenuis per litre of cell culture growing in the dark induced the production of 0.04 mg diosgenin g−1 dry cell weight. In both cases, the production of diosgenin was preceded by a transient induction of isopentenyl diphosphate isomerase activity.

Isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2) and farnesyl diphosphate (FPP) synthase (EC 2.5.1.10) activities were studied in plant cell cultures of Cinchona robusta , Morinda citrifolia , Rubia tinctorum , all belonging to the Rubiaceae, and Tabernaemontana divaricata and Catharanthus roseus , both belonging to the Apocynaceae. The presence of isoforms of IPP isomerase was detected after hydroxyapatite chromatography and by Western blotting using anti-IPP isomerase antibodies. The Rubiaceae cultures are able to accumulate anthraquinones, and for their biosynthesis isoprene C 5 -units are required. In a M. citrifolia cell line accumulating anthraquinones (AQ+), an almost two-fold higher IPP isomerase was observed compared with a cell line without accumulation of anthraquinones. In the Western blots three polypeptides were detected in the extracts from the AQ+ cells. In C. robusta cells the anthraquinone biosynthesis was induced by medium optimization and by elicitation. After both treatments, a specific isoform of IPP isomerase was induced. The C. roseus culture (not accumulating anthraquinones) expressed relatively low IPP isomerase activity, but two isoforms could be detected. The enzyme activities as observed in the different Rubiaceae cultures may indicate an efficient channelling of C 5 -units into anthraquinone biosynthesis.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn2+ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C5-precursors into phytoalexin biosynthesis.

The yeast Xanthophyllomyces dendrorhous synthesizes astaxanthin as its most prevalent xanthophyll derivative. Comparisons between the protein profiles of mutant lines of this yeast can provide insight into the carotenogenic pathway. Differently colored mutants (red, orange, pink, yellow, and white) were obtained from this yeast species, and their protein profiles were determined using two-dimensional polyacrylamide gel electrophoresis (2DE). Individual proteins differentially expressed were identified using mass spectrometry. The red mutants hyperproduced total carotenoids (mainly astaxanthin), while in white and orange mutants, mutagenesis affected the phytoene dehydrogenase activity as indicated by the accumulation of phytoene. Inactivation of astaxanthin synthase after the mutagenic treatment was evident in β-carotene accumulating mutants. Differences in the proteomic profiles of wild-type X. dendrorhous and its colored mutants were demonstrated using 2DE. Of the total number of spots detected in each gel (297–417), 128 proteins were present in all strains. The red mutant showed the greatest number of matches with respect to the wild type (305 spots), while the white and yellow mutants, which had reduced concentrations of total carotenoids, presented the highest correlation coefficient (0.6) between each other. A number of differentially expressed proteins were sequenced, indicating that tricarboxylic acid cycle and stress response proteins are closely related to the carotenogenic process.

Chemical studies on Hamelia patens (Rubiaceae) micropropagated plantlets allowed production of a new monoterpenoid oxindole alkaloid, named (-)-hameline (7), together with eight known alkaloids, tetrahydroalstonine (1), aricine (2), pteropodine (3), isopteropodine (4), uncarine F (5), speciophylline (6), palmirine (8), and rumberine (9). The structure of the new alkaloid was assigned on the basis of 1D and 2D NMR spectroscopy, mass spectrometry, and molecular modeling.

The activity and gene expression of strictosidine-related enzymes in Uncaria tomentosa root cultures exposed to oxidative stress were studied. Elicitation with 0.2 mM hydrogen peroxide (H2 O2 ) or a combination of 0.8 mM buthionine sulfoximine and 0.2 mM jasmonic acid (BSO-JA) increased peroxidase activities by twofold at Day 8 and glutathione reductase by 1.4-fold at Day 5 in H2 O2 elicited cultures respect to the control. Production of monoterpenoid oxindole alkaloids (MOA), 3α-dihydrocadambine, and dolichantoside was stimulated after H2 O2 elicitation, reaching levels of 886.4 ± 23.6, 847.7 ± 25.4, and 87.5 ± 7.2 µg/g DW, at Day 8 which were 1.7-, 2.1-, and 2.3-fold higher relative to control. BSO-JA elicited cultures produced about twice alkaloids than H2 O2 -treated cultures, following a biphasic pattern with maxima at 0.5 and 8 days. Alkaloid production was preceded by increase in strictosidine synthase (STR) and strictosidine glucosidase (SGD) activities. After elicitation with H2 O2 or BSO-JA, the STR activity (pKat/mg protein) increased by 1.9-fold (93.8 ± 17.8 at 24 h) or 2.5-fold (102.4 ± 2.2 at 6 h) and the SGD activity (pKat/mg protein) by 2.8-fold (245.2 ± 14.4 at 6 h) or 4.2-fold (421.2 ± 1.8 at 18 h) relative to control. STR and SGD transcripts were upregulated after elicitation. H2 O2 -treated roots showed higher levels of STR at 48-192 h and SGD at 24-48 h, while BSO-JA treatments showed STR increased at 12 h and SGD at 24 h. Also, LC/ESI-MS confirmed the biosynthesis of dolichantoside from N-ω-methyltryptamine and secologanin by U. tomentosa protein extracts.

The alkaloids of Uncaria tomentosa micropropagated plantlets and root cultures were isolated and identified by NMR and mass spectrometry. Plantlets yielded pteropodine (1), isopteropodine (2), mitraphylline (3), isomitraphylline (4), uncarine F (5), speciophylline (6), rhynchophylline (7) and isorhynchophylline (8). In plantlets growing under continuous light, tetracyclic alkaloids 7 and 8 decreased from 20 ± 1.8 at 2 months to 2.2 ± 0.33 mg/g dry wt at 6 months, while the pentacyclic alkaloids 1–4 increased from 7.7 ± 1.4 to 15 ± 0.05 mg/g dry wt, supporting their biogenetic conversion. Micropropagated plantlets produced four times more alkaloids (27.6 ± 3.1 mg/g dry wt) than greenhouse plants. Plantlet roots yielded 3, 4, 8 and the glucoindole alkaloids 3α-dihydrocadambine (9) and dolichantoside (10), the last one not previously found in Uncaria.

R. Marcos Soto-Hernandez, Rosario Garcia-Mateos, Ruben San Miguel-Chavez, Geoffrey Kite, Mariano Martinez-Vazquez and Ana C. Ramos-Valdivia 1Colegio de Postgraduados, Campus Montecillo Mexico, Universidad Autonoma Chapingo, Preparatoria Agricola, Mexico, 2Royal Botanic Gardens, Kew Richmond, 3Universidad Nacional Autonoma de Mexico, Instituto de Quimica Mexico, 4Centro de Investigacion y Estudios Avanzados del Instituto Politecnico Nacional, Unidad Zacatenco, 1,3,4Mexico 2UK

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.

Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate _ gavg ¼ 86s � 1 . The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 mmol H2O2 g � 1 dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 � 40 m gL � 1 at 24 h). At the stationary phase (144 h), the increase of the shear rate _ gavg up to 150 s � 1 and/or oxygen tension up to 85% generated H2O2, restoring oxi- ndole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 � 40 m gL � 1 . A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 mM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 mM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress. Biotechnol. Bioeng. 2007;98: 230-238.

Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.