Substrate Mixture Research Articles

Effect evaluation of different green wastes on food waste digestate composting and improvement of operational conditions.

This study attempted to determine the influence of diverse green wastes on food waste digestate composting and the improvement of operational conditions. Various effects of the green wastes (GW), with different types and sizes, initial substrate mixture C/N ratios, compost pile heights, and turning frequencies on the food waste digestate (FWD) composting were examined in the current work. The findings showed that the use of street sweeping green waste (SSGW) as an additive can maintain the thermophilic stage of the FWD composting for 28days, while the end-product contained the greatest amounts of total phosphorus (TP, 2.29%) and total potassium (TK, 4.61%) and the lowest moisture content (14.8%). Crushed SSGW (20mm) enabled the FWD composting to maintain the longest thermophilic period (28days), achieving the highest temperature (70.2°C) and seed germination index (GI, 100%). Adjusting the initial substrate mixture C/N ratio to 25, compost pile height to 30cm, and turning frequency to three times a day could enhance the efficiency and improve the fertilizer quality of the co-composting of the FWD and SSGW. This study suggested that co-composting of FWD and SSGW (FWD/SSGW = 2.3, wet weight) is a promising technique for the treatment of municipal solid waste and provided significant theoretical data for the application of composting.

Multiple exegetically optimization of ultrasonic pretreatment and substrate mixture for biohydrogen and biomethane improvement

The aim of this study was to optimize the bio-hydrogen and bio-methane production process by analyzing the effects of substrate mixtures (cow manure: tomato waste) and ultrasonic factors (ultrasonic power and sonication time) on exergy efficiency. A multivariate optimization technique that combined regression modeling and desirability analysis was applied. Different proportions of cow manure and tomato waste (0–100 %) were pretreated with various levels of ultrasonic power (0.08, 0.25, and 0.42 W/mL) and sonication time (15, 30, and 45 min) based on an exergy equilibrium model. The optimal conditions were 0.33 W/mL of ultrasonic power, 23.1 min of sonication time, and a mixture of 2 % cow manure and 98 % tomato waste, which resulted in the highest desirability of the process (=0.687). Under these conditions, the input exergetic parameters (chemical, physical, and total exergies) were the lowest (1.510, 0.559, and 2.062 MJ/kg, respectively), the output exergetic parameters were the highest (0.027, 0.871, and 0.749 MJ/kg, respectively), the exergy efficiency and exergetic improvement potential rate were the highest (43.64 % and 0.73 MJ/kg, respectively), and the exergy destruction was the lowest (1.26 MJ/kg). The experiments were repeated to validate the modeling and optimization processes. The results showed that increasing the ultrasonic factors enhanced exergy efficiency and exergetic potential rate but also increased exergy destruction. A weak ultrasonic pretreatment with a high percentage of cow manure in the mixtures was found to be beneficial for improving plant efficiency and reducing system destruction.

Development and Optimization of a Rapid In Vitro Micropropagation System for the Perennial Vegetable Night Lily, Hemerocallis citrina Baroni

The perennial herbaceous night lily, Hemerocallis citrina Baroni, is an important vegetable crop with an increasing production and consumption in China. The long lifecycle and slow growth of the night lily are becoming bottlenecks for the large-scale production of elite lines and various genetic and breeding studies. There is a lack of a protocol for rapid and efficient micropropagation for this crop. Here, we reported the systematic investigation and optimization of in vitro plant regeneration through tissue-culture-based organogenesis in the night lily variety ‘Datong Huanghua’. We evaluated various factors affecting the efficiency of callus induction and subculture, shoot regeneration, rooting and plantlet establishment, including explant type and age, inoculation methods, basal culture media and the type and concentration of plant growth regulator (phytohormones) in various growth media. We developed an optimized protocol, as follows. The highest efficiency of callus induction was observed on Murashige and Skoog (MS) medium supplied with 22.7 µM TDZ (thidiazuron) using the young scape (flower stem or stalk) as the explant, which was cut longitudinally in half to produce a segment approximately 0.5 cm in length. Callus subculture and proliferation were more efficient on MS medium containing 9.0 µM 2,4-D (2,4-dichlorophenoxyacetic acid) under light culture conditions. Shoot regeneration showed the highest efficiency on MS medium supplemented with 8.9 µM 6-BA (6-benzylaminopurine) + 5.4 µM NAA (α-naphthaleneacetic acid), while the best rooting medium was MS medium containing 2.7 µM NAA. After transplanting, the transplanted regenerated seedlings showed the highest survival rate (96%) on a substrate mixture with a 2:1:1 ratio of peat/perlite/vermiculite. A protocol and flowchart for the rapid in vitro micropropagation of night lily plants is proposed that will facilitate various genetic, genomic and breeding studies on this crop.

Toward the production of block copolymers in microbial cells: achievements and perspectives.

The microbial production of polyhydroxyalkanoate (PHA) block copolymers has attracted research interests because they can be expected to exhibit excellent physical properties. Although post-polymerization conjugation and/or extension have been used for PHA block copolymer synthesis, the discovery of the first sequence-regulating PHA synthase, PhaCAR, enabled the direct synthesis of PHA-PHA type block copolymers in microbial cells. PhaCAR spontaneously synthesizes block copolymers from a mixture of substrates. To date, Escherichia coli and Ralstonia eutropha have been used as host strains, and therefore, sequence regulation is not a host-specific phenomenon. The monomer sequence greatly influences the physical properties of the polymer. For example, a random copolymer of 3-hydroxybutyrate and 2-hydroxybutyrate deforms plastically, while a block copolymer of approximately the same composition exhibits elastic deformation. The structure of the PHA block copolymer can be expanded by in vitro evolution of the sequence-regulating PHA synthase. An engineered variant of PhaCAR can synthesize poly(D-lactate) as a block copolymer component, which allows for greater flexibility in the molecular design of block copolymers. Therefore, creating sequence-regulating PHA synthases with a further broadened substrate range will expand the variety of properties of PHA materials. This review summarizes and discusses the sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation. KEY POINTS: • Spontaneous monomer sequence regulation generates block copolymers • Poly(D-lactate) segment can be synthesized using a block copolymerization system • Block copolymers exhibit characteristic properties.

Can black soldier fly larvae (Hermetia illucens) be reared on waste streams for food and feed? – A safety perspective

Abstract The use of insects as feed and food can be part of the solution towards a circular economy, in case the safety of insect products is assured. Black soldier fly larvae (BSFL, Hermetia illucens) can be reared on different waste streams. However, before BSFL can be legally reared on these streams, the safety of BSFL for feed and food should be assessed thoroughly. This study aimed to investigate several food safety aspects of BSFL grown on waste streams. Therefore, BSFL were reared for 7 days on substrate mixtures of waste streams with similar protein and moisture content. These waste streams included fast food waste (FF), mushroom stem (MS), pig manure solids (PS), poultry meal (PM) and slaughter waste (SW). The substrates, BSFL and the frass were analysed for the presence of metals and veterinary drugs. The substrates and BSFL were also analysed for presence of DNA of ruminant, pig and chicken. Some of the metals accumulated in BSFL, although the concentrations in BSFL (as these would be manufactured as feed) were below maximum limits for feed. Only traces of some of the analysed veterinary drugs were found in the BFSL and no accumulation thereof was observed. DNA of ruminant and pig was traced back in BSFL samples, however, chicken was not. A good understanding of the presence of food safety hazards and possible variance thereof in potential substrates, such as waste streams, and their possible residues in insects is necessary for implementation of this circular way of insect feeding in the food chain.

Optimizing tea waste as a sustainable substrate for oyster mushroom (Pleurotus ostreatus) cultivation: a comprehensive study on biological efficiency and nutritional aspect

IntroductionIn Bangladesh, rice straw (RS) and sawdust (SD) substrates have traditionally been used in the production of oyster mushrooms (Pleurotus ostreatus). However, the rising costs of these substrates have led many to look for alternatives.ObjectivesThe present study thus focuses on the potential of waste tea leaves (WTL) for mushroom farming.MethodsWe prepared various substrate mixtures by combining WTL with SD and RS, subsequently evaluating mushroom yield and various quality parameters such as amino acid concentration, mineral content, and biological efficiency.Results and discussionOur investigation revealed that WTL alone is not a suitable substrate for mushroom (Pleurotus ostreatus) growth. However, when combined with SD at a 50% ratio, it significantly boosts mushroom yield and biological efficiency (BE). Conversely, a reduction in yield was noted when WTL was mixed with RS in all tested treatments, although BE surpassed 50%. In summary, incorporating WTL into both substrates proves economically viable from the BE standpoint. According to PCA analysis, the minerals and amino acid content varied based on the different substrate formulations involving WTL blending with both SD and RS at different ratios. Remarkably, mushroom fruiting bodies exhibited lower levels of Na and Fe despite these elements being present in higher concentrations in the growing substrates, suggesting the inability of P. ostreatus to bioaccumulate Na and Fe. Conversely, we observed higher bioaccumulation of Zn and P, even exceeding substrate levels. Importantly, our findings showed that mushrooms cultivated on WTL-based formulations consistently contained elevated Zn levels irrespective of substrate types, indicating that WTL enriched Zn in mushrooms. Additionally, the Fe level increased specifically in RS + WTL-based formulations. All essential and non-essential amino acids were detected, with the highest concentration of histidine, isoleucine, and methionine found in the WTL + SD formulation. Non-essential amino acids (NEAA) like alanine and glutamic acid were more prominent in formulations combining WTL with RS. This study represents the first documented exploration of the impact of WTL on the accumulation of intracellular metabolites including minerals and amino acids, in P. ostreatus.

Consistent biosynthesis of D-glycerate from variable mixed substrates

The use of waste streams and other renewable feedstocks in microbial biosynthesis has long been a goal for metabolic engineers. Microbes can utilize the substrate mixtures found in waste streams, though they are more technically challenging to convert to useful products compared to the single substrates of standard practice. It is difficult to achieve consistent biosynthesis in the face of the temporally changing nature of waste streams. Furthermore, the expression of all the enzymes necessary to convert mixed substrates into a product likely presents significant metabolic burden, which already plagues processes that utilize a single substrate. We developed an approach to utilize mixed feedstocks for production by activating expression of each biosynthetic pathway in the presence of its substrate. This expression control was used for two novel pathways that converted two substrates, galacturonate and gluconate, into a single product, D-glycerate. A production strain harboring both pathway plasmids produced 1.8 ± 0.3 and 1.64 ± 0.09 g L−1 of D-glycerate from galacturonate and gluconate alone, respectively. Fermentations that were fed a mixture of the two substrates, at different ratios, resulted in product titers between 1.48 ± 0.03 and 1.8 ± 0.1 g L−1. All fermentations were fed a total of 10 g L−1 substrate and there was no statistically significant difference in D-glycerate titer from the single or mixed substrate fermentations. We thus demonstrated consistent D-glycerate biosynthesis from single and mixed substrates as an example of robust conversion of complex feedstocks.

Anaerobic co-digestion of human excreta, food leftovers and kitchen residue: 1 ternary mixture design, synergistic effects and RSM approach

Anaerobic digestion of multiple substrates can generate more biogas while remaining stable, if positive synergistic effects are achieved. The type of co-digested substrates and the mixing ratio used, are the most important variables as each substrate has unique set of characteristics. Optimizing the volume ratios by testing various substrate mixing ratios is a popular method for determining the best-performing ratio of substrate mixture. The ternary mixture design has reportedly been found to quicken the process of testing different mixing ratios with high accuracy without running several experiments. Therefore, a ternary mixture design and a response surface approach are used in this work to ascertain the relationship between substrate mix and responses (biogas yield, methane yield, and synergy). The findings of the experiment revealed that R9 comprising 78.8 % human excreta, 11.8 % food leftovers and 9.4 % kitchen residue, had the highest methane production of 764.79 mLCH4/gVS and a synergistic index of 3.26. Additionally, the 3D response surface plots from the response surface model showed important and shared interactions between Human Excreta, (HE), Food Leftovers (FLO), and Kitchen Residue (KR). HE and KR had a similar positive synergistic effect on biogas yield, methane yield, and synergy, which was not the case for FLO. The response surface plots showed that the predicted responses (methane yield, biogas yield and synergy) increased with increasing HE and KR fractions and decreased with increasing FLO fractions in the substrate mixtures.

Substrato à base de resíduos agroindustriais e coloração de bancadas de cultivo influenciam a produção de Zinnia elegans

Abstract Among the various technologies that can be used to modify the environmental conditions in protected cultivation, the use of agricultural waste and the use of cultivation benches of different colors can be beneficial to plants, providing adequate plant development and giving greater sustainability to the production system. The study aimed to identify the potential for using substrate based on agro-industrial waste and colored benches as alternatives for the production of Zinnia elegans. The experiment was conducted in the experimental area of the Mato Grosso do Sul State University (UEMS), in the Unit of Cassilândia, using a completely randomized design arranged in a 3 x 5 factorial scheme (three bench colors x five substrate doses), with three replications of four plants for each treatment. The treatments consisted of three colored benches (white, red, and control - metal bench) and five substrate mixtures with different concentrations of agro-industrial waste (SBRA) and Ecosolo® (0, 25%, 50%, 75%, and 100%) in which Zinnia elegans was grown. The variables analyzed were plant height, length of internodes, the number of leaves, relative chlorophyll content (SPAD), stem diameter, flower diameter, number of petals, time required for flowering and shoot dry mass. It was found that the composition of the substrate affected most of the characteristics evaluated, except for internode length and stem diameter. The benches resulted in significant differences in relative chlorophyll content and number of petals. Adding increasing proportions of SBRA, provides greater vegetative growth and flower development in Zinnia elegans plants.

Multicomponent synthesis of functionalized pyrrolo[3,4-e]isoindole-1,3,6,8-tetraones via [2 + 2 + 2] cycloaddition of maleimides with nitroenamines

We developed a protocol for the synthesis of PIITOs from maleimides and nitroenamines. This strategy featured a [2 + 2 + 2] cycloaddition with the heating of a mixture of substrates 1 and 2 with DMF without any catalyst.

Desulfurization properties, pathways, and potential applications of two novel and efficient chemolithotrophic sulfur-oxidizing strains of Pseudomonas sp. GHWS3 and Sphingobacterium sp. GHWS5.

With the accelerated modernization of agriculture and industry, sulfides have been released into the environment as a by-products of various production processes. Elevated levels of sulfide pose a threat to organisms' health and disrupt ecosystem equilibrium. This study successfully isolated two highly efficient sulfur-oxidizing strains, namely Pseudomonas aeruginosa GHWS3 and Sphingobacterium sp. GHWS5. Neither strain exhibited hemolytic activity or pathogenicity. Additionally, GHWS3 inhibited the common aquaculture pathogen Vibrio anguillarum, while GHWS5 exhibited inhibitory effects against Vibrio harveyi. GHWS3 and GHWS5 demonstrated effective removal of sulfide under the following conditions: temperature range of 20-40°C, pH level of 4.5-8.5, salinity range of 0-50‰, C/N ratio of 5-15, and sulfide concentration of 20-200mg/L. By amplifying the key functional genes of the sulfur-oxidizing Sox and rDsr systems in both GHWS3 and GHWS5 strains, potential desulfurization pathways were analyzed. Furthermore, both strains displayed high efficiency in removing sulfides from actual aquaculture pond substrate mixtures. The findings of this study provide two promising candidate strains for sulfides removal from farm tailwater, industrial wastewater, and domestic wastewater.

Mitigating Ammonia, Methane, and Carbon Dioxide Emissions from Stored Pig Slurry Using Chemical and Biological Additives

This study addresses the challenge of mitigating ammonia and greenhouse gas (GHG) emissions from stored pig slurry using chemical and biological additives. The research employs dynamic chambers to evaluate the effectiveness of these additives. Chemical agents (sulfuric acid) and biological additives (DAB bacteria) containing specific microbial strains are tested (a mixture of Rhodopseudomonas palustris, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Nitrosomona europea, Nictobacter winogradaskyi, and nutritional substrate). Controlled experiments simulate storage conditions and measure emissions of ammonia, methane, and carbon dioxide. Through statistical analysis of the results, this study evaluates the additives’ impact on emission reduction. Sulfuric acid demonstrated a reduction of 92% in CH4, 99% in CO2, and 99% in NH3 emissions. In contrast, the biological additives showed a lesser impact on CH4, with an 8% reduction, but more substantial reductions of 71% for CO2 and 77% for NH3.These results shed light on the feasibility of employing these additives to mitigate environmental impacts in pig slurry management and contribute to sustainable livestock practices by proposing strategies to reduce the ecological consequences of intensive animal farming.

Intracellular Application of an Asparaginyl Endopeptidase for Producing Recombinant Head-to-Tail Cyclic Proteins.

Peptide backbone cyclization is commonly observed in nature and is increasingly applied to proteins and peptides to improve thermal and chemical stability and resistance to proteolytic enzymes and enhance biological activity. However, chemical synthesis of head-to-tail cyclic peptides and proteins is challenging, is often low yielding, and employs toxic and unsustainable reagents. Plant derived asparaginyl endopeptidases such as OaAEP1 have been employed to catalyze the head-to-tail cyclization of peptides in vitro, offering a safer and more sustainable alternative to chemical methods. However, while asparaginyl endopeptidases have been used in vitro and in native and transgenic plant species, they have never been used to generate recombinant cyclic proteins in live recombinant organisms outside of plants. Using dihydrofolate reductase as a proof of concept, we show that a truncated OaAEP1 variant C247A is functional in the Escherichia coli physiological environment and can therefore be coexpressed with a substrate protein to enable concomitant in situ cyclization. The bacterial system is ideal for cyclic protein production owing to the fast growth rate, durability, ease of use, and low cost. This streamlines cyclic protein production via a biocatalytic process with fast kinetics and minimal ligation scarring, while negating the need to purify the enzyme, substrate, and reaction mixtures individually. The resulting cyclic protein was characterized in vitro, demonstrating enhanced thermal stability compared to the corresponding linear protein without impacting enzyme activity. We anticipate this convenient method for generating cyclic peptides will have broad utility in a range of biochemical and chemical applications.

Versatile Applications of Brewer's Spent Grain: Solid-State Fermentation and Nutritional Added Value.

Brewer's spent grain (BSG) is a major by-product in the beer-brewing process which contributes to 85% of the entire generated by-product in the brewing process. BSG is rich in proteins, and most of the malt proteins (74-78%) remain insoluble in BSG after the mashing process. Solid-state fermentation (SSF) is a promising bioprocess that enables microorganisms to survive in environments with minimal water and has shown to enhance the nutritional composition of BSG. In this review, the potential application of protein, amino acids (proline, threonine, and serine), phenolic contents, and soluble sugars (glucose, fructose, xylose, arabinose, and cellobiose) extracted from BSG by various microorganisms using SSF is explored. Incorporation of BSG into animal feed, human diets, and as a substrate for microorganisms are the prospects that could be implemented in the industrial scale. This review also discussed various advances to improve the fermentation yield such as symbiotic fermentation, the addition of nitrogen supplements, and an optimal mixture of the agro-industrial waste substrate. Future perspectives on SSF are also addressed to provide important ideas for immediate and future studies. However, challenges include optimizing SSF conditions and design of bioreactors, and operational costs must be addressed in the future to overcome current obstacles. Overall, this mini review highlights the potential benefits of BSG utilization and SSF in a sustainable way.

Exploitation of Catalytic Dyads by Short Peptide‐Based Nanotubes for Enantioselective Covalent Catalysis

AbstractExtant enzymes with precisely arranged multiple residues in their three‐dimensional binding pockets are capable of exhibiting remarkable stereoselectivity towards a racemic mixture of substrates. However, how early protein folds that possibly featured short peptide fragments facilitated enantioselective catalytic transformations important for the emergence of homochirality still remains an intriguing open question. Herein, enantioselective hydrolysis was shown by short peptide‐based nanotubes that could exploit multiple solvent‐exposed residues to create chiral binding grooves to covalently interact and subsequently hydrolyse one enantiomer preferentially from a racemic pool. Single or double‐site chiral mutations led to opposite but diminished and even complete loss of enantioselectivities, suggesting the critical roles of the binding enthalpies from the precise localization of the active site residues, despite the short sequence lengths. This work underpins the enantioselective catalytic prowess of short peptide‐based folds and argues their possible role in the emergence of homochiral chemical inventory.

Exploitation of Catalytic Dyads by Short Peptide-Based Nanotubes for Enantioselective Covalent Catalysis.

Extant enzymes with precisely arranged multiple residues in their three-dimensional binding pockets are capable of exhibiting remarkable stereoselectivity towards a racemic mixture of substrates. However, how early protein folds that possibly featured short peptide fragments facilitated enantioselective catalytic transformations important for the emergence of homochirality still remains an intriguing open question. Herein, enantioselective hydrolysis was shown by short peptide-based nanotubes that could exploit multiple solvent-exposed residues to create chiral binding grooves to covalently interact and subsequently hydrolyse one enantiomer preferentially from a racemic pool. Single or double-site chiral mutations led to opposite but diminished and even complete loss of enantioselectivities, suggesting the critical roles of the binding enthalpies from the precise localization of the active site residues, despite the short sequence lengths. This work underpins the enantioselective catalytic prowess of short peptide-based folds and argues their possible role in the emergence of homochiral chemical inventory.

Hybrid methodology for the characterization and revalorization of the organic fraction of waste generated in a Mayan community

Low-income rural communities suffer from a lack of quality and availability of services that impact their quality of life, such as sanitation facilities or solid waste collection. From an engineering perspective, it is possible to develop solutions that comprehensively address these needs. Domestic biodigesters are a convenient method to dispose of the organic fraction of solid waste; they can function as safely managed sanitary facilities and return useful products to the community such as cooking gas and organic fertilizer. However, successful implementation requires designing a solution according to the needs and resources of each community. The objective of this research was to develop a methodology for the characterization of organic waste generated in a Mayan community located in Yaxuná, Yaxcabá, Yucatán, and to revalorize it through its co-digestion with human excreta in domestic biodigesters. The proposed hybrid methodology (the extended application of questionnaires combined with the physical inspection of a small sample) was able to detect general patterns in the generation of organic wastes that were subsequently analyzed to select those wastes that, due to their characteristics (statistical and physicochemical), can enter the anaerobic digestion process in combination with human excreta. Finally, a mixture of substrates was proposed according to the particularities of Yaxuná. It was estimated that methane production from this mixture would be 53.36 L per person per day.

Carbon dynamics during long-term starving poplar trees-the importance of older carbohydrates and a shift to lipids during survival.

Carbon (C) assimilation can be severely impaired during periods of environmental stress like drought or defoliation, making trees heavily dependent on the use of C reserve pools for survival; yet, dynamics of reserve use during periods of reduced C supply are still poorly understood. We used stem girdling in mature poplar trees (Populus tremula L. hybrids), a lipid-storing species, to permanently interrupt phloem C transport and induced C shortage in the isolated stem section below the girdle and monitored metabolic activity during three campaigns in the growing seasons of 2018, 2019, and 2021. We measured respiratory fluxes (CO2 and O2), NSC concentration, the respiratory substrate (based on isotopic analysis and CO2/O2 ratio) and the age of the respiratory substrate (based on radiocarbon analysis). Our study shows that poplar trees can survive long periods of reduced C supply from the canopy by switching in metabolism from recent carbohydrates to older storage pools with a potential mixture of respiratory substrates, including lipids. This mechanism of stress resilience can explain why tree decline may take many years until death occurs.

199 Effects of Dietary Fiber and Biotic Supplementation on Apparent Total Tract Macronutrient Digestibility, Fecal Characteristics, and Fecal Metabolites of Healthy Adult Dogs

Abstract Synbiotics, or mixtures of live microorganisms and substrates selectively utilized by host microorganisms, may confer complementary or synergistic health benefits to the host. Although synbiotics have demonstrated the potential to modulate the gut microbiome and improve gastrointestinal health in many host species, research trials in companion animal diets are limited. Therefore, this study was conducted to determine the apparent total tract macronutrient digestibility (ATTD) of diets supplemented with fibers or biotics and to evaluate their effects on the fecal characteristics, metabolites, microbiota, and immunoglobulin (Ig) A concentrations of dogs consuming them. Healthy adult female beagles (n = 12; age= 6.2 ± 1.6 yr; BW = 9.5 ± 1.1 kg) were used in a replicated 3 x 3 Latin square design to test three dietary treatments: 1) basal diet based on rice, chicken meal, tapioca starch, and cellulose and without probiotics or prebiotics + a placebo treat (control, CT); 2) diet based on rice, chicken meal, garbanzo beans, and cellulose + placebo treat (GB); 3) diet based on rice, chicken meal, and garbanzo beans, and functional fiber/prebiotic blend (mannanoligosaccharides; fructooligosaccharides, inulin, pea fiber, alfalfa meal, Saccharomyces cerevisiae fermentation product) + probiotic-containing treat (synbiotic treatment, SYN). The probiotic treat contained Bacillus subtilus and Bacillus amyloliquefaciens (2 x 109 CFU/d). In each 28-day period, a 22-day diet adaptation was followed by a 5-day fecal collection phase for measurement of ATTD and fecal characteristics, metabolites, microbiota, and IgA concentrations. Fasted blood samples were collected on day 28 for serum chemistry and complete blood count. All data were analyzed using the Mixed Models procedure of SAS 9.4, with P < 0.05 being significant. ATTD of dry matter (DM), organic matter, and energy were less and DM fecal output was greater in dogs fed SYN than those fed CT or GB, whereas ATTD of crude protein was greater in dogs fed CT and SYN than those fed GB. ATTD of fat was higher and wet fecal output was less in dogs fed CT than those fed GB or SYN. Fecal DM% was greater in dogs fed CT than those fed SYN or GB, and greater in dogs fed SYN than those fed GB. Fecal short-chain fatty acids (SCFA; acetate, propionate, total SCFA) were higher in dogs fed GB than those fed CT or SYN, and higher in dogs fed GB than those fed SYN. Fecal IgA concentrations were greater in dogs fed GB than those fed CT. Serum triglycerides were less in dogs fed GB than those fed SYN or CT. Our results demonstrate that legume-based dietary fibers, with or without prebiotics and probiotics, slightly reduce nutrient digestibility, increase stool output, beneficially shift fecal metabolites, and reduce blood lipids of adult dogs.

Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.