Production Of High-value Compounds Research Articles

Study of phenoxy radical couplings using the enzymatic secretome of Botrytis cinerea

Phenoxy radical coupling reactions are widely used in nature for the synthesis of complex molecules such as lignin. Their use in the laboratory has great potential for the production of high value compounds from the polyphenol family. While the enzymes responsible for the generation of the radicals are well known, the behavior of the latter is still enigmatic and difficult to control in a reaction flask. Previous work in our laboratory using the enzymatic secretome of B. cinerea containing laccases has shown that incubation of stilbenes leads to dimers, while incubation of phenylpropanoids leads to dimers as well as larger coupling products. Building on these previous studies, this paper investigates the role of different structural features in phenoxy radical couplings. We first demonstrate that the presence of an exocyclic conjugated double bond plays a role in the generation of efficient reactions. In addition, we show that the formation of phenylpropanoid trimers and tetramers can proceed via a decarboxylation reaction that regenerates this reactive moiety. Lastly, this study investigates the reactivity of other phenolic compounds: stilbene dimers, a dihydro-stilbene, a 4-O-methyl-stilbene and a simple phenol with the enzymatic secretome of B. cinerea. The observed efficient dimerization reactions consistently correlate with the presence of a para-phenol conjugated to an exocyclic double bond. The absence of this structural feature leads to variable results, with some compounds showing low conversion or no reaction at all. This research has allowed the development of a controlled method for the synthesis of specific dimers and tetramers of phenylpropanoid derivatives and novel stilbene derivatives, as well as an understanding of features that can promote efficient radical coupling reactions.

Current and novel approaches in yeast cell death research.

The study of cell death mechanisms in fungi, particularly yeasts, has gained substantial interest in recent decades driven by the potential for biotechnological advancements and therapeutic interventions. Examples include the development of robust yeast strains for industrial fermentations and high-value compound production, novel food preservation strategies against spoilage yeasts, and the identification of targets for treating fungal infections in the clinic. In this review, we discuss a wide range of methods to characterize cellular alterations associated with yeast cell death, noting the advantages and limitations. We describe assays to monitor reversible events versus those that mark a commitment to cell death (point-of-no-return), as these distinctions are important to decipher the underlying regulatory mechanisms. Several well-known challenges remain, including the varied susceptibilities to death within a cell population and the delineation of detailed cell death mechanisms. The identification and characterization of morphologically distinct subsets of dying yeast cells within dynamic yeast populations provides opportunities to reveal novel vulnerabilities and survival mechanisms. Elucidating the intricacies of yeast regulated cell death (yRCD) will contribute to the advancement of scientific knowledge and foster breakthrough discoveries with broad-ranging implications.

Coupling dairy wastewaters for nutritional balancing and water recycling: sustainable heterologous 2-phenylethanol production by engineered cyanobacteria.

Microalgae biotechnology is hampered by the high production costs and the massive usage of water during large-volume cultivations. These drawbacks can be softened by the production of high-value compounds and by adopting metabolic engineering strategies to improve their performances and productivity. Today, the most sustainable approach is the exploitation of industrial wastewaters for microalgae cultivation, which couples valuable biomass production with water resource recovery. Among the food processing sectors, the dairy industry generates the largest volume of wastewaters through the manufacturing process. These effluents are typically rich in dissolved organic matter and nutrients, which make it a challenging and expensive waste stream for companies to manage. Nevertheless, these rich wastewaters represent an appealing resource for microalgal biotechnology. In this study, we propose a sustainable approach for high-value compound production from dairy wastewaters through cyanobacteria. This strategy is based on a metabolically engineered strain of the model cyanobacterium Synechococcus elongatus PCC 7942 (already published elsewhere) for 2-phenylethanol (2-PE). 2-PE is a high-value aromatic compound that is widely employed as a fragrance in the food and cosmetics industry thanks to its pleasant floral scent. First, we qualitatively assessed the impact of four dairy effluents on cyanobacterial growth to identify the most promising substrates. Both tank-washing water and the liquid effluent of exhausted sludge resulted as suitable nutrient sources. Thus, we created an ideal buffer system by combining the two wastewaters while simultaneously providing balanced nutrition and completely avoiding the need for fresh water. The combination of 75% liquid effluent of exhausted sludge and 25% tank-washing water with a fine-tuning ammonium supplementation yielded 180mgL-1 of 2-PE and a biomass concentration of 0.6gDW L-1 within 10days. The mixture of 90% exhausted sludge and 10% washing water produced the highest yield of 2-PE (205mgL-1) and biomass accumulation (0.7gDW L-1), although in 16days. Through these treatments, the phosphates were completely consumed, and nitrogen was removed in a range of 74%-77%. Overall, our approach significantly valorized water recycling and the exploitation of valuable wastewaters to circularly produce marketable compounds via microalgae biotechnology, laying a promising groundwork for subsequent implementation and scale-up.

Microbial-Driven Synthesis and Hydrolysis of Neohesperidin Dihydrochalcone: Biotransformation Process and Feasibility Investigation.

A novel yeast-mediated hydrogenation was developed for the synthesis of neohesperidin dihydrochalcone (NHDC) in high yields (over 83%). Moreover, whole-cell catalytic hydrolysis was also designed to hydrolyze NHDC into potential sweeteners, hesperetin dihydrochalcone-7-O-glucoside (HDC-G) and hesperetin dihydrochalcone (HDC). The biohydrogenation was further combined with whole-cell hydrolysis to achieve a one-pot two-step biosynthesis, utilizing yeast to hydrogenate C═C in the structure, while Aspergillus niger cells hydrolyze glycosides. The conversion of NHDC and the proportion of hydrolysis products could be controlled by adjusting the catalysts, the components of the reaction system, and the addition of glucose. Furthermore, yeast-mediated biotransformation demonstrated superior reaction stability and enhanced safety and employed more cost-effective catalysts compared to the traditional chemical hydrogenation of NHDC synthesis. This research not only provides a new route for NHDC production but also offers a safe and flexible one-pot cascade biosynthetic platform for the production of high-value compounds from citrus processing wastes.

Multiplex Marker-Less Genome Integration in Pichia pastoris Using CRISPR/Cas9.

Pichia pastoris is known for its excellent protein expression ability. As an industrial methyl nutritional yeast, it can effectively utilize methanol as the sole carbon source, serving as a potential platform for C1 biotransformation. Unfortunately, the lack of synthetic biology tools in P. pastoris limits its broad applications, particularly when multigene pathways should be manipulated. Here, the CRISPR/Cas9 system is established to efficiently integrate multiple heterologous genes to construct P. pastoris cell factories. In this protocol, with the 2,3-butanediol (BDO) biosynthetic pathway as a representative example, the procedures to construct P. pastoris cell factories are detailed using the established CRISPR-based multiplex genome integration toolkit, including donor plasmid construction, competent cell preparation and transformation, and transformant verification. The application of the CRISPR toolkit is demonstrated by the construction of engineered P. pastoris for converting methanol to BDO. This lays the foundation for the construction of P. pastoris cell factories harboring multi-gene biosynthetic pathways for the production of high-value compounds.

Engineered living materials for the conversion of a low-cost food-grade precursor to a high-value flavonoid

Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, anti-bacterial, anti-cancer and other beneficial effects but are produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.

The Influence of Phytohormones on Antioxidative and Antibacterial Activities in Callus Cultures of Hypericum perforatum L.

Natural extracts of Hypericum perforatum L. are widely used in the pharmaceutical industry due to their antiviral, antioxidant, antibacterial, antidiabetic, and antidepressant activities. Plant biotechnological methods are gaining more attention as efficient and biosustainable approaches for the production of high value compounds by using plant callus cultures in vitro. The aim of this research was to evaluate the influence of phytohormones on the formation of Hypericum perforatum L. callus culture, to explore antioxidant and antibacterial activities, and to determine the amounts of phytochemicals in the analyzed extracts. Callus cultures were induced on Murashige and Skoog basal medium supplemented with phytohormones (auxins, cytokinins, and salicylic acid), and containing newly synthesized compound N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide. The antioxidant activities of callus cultures were evaluated using DPPH and FRAP assays and the enzyme antioxidants (superoxide dismutase, catalase, and ascorbate peroxidase) were evaluated. The antibacterial activity of plant extracts was screened against Escherichia coli and Bacillus subtilis bacteria by the diffusion agar method. There was a significant positive correlation between total phenolic content and high antioxidant efficiency in Hypericum perforatum L. callus cultures, which were formed on Murashige and Skoog medium supplemented with auxin, cytokinin, and salicylic acid (100 µM). Our results have demonstrated that callus cultures generated on MS medium supplemented with N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide and cytokinin BAP (0.5 mg L−1) exhibited improved antioxidant activities for the first time.

Kinetic studies of solid foam catalysts for the production of sugar alcohols: Xylitol from biomass resources

Structured catalysts, such as solid foams, represent a very promising technology for continuous and stable production of high-value compounds derived from biomass, traditionally produced with batch and semibatch technologies using suspended catalysts. However, the synthesis of structured catalysts presents additional challenges related to their structure and the generation of porous coatings with suitable properties for dispersing the catalytically active phase on the support.This work was focused on synthesizing a Ru/C solid foam catalyst and investigating its activity in the selective hydrogenation of xylose to xylitol under different operational conditions. The carbon coating, the key step of preparation, was based on the formation and pyrolysis of poly(furfuryl alcohol) in the presence of different amounts of poly(ethylene glycol) (PEG; M = 8 kDa) as a pore former, which enabled tuning the support porosity. Thus, the catalyst prepared with 5 wt% PEG presented a micro-to-mesopores volume ratio of 1, and a good dispersion of Ru nanoparticles, as well as a better stability compared to the catalyst prepared without PEG.The extensive kinetic data collected in this work were mathematically modelled using three different approaches to elucidate the reactant adsorption mode: a non-competitive adsorption model, a non-competitive adsorption model considering the effect of temperature, and a semi-competitive adsorption model. The non-competitive temperature-dependent model displayed better performance in terms of fitting and reliability of the estimated parameters and predicted the adsorption of xylose as an endothermic process. On the other hand, the semi-competitive model gave similar results in terms of fitting and a value for the competitiveness factor of 0.74, which matches the hypothesis that the larger molecules, sugars, can occupy most of the active sites, while some interstitial sites remain accessible for hydrogen adsorption. The modelling results revealed a complex mode of sugar adsorption on the catalyst surface. This modelling concept can be applied to any system in which the molecule sizes are very different.

Valorization of callus biomass from sterile medical hemp cultures for the production of high-value compounds via biorefinery processes

Valorization of callus biomass from sterile medical hemp cultures for the production of high-value compounds via biorefinery processes

PH‐getriggerte Rückgewinnung von photokatalytischen Partikeln aus organischen Polymeren zur Herstellung hochwertiger Verbindungen und verbesserter Recyclingfähigkeit

AbstractPseudohomogene polymere Photokatalysatoren sind eine neue Klasse hocheffizienter und einstellbarer photokatalytischer Materialien, bei denen die photokatalytischen Zentren leicht zugänglich sind. Die Schaffung hocheffizienter photokatalytischer Materialien, die schnell abgetrennt und zurückgewonnen werden können, ist eine der entscheidenden Herausforderungen in der photokatalytischen Chemie. Hier beschreiben wir auf den pH‐Wert reagierende photokatalytische Nanopartikel, die unter sauren Bedingungen aktiv und gut dispergiert sind, aber bei Erhöhung des pH‐Werts sofort aggregieren und so eine einfache Rückgewinnung ermöglichen. Diese auf den pH‐Wert reagierenden photokatalytischen Polymere können für verschiedene photokatalytische Umwandlungen verwendet werden, darunter die Reduktion von CrVI und die Photoredoxalkylierung von Indolderivaten. Insbesondere beschleunigt die kationische Natur des Photokatalysators die Reaktionsgeschwindigkeit eines anionischen Substrats im Vergleich zu ungeladenen Spezies. Diese photokatalytischen Partikel können leicht recycelt werden und ermöglichen mehrere aufeinanderfolgende photokatalytische Reaktionen ohne deutlichen Aktivitätsverlust.

PH-Triggered Recovery of Organic Polymer Photocatalytic Particles for the Production of High Value Compounds and Enhanced Recyclability.

Pseudo-homogeneous polymeric photocatalysts are an emerging class of highly efficient and tunable photocatalytic materials, where the photocatalytic centers are easily accessible. The creation of highly efficient photocatalytic materials that can be rapidly separated and recovered is one of the critical challenges in photocatalytic chemistry. Here, we describe pH-responsive photocatalytic nanoparticles that are active and well-dispersed under acidic conditions but aggregate instantly upon elevation of pH, enabling easy recovery. These responsive photocatalytic polymers can be used in various photocatalytic transformations, including CrVI reduction and photoredox alkylation of indole derivative. Notably, the cationic nature of the photocatalyst accelerates reaction rate of an anionic substrate compared to uncharged species. These photocatalytic particles could be readily recycled allowing multiple successive photocatalytic reactions with no clear loss in activity.

MicroRNA-Mediated Responses: Adaptations to Marine Extreme Environments

Extreme environments are characterized by peculiar conditions, such as hypoxia/anoxia, freezing/heat temperatures, and desiccation. With climate change, more and more habitats are facing extreme conditions and living communities are finding ways to adapt in order to survive. In this study, we show several species which have been shown to adapt to marine extreme conditions also via miRNA-mediated responses. miRNAs are a class of small non-coding RNAs that mediate gene regulation via interactions with transcripts. Their action can directly or indirectly regulate pathways that can result in a response to a specific condition. Furthermore, the study of these miRNA-mediated responses could help in the biotechnological field for their application in the development of environmental biomarkers of stress conditions, or in the genetic engineering of algal species for the production of high-value compounds.

Exploring Nordic microalgae as a potential novel source of antioxidant and bioactive compounds

Nordic microalgae are a group of photosynthetic organisms acclimated to growth at low temperature and in varying light conditions; the subarctic climate offers bright days with moderate temperatures during summer and cold and dark winter months. The robustness to these natural stress conditions makes the species interesting for large-scale cultivation in harsh environments and for the production of high-value compounds. The aim of this study was to explore the ability of nineteen species of Nordic microalgae to produce different bioactive compounds, such as carotenoids or polyphenols. The results showed that some of these strains are able to produce high amounts of carotenoids (over 12 mg·g-1 dry weight) and phenolic compounds (over 20 mg GAE·g-1 dry weight). Based on these profiles, six species were selected for cultivation under high light and cold stress (500 μmol·m-2·s-1 and 10 ˚C). The strains Chlorococcum sp. (MC1) and Scenedesmus sp. (B2–2) exhibited similar values of biomass productivity under standard or stress conditions, but produced higher concentrations of carotenoids (an increase of 40% and 25%, respectively), phenolic compounds (an increase of 40% and 30%, respectively), and showed higher antioxidant capacity (an increase of 15% and 20%, respectively) during stress. The results highlight the ability of these Nordic microalgae as outstanding producers of bioactive compounds, justifying their cultivation at large scale in Nordic environments.

The feruloyl esterase from Thermobacillus xylanilyticus shows broad specificity for processing pre-biotic feruloylated xylooligosaccharides at high temperatures

Ferulic acid has antioxidant properties of interest to the food industry and can be released from natural plant fibres using feruloyl esterases. Esterases active at high temperatures are highly desirable but currently underrepresented. Here we report the biochemical characterization of the feruloyl esterase from Thermobacillus xylanilyticus. Specific activity of recombinant Tx-Est1 with ethyl ferulate was 29.2 ± 2.9 U mg−1, with a catalytic efficiency (Kcat/Km) of 393.7 ± 9.8 s-1mM−1. The temperature and pH optima were 60 °C and 7.5, whereby Tx-Est1 retains 70% activity after 25 h at 40 °C. MALDI-TOF MS revealed Tx-ESTI released ferulic acid from xylooligosaccharides with DP4-DP13, and from DP6-8 containing two ferulic acid groups. HPLC demonstrated ferulic acid release from destarched wheat bran was strongly potentiated by co-incubation with xylanase. These properties, especially the high activity at elevated temperatures, suggest Tx-Est1 can be employed for production of high-value compounds from agricultural waste or during plant polysaccharide saccharification.

Transcriptional deregulation of stress-growth balance in Nicotiana benthamiana biofactories producing insect sex pheromones.

Plant biofactories are a promising platform for sustainable production of high-value compounds, among which are insect sex pheromones, a green alternative to conventional insecticides in agriculture. Recently, we have constructed transgenic Nicotiana benthamiana plants ("Sexy Plants", SxP) that successfully produce a blend of moth (Lepidoptera) sex pheromone compounds (Z)-11-hexadecen-1-ol and (Z)-11-hexadecenyl acetate. However, efficient biosynthesis of sex pheromones resulted in growth and developmental penalty, diminishing the potential for commercial use of SxP in biomanufacturing. To gain insight into the underlying molecular responses, we analysed the whole-genome transcriptome and evaluated it in relation to growth and pheromone production in low- and high-producing transgenic plants of v1.0 and v1.2 SxP lines. In our study, high-producing SxPv1.2 plants accumulated the highest amounts of pheromones but still maintained better growth compared to v1.0 high producers. For an in-depth biological interpretation of the transcriptomic data, we have prepared a comprehensive functional N. benthamiana genome annotation as well as gene translations to Arabidopsis thaliana, enabling functional information transfer by using Arabidopsis knowledge networks. Differential gene expression analysis, contrasting pheromone producers to wild-type plants, revealed that while only a few genes were differentially regulated in low-producing plants, high-producing plants exhibited vast transcriptional reprogramming. They showed signs of stress-like response, manifested as downregulation of photosynthesis-related genes and significant differences in expression of hormonal signalling and secondary metabolism-related genes, the latter presumably leading to previously reported volatilome changes. Further network analyses confirmed stress-like response with activation of jasmonic acid and downregulation of gibberellic acid signalling, illuminating the possibility that the observed growth penalty was not solely a consequence of a higher metabolic burden imposed upon constitutive expression of a heterologous biosynthetic pathway, but rather the result of signalling pathway perturbation. Our work presents an example of comprehensive transcriptomic analyses of disadvantageous stress signalling in N. benthamiana biofactory that could be applied to other bioproduction systems.

Nutrient Starvation Exposure Induced the Down-Regulation of Stress Responsive Genes and Selected Bioactive Metabolic Pathways in Phaeodactylum tricornutum

The microalga Phaeodactylum tricornutum is considered a model diatom. It is the second diatom whose genome was sequenced and the first one genetically engineered. This permits its use as a cell factory for the production of high-value compounds for nutraceutical, cosmeceutical, pharmaceutical, biodiesel, and bioplastic applications. This study is focused on analyzing expression levels of enzymes involved in the synthesis of sulfoglycolipids and monogalactosyldiacylglycerols, compounds known to have anticancer and immunomodulatory activities, and genes coding antioxidant, heat shock and stress-responsive proteins, in various culturing conditions. Our data showed that both nutrient starvation and senescence induced the down-regulation of both sulfoglycolipid and monogalactosyldiacylglycerol synthesis-related genes and stress-responsive genes (compared to the replete condition), suggesting that the control condition, consisting of cells in the exponential phase in replete medium, is the condition with the highest expression of the genes of interest and worth of further bioactivity screening and chemical analyses for drug discovery and biotechnological applications.

Controlling Light to Optimize Growth and Added Value of the Green Macroalga Codium tomentosum

Codium tomentosumis a recently domesticated green macroalga, being currently cultured as an extractive species in integrated multi-trophic aquaculture (IMTA). Optimization of light requirements in outdoor systems must be achieved to increase the market value of cultivated algal biomass. The present study addresses the seasonal effects of light intensity and wavelength on productivity, pigment composition and epiphyte overgrowth inC. tomentosumcultured in a land-based IMTA system. Exposure to high light (non-filtered sun light) lead to higher net productivities in spring. However, non-filtered sun light caused significantly reduced productivities during summer when compared to filtered sun light (~ 4x lower irradiance levels). Furthermore, lower photosynthetic capacity (Fv/Fm) was observed in macroalgae cultured under high light during summer, indicating photoinhibition. Treatments with filtered sun light (low and red light) showed intermediate and more stable productivities. Epiphyte biomass was higher under high light and the lowest epiphyte overgrowth was recorded under red light. Concentrations of light-harvesting pigments were lower in summer than in spring, indicating a seasonal photoacclimation of macroalgae. An opposite seasonal trend was observed for accessory xanthophylls, as the main role of these pigments is photoprotection. Higher all-trans-neoxanthin and violaxanthin concentrations were found in high light than in low or red light treatments, confirming the important role of these biomolecules in the photoprotection ofC. tomentosum. This study underlines the importance of controlling light to optimize algal growth outdoors and enhance the production of high-value compounds (i.e., pigments). Additionally, this practice can also reduce epiphyte overgrowth, thus enhancing the valorization of macroalgal biomass derived fromC. tomentosumaquaculture.

Co-cultivation of Isochrysis galbana and Marinobacter sp. can enhance algal growth and docosahexaenoic acid production

Isochrysis galbana, an important diet microalgal species, is widely used in aquaculture and rich in docosahexaenoic acid (DHA). Currently, no efficient strategies are available to steadily enhance the production of its biomass and DHA. It has been reported that algae-associated bacteria can affect the yield of algae and the production of high-value compounds. Here, we identified a growth-promoting bacterial strain Marinobacter sp., which could enhance the growth, the content of chlorophyll a, the maximal photochemical efficiency of PS II (Fv/Fm), and soluble protein content of I. galbana when they were co-cultured, while superoxide dismutase (SOD) activity was decreased. Besides, Marinobacter sp. promoted the production of DHA and up-regulated the expressions of genes involved in DHA synthesis pathway of I. galbana. Our study clearly suggested that co-cultivation of I. galbana and Marinobacter sp. could effectively enhance the quality and quantity of microalgae, showing promising applications in improving productivity and sustainability of aquaculture algal rearing systems.

Use of Different Organic Carbon Sources in Cynara cardunculus Cells: Effects on Biomass Productivity and Secondary Metabolites.

Cynara cardunculus (Asteraceae family) is a perennial plant native to Mediterranean regions. This plant represents a source of high-value compounds, such as polyphenols and fatty acids that have several industrial applications. However, in vitro plant cell cultures can represent a valid alternative to in-field cultivation and facilitate the extraction of metabolites of commercial interest. Generally, sucrose is the main sugar used for plant cell cultures, but other carbon sources can be considered. Here, we investigated the potential use of alternative organic carbon sources, such as galactose, maltose, glucose, glycerol, fructose, lactose, and starch, for the cultivation of C. cardunculus cells. Moreover, cardoon cells were collected, and an extraction of polyphenols and oils was performed to study the effects of different carbon sources on the production of bioactive molecules. This study provided evidence that cardoon cell growth can be supported by carbon sources other than sucrose. However, the carbon source inducing optimum growth, did not necessarily induce the highest production of high-value compounds.

Photocatalytic polymer nanomaterials for the production of high value compounds

Nanotechnology has provided a platform for producing new photocatalytic materials, where the reduction in length scales has been used to amplify the efficiency of these light active materials. The progression to nano-based photocatalysts has been driven by the increase in surface area that is achieved. Furthermore, nanophotocatalysts based on porous polymers or gel materials are often more active as reagents can more easily partition across the whole photocatalyst. Here, reducing the diffusional path length for substrates across the porous/gel material increases the quantity of accessible active sites in the photocatalytic material. The formation of nanophotocatalytic materials has also enabled the formation of functional nanoparticles that can be used in different conditions traditionally inaccessible to bulk catalysts. Specifically, aqueous compatible nanophotocatalytic materials have been reported, enabling greener reaction conditions and new applications of photocatalysts.