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Poly-hydroxybutyrate (PHB) is an environmentally friendly alternative for conventional fossil fuel-based plastics that is produced by various microorganisms. Large-scale PHB production is challenging due to the comparatively higher biomanufacturing costs. A PHB overproducer is the haloalkaliphilic bacterium Halomonas campaniensis, which has low nutritional requirements and can grow in cultures with high salt concentrations, rendering it resistant to contamination. Despite its virtues, the metabolic capabilities of H. campaniensis as well as the limitations hindering higher PHB production remain poorly studied. To address this limitation, we present HaloGEM, the first high-quality genome-scale metabolic network reconstruction, which encompasses 888 genes, 1528 reactions (1257 gene-associated), and 1274 metabolites. HaloGEM not only displays excellent agreement with previous growth data and experiments from this study, but it also revealed nitrogen as a limiting nutrient when growing aerobically under high salt concentrations using glucose as carbon source. Among different nitrogen source mixtures for optimal growth, HaloGEM predicted glutamate and arginine as a promising mixture producing increases of 54.2% and 153.4% in the biomass yield and PHB titer, respectively. Furthermore, the model was used to predict genetic interventions for increasing PHB yield, which were consistent with the rationale of previously reported strategies. Overall, the presented reconstruction advances our understanding of the metabolic capabilities of H. campaniensis for rationally engineering this next-generation industrial biotechnology platform. KEY POINTS: A comprehensive genome-scale metabolic reconstruction of H. campaniensis was developed. Experiments and simulations predict N limitation in minimal media under aerobiosis. In silico media design increased experimental biomass yield and PHB titer.

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Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

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Petroleum-derived plastics are materials of great importance for the contemporary lifestyle, and are widely used commercially because they are low cost, resistant, malleable, and weightless, in addition to their hydrophobic character. However, some factors that confer the qualities of these materials also cause problems, mainly environmental, associated with their use. The COVID-19 pandemic aggravated these impacts due to the high demand for personal protective equipment and the packaging sector. In this scenario, bioplastics are environmentally positive alternatives to these plastics due to their applicability in several areas ranging from packaging, to biomedicine, to agriculture. Polyhydroxyalkanoates (PHAs) are biodegradable biopolymers usually produced by microorganisms as an energy reserve. Their structural variability provides a wide range of applications, making them a viable option to replace polluting materials. PHAs can be applied in various biotechnology sectors, such as producing drug carriers and scaffolds for tissue engineering. This review aimed to survey works published in the last five years on the study and biotechnological application of PHAs in the biomedical sector, exploring the versatility and advantages of their use and helping to understand how to enhance their application.

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Microbial polyhydroxyalkanoates (PHAs) are bio-based aliphatic biopolyester produced by bacteria as an intracellular storage material of carbon and energy under stressed conditions. PHAs have been paid attention to due to their unique and impressive biological properties including high biodegradability, biocompatibility, low cytotoxicity, and different mechanical properties. Under this context, the development of drug-delivery nanosystems based on PHAs has been revealed to have numerous advantages compared with synthetic polymers that included biocompatibility, biodegradability, non-toxic, and low-cost production, among others. In this review article, we present the available state of the art of PHAs. Moreover, we discussed the potential benefits, weaknesses, and perspectives of PHAs to the develop drug delivery systems.

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Polyhydroxyalkanoate (PHA), a biodegradable polymer obtained from microorganisms and plants, have been widely used in biomedical applications and devices, such as sutures, cardiac valves, bone scaffold, and drug delivery of compounds with pharmaceutical interests, as well as in food packaging. This review focuses on the use of polyhydroxyalkanoates beyond the most common uses, aiming to inform about the potential uses of the biopolymer as a biosensor, cosmetics, drug delivery, flame retardancy, and electrospinning, among other interesting uses. The novel applications are based on the production and composition of the polymer, which can be modified by genetic engineering, a semi-synthetic approach, by changing feeding carbon sources and/or supplement addition, among others. The future of PHA is promising, and despite its production costs being higher than petroleum-based plastics, tools given by synthetic biology, bioinformatics, and machine learning, among others, have allowed for great production yields, monomer and polymer functionalization, stability, and versatility, a key feature to increase the uses of this interesting family of polymers.

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Solid-state fermentation (SSF) is a type of fermentation process with potential to use agro-industrial by-products as a carbon source. Nonetheless, there are few studies evaluating SSF compared to submerged fermentation (SmF) to produce polyhydroxyalkanoates (PHAs). Different methodologies are available associating the two processes. In general, the studies employ a 1st step by SSF to hydrolyze the agro-industrial by-products used as a carbon source, and a 2nd step to produce PHA that can be carried out by SmF or SSF. This paper reviewed and compared the different methodologies described in the literature to assess their potential for use in PHA production. The studies evaluated showed that highest PHA yields (86.2% and 82.3%) were achieved by associating SSF and SmF by Cupriavidus necator. Meanwhile, in methodologies using only SSF, Bacillus produced the highest yields (62% and 56.8%). Since PHA (%) does not necessarily represent a higher production by biomass, the productivity parameter was also compared between studies. We observed that the highest productivity results did not necessarily represent the highest PHA (%). C. necator presented the highest PHA yields associating SSF and SmF, however, is not the most suitable microorganism for PHA production by SSF. Concomitant use of C. necator and Bacillus is suggested for future studies in SSF. Also, it discusses the lack of studies on the association of the two fermentation methodologies, and on the scaling of SSF process for PHA production. In addition to demonstrating the need for standardization of results, for comparison between different methodologies.

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Water management and treatment are high concern fields with several challenges due to increasing pollutants produced by human activity. It is imperative to find integral solutions and strategic measures with robust remediation. Landfill leachate production is a high concern emerging problem. Especially in low middle-income countries due to no proper local waste disposition regulation and non-engineered implemented methods to dispose of urban waste. These landfills can accumulate electronic waste and release heavy metals during the degradation process. Similar phenomena include expired pharmaceuticals like antibiotics. All these pollutants accumulated in leachate made it hard to dispose of or treat. Leachate produced in non-engineered landfills can permeate soils and reach groundwater, dragging different contaminants, including antibiotics and heavy metals, which eventually can affect the environment, changing soil properties and affecting wildlife. The presence of antibiotics in the environment is a problem with particular interest to solve, mainly to avoid the development of antibiotic-resistant microorganisms, which represent a future risk for human health with possible epidemic implications. It has been reported that the use of contaminated water with heavy metals to produce and grow vegetables is a risk for consumers, heavy metals effects in humans can include carcinogenic induction. This work explores the opportunities to use leachate as a source of nutrients to grow microalgae. Microalgae stand out as an alternative to bioremediate leachate, at the same time, microalgae produce high-value compounds that can be used in bioplastic, biofuels, and other industrial applications.

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In the last two decades, artificial scaffolds for nerve regeneration have been produced using a variety of polymers. Polyhydroxybutyrate (PHB) is a natural polyester that can be easily processed and offer several advantages; hence, the purpose of this review is to provide a better understanding of the efficacy of therapeutic approaches involving PHB scaffolds in promoting peripheral nerve regeneration following nerve dissection in animal models. A systematic literature review was performed following the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) criteria. The revised databases were: Pub-Med/MEDLINE, Web of Science, Science Direct, EMBASE, and SCOPUS. Sixteen studies were included in this review. Different animal models and nerves were studied. Extension of nerve gaps reconnected by PHB scaffolds and the time periods of analysis were varied. The additives included in the scaffolds, if any, were growth factors, neurotrophins, other biopolymers, and neural progenitor cells. The analysis of the quality of the studies revealed good quality in general, with some aspects that could be improved. The analysis of the risk of bias revealed several weaknesses in all studies. The use of PHB as a biomaterial to prepare tubular scaffolds for nerve regeneration was shown to be promising. The incorporation of additives appears to be a trend that improves nerve regeneration. One of the main weaknesses of the reviewed articles was the lack of standardized experimentation on animals. It is recommended to follow the currently available guidelines to improve the design, avoid the risk of bias, maximize the quality of studies, and enhance translationality.

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Burkholderia sensu lato (s.l.) species have a versatile metabolism. The aims of this review are the genomic reconstruction of the metabolic pathways involved in the synthesis of polyhydroxyalkanoates (PHAs) by Burkholderia s.l. genera, and the characterization of the PHA synthases and the pha genes organization. The reports of the PHA synthesis from different substrates by Burkholderia s.l. strains were reviewed. Genome-guided metabolic reconstruction involving the conversion of sugars and fatty acids into PHAs by 37 Burkholderia s.l. species was performed. Sugars are metabolized via the Entner-Doudoroff (ED), pentose-phosphate (PP), and lower Embden-Meyerhoff-Parnas (EMP) pathways, which produce reducing power through NAD(P)H synthesis and PHA precursors. Fatty acid substrates are metabolized via ß-oxidation and de novo synthesis of fatty acids into PHAs. The analysis of 194 Burkholderia s.l. genomes revealed that all strains have the phaC, phaA, and phaB genes for PHA synthesis, wherein the phaC gene is generally present in ≥2 copies. PHA synthases were classified into four phylogenetic groups belonging to class I II and III PHA synthases and one outlier group. The reconstruction of PHAs synthesis revealed a high level of gene redundancy probably reflecting complex regulatory layers that provide fine tuning according to diverse substrates and physiological conditions.

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Conventional petrochemical plastics have become a serious environmental problem. Its unbridled use, especially in non-durable goods, has generated an accumulation of waste that is difficult to measure, threatening aquatic and terrestrial ecosystems. The replacement of these plastics with cleaner alternatives, such as polyhydroxyalkanoates (PHA), can only be achieved by cost reductions in the production of microbial bioplastics, in order to compete with the very low costs of fossil fuel plastics. The biggest costs are carbon sources and nutrients, which can be appeased with the use of photosynthetic organisms, such as cyanobacteria, that have a minimum requirement for nutrients, and also using agro-industrial waste, such as the livestock industry, which in turn benefits from the by-products of PHA biotechnological production, for example pigments and nutrients. Circular economy can help solve the current problems in the search for a sustainable production of bioplastic: reducing production costs, reusing waste, mitigating CO2, promoting bioremediation and making better use of cyanobacteria metabolites in different industries.

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Under conditions of carbon starvation or thermal, osmotic, or oxidative shock, mutants affected in the synthesis or mobilization of poly-3-hydroxybutyrate (PHB) are known to survive less well. It is still unclear if the synthesis and accumulation of PHB are sufficient to protect bacteria against stress conditions or if the stored PHB has to be mobilized. Here, we demonstrated that mobilization of PHB in Herbaspirillum seropedicae SmR1 was heat-shock activated at 45°C. In situ proton (1H) nuclear magnetic resonance spectroscopy (i.e., 1H-nuclear magnetic resonance) showed that heat shock increased amounts of 3-hydroxybutyrate (3HB) only in H. seropedicae strains able to synthesize and mobilize PHB. H. seropedicae SmR1 mutants unable to synthesize or mobilize PHB were more susceptible to heat shock and survived less well than the parental strain. When 100 mM 3-hydroxybutyrate was added to the medium, the ΔphaC1 strain (an H. seropedicae mutant unable to synthesize PHB) and the double mutant with deletion of both phaZ1 and phaZ2 (i.e., ΔphaZ1.2) (unable to mobilize PHB) showed partial rescue of heat adaptability (from 0% survival without 3HB to 40% of the initial viable population). Addition of 200 mM 3HB before the imposition of heat shock reduced protein aggregation to 15% in the ΔphaC1 mutant and 12% in the ΔphaZ1.2 mutant. We conclude that H. seropedicae SmR1 is naturally protected by 3HB released by PHB mobilization, while mutants unable to generate large amounts of 3HB under heat shock conditions are less able to cope with heat damage.IMPORTANCE Bacteria are subject to abrupt changes in environmental conditions affecting their growth, requiring rapid adaptation. Increasing the concentration of some metabolites can protect bacteria from hostile conditions that lead to protein denaturation and precipitation, as well as damage to plasma membranes. In this work, we demonstrated that under thermal shock, the bacterium Herbaspirillum seropedicae depolymerized its intracellular stock polymer known as poly-3-hydroxybutyrate (PHB), rapidly increasing the concentration of 3-hydroxybutyrate (3HB) and decreasing protein precipitation by thermal denaturation. Mutant H. seropedicae strains unable to produce or depolymerize PHB suffered irreparable damage during thermal shock, resulting in fast death when incubated at 45°C. Our results will contribute to the development of bacteria better adapted to high temperatures found either in natural conditions or in industrial processes. In the case of H. seropedicae and other bacteria that interact beneficially with plants, the understanding of PHB metabolism can be decisive for the development of more-competitive strains and their application as biofertilizers in agriculture.

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BACKGROUND: Poly-3-hydroxybutyrate (PHB) can be efficiently produced in recombinant Escherichia coli by the overexpression of an operon (NphaCAB) encoding PHB synthetase. Strain improvement is considered to be one of critical factors to lower the production cost of PHB in recombinant system. In this study, one of key regulators that affect the cell growth and PHB content was confirmed and analyzed. RESULT: S17-3, a mutant E. coli strain derived from S17-1, was found to be able to achieve high cell density when expressing NphaCAB with the plasmid pBhya-CAB. Whole genome sequencing of S17-3 revealed genetic alternations on the upstream regions of csrA, encoding a global regulator cross-talking between stress response, catabolite repression and other metabolic activities. Deletion of csrA or expression of mutant csrA resulted in improved cell density and PHB content. CONCLUSION: The impact of gene deletion of csrA was determined, dysfunction of the regulators improved the cell density of recombinant E. coli and PHB production, however, the detail mechanism needs to be further clarified.

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Concerns about microplastics (MPs) environmental behavior and accumulation are growing at global scale and meanwhile, the attention to employ bioplastics for replacing conventional plastics is increasing. The research priority for a better understanding of the fate and potential impacts of MPs from bioplastics is of utmost importance. However, the investigations on the effects of bioplastics in terms of MPs are still limited and largely unknown. In this discussion, the current knowledge of MPs is timely highlighted to incorporate biodegradable MPs in the ongoing researches. Recent studies have identified that some biodegradable MPs exhibit same effect as conventional type MPs. Furthermore, we performed a simple degradation experiment and found that polyhydroxyalkanoate films formed MPs in water environment alike other biodegradable and conventional plastics sharing common research interests. In an effort to promote investigations, we recommend the knowledge gaps identified on bioplastics MPs: understanding the timeframe of disintegration and degradation of developing bioplastics; ensuring degradability and less persistence; promoting toxicity tests and potential effects on a wide variety of organisms; promoting attempts to assess the impacts on ecosystems; evaluating the interaction of microorganisms and MPs; working towards identifying novel disposal and collection methods from public to ease recycling and degradation processes.

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Poly(hydroxybutyrate-co-hydroxyvalerate) (P(HB-co-HV)) is a prominent biopolymer as a potential candidate for use in the biomedical area. Several Bacillus spp. strains show promising characteristics in the use of several carbon sources and are an interesting alternative for the production of P(HB-co-HV). Sewage from the agricultural and food processing industries can be used to obtain abundantly starch as a carbon source for PHA production. The aim of the present study was to optimize by response surface methodology and desirability, the production of PHA by a Bacillus megaterium strain using starch as the sole carbon source. Two optimal conditions were determined without sporulation and were used to perform new experiments to calibrate and validate a mechanistic model, developed to simulate the dynamics of PHA and biomass production. The developed model successfully represents the kinetics of the microorganism. Employing different characterization techniques, it was determined that the PHA produced by the strain is a copolymer composed of different HB:HV proportions. Using starch as the sole carbon source in a minimal salt medium, this work shows the first reports in the literature of: 1) a mathematical model for predicting growth kinetic and PHA production for B. megaterium strain and 2) a Bacillus spp. producing P(HB-co-HV) copolymer.

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Enhanced biological phosphorus removal (EBPR) is a sustainable and promising technology for phosphorus removal from wastewater. The efficiency of this technology, however, is often discounted due to the insufficient carbon sources in influent. In this work, the effect of acetate to glycerol ratio on the EBPR performance was evaluated. The experimental results showed when the ratio of acetate to glycerol decreased from 100/0% to 50/50%, the EBPR efficiency increased from 90.2% to 96.2%. Further decrease of acetate to glycerol ratio to 0/100% decreased the efficiency of EBPR to 30.5%. Fluorescence in situ hybridization analysis demonstrated appropriate increase of glycerol benefited to increase the relative abundance of phosphate accumulating organisms. Further investigation revealed the proper addition of glycerol increased the amount of polyhydroxyalkanoates synthesis, and then produced sufficient energy for oxic luxury phosphorus in the subsequent oxic phase.

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The aim of this study was to use petrochemical wastewater as the source of carbon for the production of polyhydroxyalkanoates (PHA) in an effort to decrease its cost of production. For this purpose, PHA producing bacteria were isolated from the petrochemical wastewater of Bandar Imam, Iran. The purified colonies were screened for PHA by Sudan Black B and Nile Blue A staining. Among positively stained bacteria, the best PHA producer was selected on the basis of cell growth, PHA content and the monomer composition of PHA. The phenotypic and genotypic identification this isolate showed it to be Bacillus axaraqunsis. The PHA was produced at a cell density of about 9.46 g/l of maximum concentration of 6.33g/l l, corresponding to 66% of cell dry weight. These results showed that B. axaraqunsis BIPC01 could be a potent PHA producer using wastewater for industrial purpose and simultaneously reducing the environmental pollution.

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Petroleum-based plastics constitute a major environmental problem due to their low biodegradability and accumulation in various environments. Therefore, searching for novel biodegradable plastics is of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable plastics. Life cycle assessment indicates that PHB is more beneficial than petroleum-based plastics. In this report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microorganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Staining with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs produced were characterized by GC-MS. Diverse substrates for the production of PHAs in strain LB400 were analyzed.

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AIM: Evaluation of the capability of Burkholderia sacchari to incorporate different monomers into polyhydroxyalkanoates (PHA). METHODS AND RESULTS: Thirty different carbon sources were evaluated as cosubstrates for B. sacchari growing on glucose with the intention to promote the incorporation of different monomers into the PHA produced by this species. With odd-numbered fatty acids, incorporation of the 3HV monomer was achieved, up to 65 mol% in the case of valerate. With 4-hydroxybutyrate, incorporation of 4HB was obtained, representing 9·1 mol%. With hexanoic acid, the production of P3HB-co-3HHx was achieved, containing up to 1·6 mol% of 3HHx. The molar fraction of 3HHx was found to be dependent on the ratio of glucose to hexanoic acid supplied. Metabolic flux analysis revealed a high efficiency of B. sacchari in converting carbon sources into P3HB-co-3HHx. Nevertheless, hexanoic acid was only poorly converted to 3HHx. CONCLUSIONS: Burkholderia sacchari is able to incorporate 3HV, 4HB and 3HHx in PHA containing mainly 3HB. The 3HHx content of P3HB-co-3HHx can be controlled by varying the glucose to hexanoic acid ratio. Burkholderia sacchari is highly efficient in converting carbon sources into PHA; however, only 2% of the hexanoic acid supplied could be converted to 3HHx. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing an approach to modulate the composition of P3HB-co-3HHx produced by bacteria using mixtures of carbohydrate and hexanoic acid as carbon source.

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Polyhydroxyalkanoate (PHA) synthase, the key enzyme in polyester biosynthesis of bacteria, has been targeted to various organelles in yeasts and plants using respective signal peptides. Here, we report that the sequences derived from SNARE domains efficiently target and integrate the PHA synthase from Pseudomonas putida CA-3 to the membrane of secretory vesicles in Saccharomyces cerevisiae. The studies with the enhanced green fluorescent protein confirm the localization of synthase enzyme in the vesicles of S. cerevisiae.

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Polyhydroxyalkanoates (PHA) and -amylase (-1,4 glucan-4-glucanohydrolase, E.C. 3.2.1.1) were co-produced by Bacillus sp. CFR-67 using unhydrolysed corn starch as a substrate. Bacterial growth and polymer production were enhanced with the supplementation of hydrolysates of wheat bran (WBH) or rice bran (RBH) individually or in combination (5-20 g L-1, based on weight of soluble substrates-SS). In batch cultivation, a mixture of WBH and RBH (1:1, 10 g L-1 of SS) along with ammonium acetate (1.75 g L-1) and corn starch (30 g L-1) produced maximum quantity of biomass (10 g L-1) and PHA (5.9 g L-1). The polymer thus produced was a copolymer of polyhydroxybutyrate-co-hydroxyvalerate of 95:5 to 90:10 mol%. Presence of WBH and corn starch (10-50 g L-1) in the medium enhanced fermentative yield of -amylase (2-40 U mL-1 min-1). The enzyme was active in a wide range of pH (4-9) and temperature (40-60ºC). This is the first report on simultaneous production of copolymer of bacterial PHA and -amylase from unhydrolysed corn starch and agro-industrial residues as substrates.