Substrate Supplementation Research Articles

Sulfur disproportionation realizes an organic-free sulfidogenic process for sustainable treatment of acid mine drainage

Biological sulfidogenic processes (BSPs) have been considered effective biotechnologies for the treatment of organic-deficit acid mine drainage (AMD) and heavy metal recovery. However, high-rate sulfide production relies on the continuous addition of exogenous organic substrates as electron donors to facilitate dissimilatory sulfate reduction, which substantially increases the operational cost and CO2 emission and also limits the wide application of BSPs in AMD treatment. In this study, we proposed a novel chemoautotrophic elemental sulfur disproportionation (SD) process as an alternative to conventional BSPs for treating AMD, in which sulfur-disproportionating bacteria (SDB) disproportionates sulfur to sulfide and sulfate without organic substrate supplementation. During the 393-day lab-scale test, we observed that the sulfur-disproportionating reactor (SDR) achieved a stable high-rate sulfide production, with a maximal rate of 21.10 mg S/L-h at an organic-substrate-free condition. This high rate of sulfide production suggested that the SD process could provide sufficient sulfide to precipitate metal ions from AMD. Thermodynamics analysis and batch tests further revealed that alkalinity rather than sulfate was the critical factor influencing the SD process, suggesting that the abundant sulfate present in AMD would not inhibit the SD process. The critical condition of SD in the SDR was therefore determined. Microbial community analysis showed that Dissulfurimicrobium sp. was the dominant SDB during the long-term operation regardless of dynamic sulfate and/or alkalinity concentrations, which provides evidence that SDB can be employed for sustainable and high-rate sulfide production for engineering purposes. A multi-stage AMD treatment system equipped with a SDR removed over 99% of the influent metals (i.e., Fe, Al, Zn, Cu, Pb) from AMD except for Mn. This study demonstrated that the novel SD process is a green and promising biotechnology for the sustainable treatment of organic-deficient metal-laden wastewater, such as AMD.

Enhancing simultaneous electrosynthesis of CO2 and nitrogen removal in microbial fuel cell (MFC) cathode compartment by adding Fe–C/biochar compound substrates

A denitrifying microbial fuel cell (MFC) based on CO2 regulation and Fe–C/biochar compound substrate supplementation was constructed to investigate the MFC performance, CO2 fixation and microbial mechanism under different experimental conditions. The results showed that the addition of CO2 and Fe–C/biochar compound substrate improved the electricity generation (397.8 mV, 88.3 mW/m2), denitrification rate and organic carbon removal performance (93.5%) of the system by promoting the generation of soluble microbial products (SMP), extracellular polymeric substances (EPS), volatile fatty acids (VFAs), polyhydroxyalkanoate (PHA) and glycogen in the cathode reactors. Microbial community analysis and metagenomic results revealed that both CO2 and Fe–C/biochar compound substrates constitute a degree of positive feedback to denitrification and carbon fixation. Nevertheless, there were a large number of exoelectrogenic bacteria (Geobacter) attached to the biochar which merely had positive correlation with heterotrophic denitrifiers (Sulfurimonas and Methylophilus). Thermomonas and Thiobacillus were positively correlated with carbon felt rather than Fe–C.

Metabolic substrates, histone modifications, and heart failure

Histone epigenetic modifications are chemical modification changes to histone amino acid residues that modulate gene expression without altering the DNA sequence. As both the phenotypic and causal factors, cardiac metabolism disorder exacerbates mitochondrial ATP generation deficiency, thus promoting pathological cardiac hypertrophy. Moreover, several concomitant metabolic substrates also promote the expression of hypertrophy-responsive genes via regulating histone modifications as substrates or enzyme-modifiers, indicating their dual roles as metabolic and epigenetic regulators. This review focuses on the cardiac acetyl-CoA-dependent histone acetylation, NAD+-dependent SIRT-mediated deacetylation, FAD+-dependent LSD-mediated, and α-KG-dependent JMJD-mediated demethylation after briefly addressing the pathological and physiological cardiac energy metabolism. Besides using an “iceberg model” to explain the dual role of metabolic substrates as both metabolic and epigenetic regulators, we also put forward that the therapeutic supplementation of metabolic substrates is promising to blunt HF via re-establishing histone modifications.

Use of fermented spent coffee grounds as a substrate supplement for rearing black soldier fly larvae, Hermetia illucens (L), (Diptera: Stratiomyidae).

Spent coffee grounds (SCG), an increasingly abundant waste product with environmental disposal problems, has been used as a dietary supplement for many animals and have the potential to be used as a dietary supplement for black soldier fly (BSF) larvae; however, its effective use is still under scrutiny. To date, no studies have considered the use of SCG after microbial fermentation (fSCG) and its effects on BSF life history. A mixture of fruit and vegetable pulp residue supplemented with one of six different fSCG percentages (0%, 20%, 40%, 60%, 80%, and 100% by weight) were provided as a diet substrate in order to evaluate the effect of the fSCG quantity on BSF growth, yield, and conversion ability. The addition of fSCG to the pulp diet prolonged larval development times, while 100% fSCG affected the larval survival rate and resulted in a male-biased adult sex ratio. The 20-40% fSCG and 40-60% fSCG treatments supported the largest prepupal and mature larval sizes, respectively. The highest waste reduction efficiency and feed conversion rate by BSF larvae was found with 20% fSCG, similar to the control (0% fSCG). From the short rearing time, high yield, and high bioconversion efficiency, a 20% fSCG supplementation of the mixed pulp was recommended for rearing BSF larvae. These data are valuable for coffee by-product waste management in urban areas.

Bioconversion of Agro-Residues into Microbial Oil-Based Oleochemicals Employing Packed Bed Bioreactor

Lignocellulosic waste-streams are resources worth investigating to produce value-added bioproducts due to their low-cost, renewability, versatility, and abundance. This study evaluated five Zygomycota strains as potential oleaginous microorganisms for direct bioconversion of cakes derived from the vegetable-oil-milling industry into microbial oil. Solid-state fermentation (SSF) conditions in tray bioreactors revealed that temperature, moisture content, and substrate affected lipid production and fatty acids composition. Employing SSF, under optimal conditions (30 °C, 65% initial moisture content), and substrate supplementation with nitrogen sources increased lipid productivity 1.1-fold on palm kernel cake (PKC) substrate. Supplementation strategies using glycerol, molasses, or mineral solution did nοt favor lipid production. When cotton seed cake (CoSC) was applied, C. echinulata was able to produce 304.1 mg/gfs of biomass with a lipid content of 40.2% (w/w) in tray bioreactors. Scaling-up SSF using CoSC in packed bed reactors led to the highest biomass production (320.5 mg/gfs), while lipids production was unaffected. Then, microbial oil was enzymatically converted into polyol esters achieving the highest conversion yield of 80% after 2 h. Physicochemical properties of polyol esters demonstrated their potential utilization as biolubricants. The present study showed the perspective of bioconverting industrial side streams into microbial oil and a route for sustainable synthesis of oleochemicals.

Screening of ligninolytic enzymes produced by Ganoderma lucidum in solid residues from the Amazon

White rot fungi have one of the most efficient oxidative lignin degradation systems, and present an enzymatic complex that is responsible for digesting lignocellulosic matter. The aim of this study was to evaluate the production of laccase and total peroxidases by Ganoderma lucidum via solid-state fermentation using açaí seeds and marupá (Simarouba amara) sawdust unsupplemented and supplemented with wheat, corn and rice bran. G. lucidum was inoculated in substrates prepared with the residues and incubated at 25 ºC. Enzymatic extractions were performed on the culture substrates every 2 days for 30 days. The enzymatic activities were determined using ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), with the addition of the enzymatic cofactor H2O2 for total peroxidases. The laccase activity was higher in the supplemented residues, with emphasis on the açaí-based substrate on the 16th day of cultivation, while in marupá the maximum activity was on the 6th day. In the unsupplemented açaí residue, the maximum peak of activity was on the 8th day and, in marupá, no fungal growth was observed. As for total peroxidases, G. lucidum cultivated in the supplemented açaí substrates showed activity peaks on the 8th, 12th, 16th and 28th day, and on 6th and 12th day under unsupplemented conditions. While, in the marupá, total peroxidase activity was observed only on the 6th day of cultivation. Thus, G. lucidum showed potential for producing laccases and total peroxidases, with the substrate supplementation inducing the synthesis of these enzymes.

Heterologous biosynthesis of isobavachalcone in tobacco based on in planta screening of prenyltransferases.

Isobavachalcone (IBC) is a prenylated chalcone mainly distributed in some Fabaceae and Moraceae species. IBC exhibits a wide range of pharmacological properties, including anti-bacterial, anti-viral, anti-inflammatory, and anti-cancer activities. In this study, we attempted to construct the heterologous biosynthesis pathway of IBC in tobacco (Nicotiana tabacum). Four previously reported prenyltransferases, including GuILDT from Glycyrrhiza uralensis, HlPT1 from Humulus lupulus, and SfILDT and SfFPT from Sophora flavescens, were subjected to an in planta screening to verify their activities for the biosynthesis of IBC, by using tobacco transient expression with exogenous isoliquiritigenin as the substrate. Only SfFPT and HlPT1 could convert isoliquiritigenin to IBC, and the activity of SfFPT was higher than that of HlPT1. By co-expression of GmCHS8 and GmCHR5 from Glycine max, endogenous isoliquiritigenin was generated in tobacco leaves (21.0 μg/g dry weight). After transformation with a multigene vector carrying GmCHS8, GmCHR5, and SfFPT, de novo biosynthesis of IBC was achieved in transgenic tobacco T0 lines, in which the highest amount of IBC was 0.56 μg/g dry weight. The yield of IBC in transgenic plants was nearly equal to that in SfFPT transient expression experiments, in which substrate supplement was sufficient, indicating that low IBC yield was not attributed to the substrate supplement. Our research provided a prospect to produce valuable prenylflavonoids using plant-based metabolic engineering.

Production of quinolone derivatives in Escherichia coli

Alkyl-4-quinolones (AQs) are natural compounds synthesized by bacteria. Members of this group are known quorum-sensing molecules. Other biological functions, such as anti-bacterial, anti-algal, antifungal, and anti-malaria activities have also been reported. The synthetic pathways of AQs have been validated in Pseudomonas aeruginosa. Five genes (pqsA–E) are involved in the synthesis of 2-heptyl-4(1H)-quinolone (HHQ). To synthesize HHQ in a microbial system, pqsA–E genes were introduced into Escherichia coli and HHQ and 2-methyl-4(1H)-quinolone (MHQ) were synthesized. After the copy number, construct promoters, and substrate supplements were optimized, 141.3 mg/L MHQ and 242.8 mg/L HHQ were synthesized.

Exploitation of wasted bread as substrate for polyhydroxyalkanoates production through the use of Haloferax mediterranei and seawater.

The use of the halophile microorganism Haloferax mediterranei, able to synthesize poly(hydroxybutyrate-hydroxyvalerate) (PHBV), is considered as a promising tool for the industrial production of bioplastic through bioprocessing. A consistent supplementation of the growth substrate in carbohydrates and minerals is overall necessary to allow its PHBV production. In this work, wasted bread was used as substrate for bioplastic production by microbial fermentation. Instead of the consistent and expensive minerals supplement required for Hfx. mediterranei DSM1411 growth, microfiltered seawater was added to the wasted bread-derived substrate. The suitable ratio of wasted bread homogenate and seawater, corresponding to 40:60, was selected. The addition of proteases and amylase to the bread homogenate promoted the microbial growth but it did not correspond to the increase of bioplastic production by the microorganism, that reach, under the experimental conditions, 1.53 g/L. An extraction procedure of the PHBV from cells, based on repeated washing with water, followed or not by a purification through ethanol precipitation, was applied instead of the conventional extraction with chloroform. Yield of PHBV obtained using the different extraction methods were 21.6 ± 3.6 (standard extraction/purification procedure with CHCl3:H2O mixture), 24.8 ± 3.0 (water-based extraction), and 19.8 ± 3.3 mg PHAs/g of wasted bread (water-based extraction followed by ethanol purification). Slightly higher hydroxyvalerate content (12.95 vs 10.78%, w/w) was found in PHBV obtained through the water-based extraction compared to the conventional one, moreover, the former was characterized by purity of 100% (w/w). Results demonstrated the suitability of wasted bread, supplemented with seawater, to be used as substrate for bioplastic production through fermentation. Results moreover demonstrated that a solvent-free extraction, exclusively based on osmotic shock, could be used to recover the bioplastic from cells.

Identification of a cytochrome P450 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzes polpunonic acid formation in celastrol biosynthesis.

Tripterygium hypoglaucum (Levl.) Hutch, a traditional Chinese medicinal herb with a long history of use, is widely distributed in China. One of its main active components, celastrol, has great potential to be developed into anti-cancer and anti-obesity drugs. Although it exhibits strong pharmacological activities, there is a lack of sustainable sources of celastrol and its derivatives, making it crucial to develop novel sources of these drugs through synthetic biology. The key step in the biosynthesis of celastrol is considered to be the cyclization of 2,3-oxidosqualene into friedelin under the catalysis of 2,3-oxidosqualene cyclases. Friedelin was speculated to be oxidized into celastrol by cytochrome P450 oxidases (CYP450s). Here, we reported a cytochrome P450 ThCYP712K1 from Tripterygium hypoglaucum (Levl.) Hutch that catalyzed the oxidation of friedelin into polpuonic acid when heterologously expressed in yeast. Through substrate supplementation and in vitro enzyme analysis, ThCYP712K1 was further proven to catalyze the oxidation of friedelin at the C-29 position to produce polpunonic acid, which is considered a vital step in the biosynthesis of celastrol, and will lay a foundation for further analysis of its biosynthetic pathway.

Potential Uses of Scallop Shell Powder as a Substrate for the Cultivation of King Oyster Mushroom (Pleurotus eryngii)

Scallop shells are currently a major form of waste generated in the Chinese fishing industry. However, they have the potential to be used as important industrial products. This study was conducted to assess the utility of scallop shell powder (SSP) supplementation in improving the growth of king oyster mushrooms (Pleurotus eryngii) grown on sawdust and sugarcane bagasse substrates. The outcomes of interest included mycelial growth, yield, biological efficiency, fruiting body traits, nutrient supply, and the mineral composition of P. eryngii. Supplementation with SSP accelerated the mycelial growth of P. eryngii. The yield of fruiting bodies (399.5 g/bag) and the biological efficiency (84.6%) were 14% higher after supplementation of the substrate with 2% SSP compared with those of mushrooms grown on substrates not supplemented with SSP (349.8 g/bag and 74.0%, respectively). Moreover, the crude protein and fiber contents of the fruiting bodies significantly improved after growth with SSP. Furthermore, supplementation with 2% SSP increased the calcium (Ca) content of the fruiting bodies of P. eryngii by 64% (to 67.2 ± 15.7 mg kg−1) compared with that of mushrooms grown on a control substrate (41.0 mg kg−1). This study revealed that P. eryngii can efficiently use the Ca provided by raw SSP, generating higher Ca contents in their fruiting bodies. Our results demonstrate that the supplementation of substrates with SSP can be useful for enhancing both the yield and nutritional content of P. eryngii.

Antihyperglycemic activity of L-norvaline and L-arginine in high-fat diet and streptozotocin-treated male rats

BackgroundA decrease in nitric oxide (NO) bioavailability has been shown to cause hyperglycemia, type II diabetes mellitus (DM), and chronic cardio-metabolic complications. In turn, hyperglycemia and hypercholesterolemia are associated with increased oxidative stress that leads to reduced nitric oxide bioavailability through disruption of L-arginine transport into cells, inactivation of nitric oxide synthase, and activation of arginase. Upregulation of arginase has been demonstrated in both diabetic patients and animal models of hyperglycemia and type 2 diabetes. L-norvaline is a nonselective inhibitor of arginase that increases NO production and promotes the normal functioning of the vascular endothelium. Another means of increasing NO bioavailability in the cardiovascular system is L-arginine supplementation. Whether L-norvaline and L-arginine have antihyperglycemic effects has not been studied. HypothesisWe hypothesized that inhibition of arginase will provide an antihyperglycemic effect and, as a result of the recovery of NO bioavailability, will protect against oxidative stress and hypercholesterolemia. MethodsRats were fed a high-fat diet (HFD) for three weeks concomitant with the two-time injection of 30 mg/kg of streptozotocin (STZ) to induce stable hyperglycemia. We studied the antihyperglycemic properties of arginase inhibition (via L-norvaline) and its combination with NOS substrate supplementation (via L-arginine). ResultsTreatment of HFD/STZ mice with L-norvaline and L-arginine reduced fasting blood glucose levels by 27.1% vs. untreated HFD/STZ rats (p < 0.001). Blood levels of total cholesterol, low-density lipoprotein (LDL), and malondialdehyde (MDA), a marker for oxidative stress, were significantly decreased in both L-norvaline- and L-norvaline+L-arginine-treated HFD/STZ rats when compared with untreated rats. In addition, administration of L-norvaline and L-arginine reversed the progression of pancreatic and kidney pathology in HFD/STZ rats as assessed by histology (p < 0.001). ConclusionsBoth L-norvaline and L-arginine act as potent antihyperglycemic agents and can represent alternative therapeutic tools in individuals with hyperglycemia and pre-diabetes.

Concurrent anaerobic chromate bio-reduction and pentachlorophenol bio-degradation in a synthetic aquifer

Chromate [Cr(VI)] and pentachlorophenol (PCP) coexist widely in the environment and are highly toxic to public health. However, whether Cr(VI) bio-reduction is accompanied by PCP bio-degradation and how microbial communities can keep long-term stability to mediate these bioprocesses in aquifer remain elusive. Herein, we conducted a 365-day continuous column experiment, during which the concurrent removals of Cr(VI) and PCP were realized under anaerobic condition. This process allowed for complete Cr(VI) bio-reduction and PCP bio-degradation at an efficiency of 92.8 ± 4.2% using ethanol as a co-metabolic substrate. More specifically, Cr(VI) was reduced to insoluble chromium (III) and PCP was efficiently dechlorinated with chloride ion release. Collectively, Acinetobacter and Spirochaeta regulated Cr(VI) bio-reduction heterotrophically, while Pseudomonas mediated not only Cr(VI) bio-reduction but also PCP bio-dechlorination. The bio-dechlorinated products were further mineralized by Azospira and Longilinea. Genes encoding proteins for Cr(VI) bio-reduction (chrA and yieF) and PCP bio-degradation (pceA) were upregulated. Cytochrome c and intracellular nicotinamide adenine dinucleotide were involved in Cr(VI) and PCP detoxification by promoting electron transfer. Taken together, our findings provide a promising bioremediation strategy for concurrent removal of Cr(VI) and PCP in aquifers through bio-stimulation with supplementation of appropriate substrates.

The protective role of selenium against uptake and accumulation of cadmium and lead in white oyster (Pleurotus ostreatus) and pink oyster (Pleurotus djamor) mushrooms

ABSTRACT Mushrooms are bioaccumulators and have been used to produce Se-enriched foods. However, these fungi can also bioaccumulate potentially toxic metals, producing food dangerous to human health. It is known that co-exposure to Se plays a protective role against metal accumulation and toxicity in some organisms due to its antioxidant properties. Thus, this study aimed to evaluate the protective effect of Se(IV) and Se(VI) on elemental uptake and accumulation as well as proteins and protein-bound Se, Cd, and Pb distribution in Pleurotus mushrooms. Pink oyster and white oyster mushrooms showed high ability to bioaccumulate Se (19–205 µg g−1), Cd (4.5 to 18.8 µg g−1), and Pb (1.6 to 7.0 µg g−1). Growth substrate supplementation with Se(IV) or Se(VI) decreased the Cd total concentration in mushrooms by 4 to 89%, while Se(VI) increased the Pb total concentration by 9% to 187%, compared to growth in absence of Se. It was found that despite molecular weights distributions of mushrooms grown on Se(IV) and Se(VI)-supplemented substrates being similar, Se(VI) supplementation favoured Se interaction with proteins of medium molecular weight (17–44 kDa), when compared to supplementation with Se(IV). Therefore, we propose the supplementation of growth substrates with Se(VI) to reduce eventual Cd accumulation and produce Se-enriched oyster mushrooms.

Towards Functional Insect Feeds: Agri-Food By-Products Enriched with Post-Distillation Residues of Medicinal Aromatic Plants in Tenebrio molitor (Coleoptera: Tenebrionidae) Breeding.

Sustainability, circular economy and alternative production systems are urgent imperatives for humanity and animal husbandry. Unless wasted, agri-food by-products can offer a promising source of high value. We evaluated the effect of rice bran (RB), corncob (CC), potato peels (PP), solid biogas residues (BR), and olive-oil processing residuals (OR), as alternative substrates to wheat bran (WB as control), on the growth and nutritional value of Tenebrio molitor during its breeding for animal feeds and/or human consumption. Innovation-wise, we further investigated the substrate supplementation (0, 10, 20%) with post-distillation residues of Mediterranean aromatic-medicinal plants (MAPs: lavender, Greek oregano, rosemary, olive; 1:1:1:1 ratio). Tenebrio molitor larvae (TML) were reared in all the studied substrates, and TML and diets’ proximate and fatty acid compositions as well as total phenol and flavonoid content and antioxidant potential were assessed using standard procedures. After statistical analysis of correlations, we observed that CC promoted oviposition and progeny survival; larval weight and dry matter were positively affected mainly by dietary energy and fat content; number of TML and/or larval weight increased using 10% MAPs inclusion in WB, RB and OR or RB, OR, BR and PP, respectively, which did not affect protein content; TML fatty acid composition decreased the content of saturated ones and increased that of mono-unsaturated ones; MAPs residues had an apparent favorable impact on total phenolic content and antioxidant activity of each substrate, with RB displaying the highest capacity and content. These findings indicate that alternative substrates can be exploited and their enrichment with natural phenolics is able to influence T. molitor growth, offering highly beneficial and nutritional value.

Analyzing the effect of storage conditions on anammox recovery performance from the perspectives of time, temperature and biomass form

In this study, the effects of different storage conditions, such as temperature, storage time and biomass form, on the properties of anaerobic ammonia oxidation (anammox) were investigated along with the identification of the process mechanism. The results showed that the influence of storage time on anammox properties was stronger than that of storage temperature and biomass form. Also, the anammox recovery activity at 15 °C was better than that at 4 °C, and the anammox recovery activity of immobilized filler was better than that of anammox granular sludge (AnGS). Although cryogenic storage severely damaged anammox activity, lower loss of extracellular polymeric substances maintained the AnGS structure. The maximum recovery of specific anammox activity at 15 °C for the immobilized filler was observed to be 109%. In addition, intermittent substrate supplementation weakened the adverse effect of long-term storage on anammox activity, and was conducive to maintaining stable flora composition and promoting regeneration of anammox bacteria (AnAOB). High-throughput sequencing analysis showed that starvation resulted in increased community diversity, and the functional bacteria Candidatus Brocadia was observed to be more tolerant to starvation than Candidatus Kuenenia. Finally, principal component analysis was used to explain the complex relationship between process performance and preservation conditions. Based on the results of this work, it is recommended to preserve AnAOB in the form of immobilized filler at 15 °C and supplement substrate intermittently during long term storage. This study provides an economical and robust strategy for the short-term and long-term preservation of AnAOB.

Chronic Kidney Disease-Induced Arterial Media Calcification in Rats Prevented by Tissue Non-Specific Alkaline Phosphatase Substrate Supplementation Rather Than Inhibition of the Enzyme.

Patients with chronic kidney disease (CKD) suffer from arterial media calcification and a disturbed bone metabolism. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the calcification inhibitor pyrophosphate (PPi) into inorganic phosphate (Pi) and thereby stimulates arterial media calcification as well as physiological bone mineralization. This study investigates whether the TNAP inhibitor SBI-425, PPi or the combination of both inhibit arterial media calcification in an 0.75% adenine rat model of CKD. Treatments started with the induction of CKD, including (i) rats with normal renal function (control diet) treated with vehicle and CKD rats treated with either (ii) vehicle, (iii) 10 mg/kg/day SBI-425, (iv) 120 µmol/kg/day PPi and (v) 120 µmol/kg/day PPi and 10 mg/kg/day SBI-425. All CKD groups developed a stable chronic renal failure reflected by hyperphosphatemia, hypocalcemia and high serum creatinine levels. CKD induced arterial media calcification and bone metabolic defects. All treatments, except for SBI-425 alone, blocked CKD-related arterial media calcification. More important, SBI-425 alone and in combination with PPi increased osteoid area pointing to a less efficient bone mineralization. Clearly, potential side effects on bone mineralization will need to be assessed in any clinical trial aimed at modifying the Pi/PPi ratio in CKD patients who already suffer from a compromised bone status.

Effects of rearing system and microbial inoculation on black soldier fly larvae growth and microbiota when reared on agri-food by-products

Black soldier fly larvae (BSFL) are widely used in recycling and upcycling of nutrients in agri-food by-products, but low and inconsistent BSFL rearing performance (i.e. larval growth, bioconversion rate, and substrate reduction) has been identified as a key challenge. The aims of this research were two-fold: (1) validate an existing closed rearing system design; and (2) assess whether a microbial inoculum derived from the rearing residue increases rearing performance. In controlled bench-scale experiments, BSFL were reared on tomato pomace (TP) and white wine pomace (WWP), along with food waste as control substrate. The two aims were assessed based on the following response variables: larval mass, substrate reduction, residue properties (i.e. pH, temperature, moisture content), and larval intestinal and residue microbiota. Higher BSFL mass (by 5.1 mg dry mass) at harvest on WWP and substrate reduction on TP (by 11.7% dry mass) in the closed system compared to the open system confirmed the potential of closed systems for rearing performance improvements of agri-food by-products. The rearing system also affected the residual moisture content and temperature, but only had a small effect on microbiota. Performance improvements by the closed rearing system design may be outweighed by insufficient aeration with pasty substrates and higher operational efforts for aeration and larval separation from the high-moisture residues. In contrast to the rearing system design, addition of the residue-derived microbial inoculum did not result in improved performance, nor did it alter intestinal and residue microbiota. Missing performance improvements could have been due to absent or low numbers of probiotic bacteria. The success of microbial substrate supplementation could be improved by studying effects of larval-associated microbes and developing cultivation methods that selectively amplify the beneficial (yet unknown) members of the microbial community. Our investigations aimed to increase the valorisation of low-value agri-food by-products in BSFL rearing.

Impact of substrate supplemented with CaCO3 on mycelial growth, yield, morphological features and storability of fruiting bodies of black poplar mushroom Agrocybe cylindracea (DC.) Marie.

Black poplar mushroom, Agrocybe cylindracea deserves special attention, due to its medicinal properties. Water and alcohol extracts from fruiting bodies of the fungus have an anti-oxidant, anti-cancer, anti-fungal, cholesterol and triglycerides blood level lowering abilities. This mushroom is rich in proteins and vitamins, mineral elements and low in fat. The aim of the experiments was to determine effect of a substrate supplementation with CaCO3 on mycelial growth, yield, morphological features and storability of fruiting bodies of four strains of A. cylindracea (DC.) Marie. The amount of additive to sawdust substrate affects rate of mycelial growth and yield of investigated strains. A. cylindracea mycelial growth was not affected by addition of CaCO3 to substrate, however a significant effect of this additive was found on yield, which was the highest with CaCO3 addition in an amount of 8 g/100 g of substrate. Carpophores characterized with the largest caps diameter, and the largest individual mass obtained of substrate enriched with CaCO3 addition of 8 g/100 g of substrate. In addition, it was found that supplementation with CaCO3 affect storability of A. cylindracea. The lowest weight loss of fruiting bodies after 3 and 7 days of storage was found with addition of CaCO3 to substrate in an amount of 4 g/100 g of substrate.

Impaired anaplerosis is a major contributor to glycolysis inhibitor toxicity in glioma

BackgroundReprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates and must be replenished (anaplerosis), mainly from pyruvate and glutamine. We recently described a novel enolase inhibitor, HEX, and its pro-drug POMHEX. Since glycolysis inhibition would deprive the cell of a key source of pyruvate, we hypothesized that enolase inhibitors might inhibit anaplerosis and synergize with other inhibitors of anaplerosis, such as the glutaminase inhibitor, CB-839.MethodsWe analyzed polar metabolites in sensitive (ENO1-deleted) and resistant (ENO1-WT) glioma cells treated with enolase and glutaminase inhibitors. We investigated whether sensitivity to enolase inhibitors could be attenuated by exogenous anaplerotic metabolites. We also determined the synergy between enolase inhibitors and the glutaminase inhibitor CB-839 in glioma cells in vitro and in vivo in both intracranial and subcutaneous tumor models.ResultsMetabolomic profiling of ENO1-deleted glioma cells treated with the enolase inhibitor revealed a profound decrease in the TCA cycle metabolites with the toxicity reversible upon exogenous supplementation of supraphysiological levels of anaplerotic substrates, including pyruvate. ENO1-deleted cells also exhibited selective sensitivity to the glutaminase inhibitor CB-839, in a manner rescuable by supplementation of anaplerotic substrates or plasma-like media PlasmaxTM. In vitro, the interaction of these two drugs yielded a strong synergistic interaction but the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood-brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors.ConclusionTogether, these data suggest that at least for ENO1-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of in vitro culture and suggest that cell culture media nutrient composition more faithful to the in vivo environment will more accurately predict in vivo efficacy of metabolism targeting drugs.