Cauliflower mosaic virus protein P6 is a multivalent node for RNA granule proteins and interferes with stress granule responses during plant infection.

Biomolecular condensation is a multipurpose cellular process that viruses use ubiquitously during their multiplication. Cauliflower mosaic virus replication complexes are condensates that differ from those of most viruses, as they are nonmembranous assemblies that consist of RNA and protein, mainly the viral protein P6. Although these viral factories (VFs) were described half a century ago, with many observations that followed since, functional details of the condensation process and the properties and relevance of VFs have remained enigmatic. Here, we studied these issues in Arabidopsis thaliana and Nicotiana benthamiana. We observed a large dynamic mobility range of host proteins within VFs, while the viral matrix protein P6 is immobile, as it represents the central node of these condensates. We identified the stress granule (SG) nucleating factors G3BP7 and UBP1 family members as components of VFs. Similarly, as SG components localize to VFs during infection, ectopic P6 localizes to SGs and reduces their assembly after stress. Intriguingly, it appears that soluble rather than condensed P6 suppresses SG formation and mediates other essential P6 functions, suggesting that the increased condensation over the infection time-course may accompany a progressive shift in selected P6 functions. Together, this study highlights VFs as dynamic condensates and P6 as a complex modulator of SG responses.

Arabidopsis RNA processing body components LSM1 and DCP5 aid in the evasion of translational repression during Cauliflower mosaic virus infection.

Viral infections impose extraordinary RNA stress, triggering cellular RNA surveillance pathways such as RNA decapping, nonsense-mediated decay, and RNA silencing. Viruses need to maneuver among these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells, with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigated the role of Arabidopsis thaliana PBs during Cauliflower mosaic virus (CaMV) infection. We found that several PB components are co-opted into viral factories that support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we established that PB components are helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, dysfunctions in PB components expose the virus to this pathway, which is similar to previous observations for transgenes. Transgenes, however, undergo RNA quality control-dependent RNA degradation and transcriptional silencing, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence among PBs, RNA silencing, and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.

Genome-centric metagenomics and methanogenic pathway analysis for acclimated anaerobic digestion of chicken manure with high ammonia stressed under thermophilic condition.

Thermophilic anaerobic digestion (AD) of animal manure offers various environmental benefits but the process requires a microbial community acclimatized to high ammonia. In current study, a lab-scale continuous stirred tank reactor (CSTR) fed with chicken manure was operated under thermophilic condition for 450 days in total. Results showed that the volumetric methane production decreased from 445 to 328 and sharply declined to 153 mL L-1·d-1 with feeding total solid (TS) step increased from 5% to 7.5% and 10%, respectively. While, after a long-term stop feeding for 80 days, highly disturbed reactor was able to recover methane generation to 739 mL L-1·d-1 at feeding TS of 10%. Isotope analysis indicted acetate converted to methane through the syntrophic acetate oxidation and hydrogenotrophic methanogenesis (SAO-HM) pathway increased from 33% to 63% as the concentration of ammonium increased from 2493 to 6258 mg L-1. Significant different in the genome expression of the SAO bacterial from 0.09% to 1.23%, combining with main hydrogenotrophic partners (Methanoculleus spp. and Methanothermobacter spp.) contented of 2.1% and 99.9% during inhibitory and recovery stages, respectively. The highly expressed KEGG pathway in level 3 (enzyme genes) for the Recovery sludge combining with the extraordinary high abundance of genera Halocella sp. suggested that Halocella sp. might be a highly efficient hydrolytic and acidogenic microorganism and enhance the process of SAO during carbon metabolic flow to methane. This report will be a basis for further study of AD studies on high nitrogen content of poultry manure.

CELLULOSE SYNTHASE INTERACTING 1 is required for wood mechanics and leaf morphology in aspen.

Cellulose microfibrils synthesized by CELLULOSE SYNTHASE COMPLEXES (CSCs) are the main load-bearing polymers in wood. CELLULOSE SYNTHASE INTERACTING1 (CSI1) connects CSCs with cortical microtubules, which align with cellulose microfibrils. Mechanical properties of wood are dependent on cellulose microfibril alignment and structure in the cell walls, but the molecular mechanism(s) defining these features is unknown. Herein, we investigated the role of CSI1 in hybrid aspen (Populus tremula × Populus tremuloides) by characterizing transgenic lines with significantly reduced CSI1 transcript abundance. Reduction in leaves (50-80%) caused leaf twisting and misshaped pavement cells, while reduction (70-90%) in developing xylem led to impaired mechanical wood properties evident as a decrease in the elastic modulus and rupture. X-ray diffraction measurements indicate that microfibril angle was not impacted by the altered CSI1 abundance in developing wood fibres. Instead, the augmented wood phenotype of the transgenic trees was associated with a reduced cellulose degree of polymerization. These findings establish a function for CSI1 in wood mechanics and in defining leaf cell shape. Furthermore, the results imply that the microfibril angle in wood is defined by CSI1 independent mechanism(s).

The Effect of Calcium/Magnesium Ratio on the Biomass Production of a Novel Thermoalkaliphilic Aeribacillus pallidus Strain with Highly Heat-Resistant Spores.

Hot springs are fascinating extreme environments for the isolation of polyextremophilic microorganisms with extraordinary characteristics. Since polyextremophilic bacterial growth are not as high as routine bacteria, the objective of this study was to investigate the effect of some environmental factors on biomass and metabolites productions in the newly isolated strain, from Larijan hot spring in Iran. The strain was identified as Aeribacillus pallidus Lhs-10 and deposited as CCUG 72355 and IBRC-M 11202 in Sweden and Iran, respectively. This thermoalkaliphilic strain can grow best at 50 °C, pH 8 and in the presence of 25 g/l NaCl. The physiological characterization of this strain show that [Ca/Mg] ratio affect its growth and biomass production with the best results obtained at the ratio of 2.5. Moreover, lactic and acetic acids production by this strain was affected by pH, aeration, and temperature, where a metabolic shift was detected from lactate to acetate production when the culture was aerated. Besides, its spores could tolerate heating at 80, 85, 90, 95 and 98 °C for 30 min without any reduction in the initial spore population, whereas D-value was defined 50 min at 98 °C. This newly lactic acid-producing strain of A. pallidus can be a promising strain that can be used in the harsh conditions in industrial processes.

Sucrose transport and carbon fluxes during wood formation.

Wood biosynthesis defines the chemical and structural properties of wood. The metabolic pathways that produce the precursors of wood cell wall polymers have a central role in defining wood properties. To make rational design of wood properties feasible, we need not only to understand the cell wall biosynthetic machinery, but also how sucrose transport and metabolism in developing wood connect to cell wall biosynthesis and how they respond to genetic and environmental cues. Here, we review the current understanding of the sucrose transport and primary metabolism pathways leading to the precursors of cell wall biosynthesis in woody plant tissues. We present both old, persistent questions and new emerging themes with a focus on wood formation in trees and draw upon evidence from the xylem tissues of herbaceous plants when it is relevant.

Spatially resolved metabolic analysis reveals a central role for transcriptional control in carbon allocation to wood.

The contribution of transcriptional and post-transcriptional regulation to modifying carbon allocation to developing wood of trees is not well defined. To clarify the role of transcriptional regulation, the enzyme activity patterns of eight central primary metabolism enzymes across phloem, cambium, and developing wood of aspen (Populus tremula L.) were compared with transcript levels obtained by RNA sequencing of sequential stem sections from the same trees. Enzymes were selected on the basis of their importance in sugar metabolism and in linking primary metabolism to lignin biosynthesis. Existing enzyme assays were adapted to allow measurements from ~1 mm3 sections of dissected stem tissue. These experiments provided high spatial resolution of enzyme activity changes across different stages of wood development, and identified the gene transcripts probably responsible for these changes. In most cases, there was a clear positive relationship between transcripts and enzyme activity. During secondary cell wall formation, the increases in transcript levels and enzyme activities also matched with increased levels of glucose, fructose, hexose phosphates, and UDP-glucose, emphasizing an important role for transcriptional regulation in carbon allocation to developing aspen wood. These observations corroborate the efforts to increase carbon allocation to wood by engineering gene regulatory networks.

13C Tracking after 13CO2 Supply Revealed Diurnal Patterns of Wood Formation in Aspen.

Wood of trees is formed from carbon assimilated in the photosynthetic tissues. Determining the temporal dynamics of carbon assimilation, subsequent transport into developing wood, and incorporation to cell walls would further our understanding of wood formation in particular and tree growth in general. To investigate these questions, we designed a (13)CO2 labeling system to study carbon transport and incorporation to developing wood of hybrid aspen (Populus tremula × tremuloides). Tracking of (13)C incorporation to wood over a time course using nuclear magnetic resonance spectroscopy revealed diurnal patterns in wood cell wall biosynthesis. The dark period had a differential effect on (13)C incorporation to lignin and cell wall carbohydrates. No (13)C was incorporated into aromatic amino acids of cell wall proteins in the dark, suggesting that cell wall protein biosynthesis ceased during the night. The results show previously unrecognized temporal patterns in wood cell wall biosynthesis, suggest diurnal cycle as a possible cue in the regulation of carbon incorporation to wood, and establish a unique (13)C labeling method for the analysis of wood formation and secondary growth in trees.

Aspen SUCROSE TRANSPORTER3 allocates carbon into wood fibers.

Wood formation in trees requires carbon import from the photosynthetic tissues. In several tree species, including Populus species, the majority of this carbon is derived from sucrose (Suc) transported in the phloem. The mechanism of radial Suc transport from phloem to developing wood is not well understood. We investigated the role of active Suc transport during secondary cell wall formation in hybrid aspen (Populus tremula × Populus tremuloides). We show that RNA interference-mediated reduction of PttSUT3 (for Suc/H(+) symporter) during secondary cell wall formation in developing wood caused thinner wood fiber walls accompanied by a reduction in cellulose and an increase in lignin. Suc content in the phloem and developing wood was not significantly changed. However, after (13)CO2 assimilation, the SUT3RNAi lines contained more (13)C than the wild type in the Suc-containing extract of developing wood. Hence, Suc was transported into developing wood, but the Suc-derived carbon was not efficiently incorporated to wood fiber walls. A yellow fluorescent protein:PttSUT3 fusion localized to plasma membrane, suggesting that reduced Suc import into developing wood fibers was the cause of the observed cell wall phenotype. The results show the importance of active Suc transport for wood formation in a symplasmically phloem-loading tree species and identify PttSUT3 as a principal transporter for carbon delivery into secondary cell wall-forming wood fibers.

Biovalorization of brewer's spent grain as single-cell protein through coupling organosolv pretreatment and fungal cultivation.

Brewer's spent grain (BSG) is a clean byproduct from the food sector, comprising 85% of the brewing process solid byproducts. BSG is mainly used as low-quality animal feed and often ends up in landfills due to its short shelf life. However, considering its abundant availability and high nutritional content, BSG holds the potential for biorefineries to produce valuable products. The recalcitrant nature of BSG poses a challenge, requiring pretreatment steps. Therefore, this study focused on valorizing BSG obtained from organosolv pretreatment by producing food- and feed-grade single-cell protein (SCP). The BSG was subject to organosolv pretreatment at 180C for 2 h with 50% v/v ethanol as solvent. Filamentous fungi N. intermedia and A. oryzae were cultivated on as-received and different fractions of organosolv-treated BSG to evaluate the effect of factors such as pretreatment, fungal strain, pretreated fraction content, and substrate loading on fungal biomass yield, biomass composition (protein content), and metabolite production. A. oryzae cultivation on all tested substrates yielded 7%-40% more biomass than N. intermedia. Cultivating A. oryzae on organosolv liquor resulted in the highest biomass protein content (44.8% ± 0.7%) with a fungal biomass concentration of 5.1 g/L. A three-fold increase in the substrate loading increased the ethanol-to-substrate yield by 50%, while protein content was decreased by 23%. Finally, a biorefinery concept was proposed to integrate the organosolv pretreatment of BSG with fungal cultivation for maximum yield of SCP while obtaining other products such as lignin and ethanol, providing a sustainable rout for managing BSG.

Membrane bioreactor assisted volatile fatty acids production from agro-industrial residues for ruminant feed application.

Volatile fatty acids (VFAs) supplementation in ruminants' diet as a source of energy and chemical precursors and their effect on animal's physiology and well-being has long been of scientific interest. Production of VFAs through anaerobic digestion of agro-industrial residues not only creates value but also presents an alternative sustainable approach for ruminant feed supplementation. Therefore, this study aimed to investigate the bioconversion of agro-industrial residues produced in large quantities such as apple pomace (AP), thin stillage (Ts), and potato protein liquor (PPL) to VFAs, fully complying to regulations set for ruminant feed supplement production. In this regard, batch acidogenic fermentation assays (pH 6-10) and semi-continuous immersed membrane bioreactor (iMBR) were applied. In batch assays, at pH 10 the co-digestion of Ts and PPL produced the highest VFAs concentration (14.2 g/L), indicating a yield of 0.85 g CODVFAs/g volatile solids (VS)added. The optimum batch condition was then applied in the iMBR for in situ fermentation and recovery of VFAs at different organic loading rates (OLR). With increasing the OLR to 3.7 gVS/L.day, the highest VFAs concentration of 28.6 g/L (1,2 g CODVFAs /gVSadded) was achieved. Successful long-term (114 days) membrane filtration was conducted in a media with a maximum of 40 g/L of total solids (TS), facing irreversible membrane fouling in the final stages. Acidogenic fermentation using an iMBR has the potential to play an important role in the future of feed additive provision through the biorefining of agro-industrial wastes via the carboxylate platform, given the role of VFAs production from organic residues.

Application of Immersed Membrane Bioreactor for Semi-Continuous Production of Polyhydroxyalkanoates from Organic Waste-Based Volatile Fatty Acids.

Volatile fatty acids (VFAs) appear to be an economical carbon feedstock for the cost-effective production of polyhydroxyalkanoates (PHAs). The use of VFAs, however, could impose a drawback of substrate inhibition at high concentrations, resulting in low microbial PHA productivity in batch cultivations. In this regard, retaining high cell density using immersed membrane bioreactor (iMBR) in a (semi-) continuous process could enhance production yields. In this study, an iMBR with a flat-sheet membrane was applied for semi-continuous cultivation and recovery of Cupriavidus necator in a bench-scale bioreactor using VFAs as the sole carbon source. The cultivation was prolonged up to 128 h under an interval feed of 5 g/L VFAs at a dilution rate of 0.15 (d-1), yielding a maximum biomass and PHA production of 6.6 and 2.8 g/L, respectively. Potato liquor and apple pomace-based VFAs with a total concentration of 8.8 g/L were also successfully used in the iMBR, rendering the highest PHA content of 1.3 g/L after 128 h of cultivation. The PHAs obtained from both synthetic and real VFA effluents were affirmed to be poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a crystallinity degree of 23.8 and 9.6%, respectively. The application of iMBR could open an opportunity for semi-continuous production of PHA, increasing the feasibility of upscaling PHA production using waste-based VFAs.

Evaluation of in vitro digestibility of Aspergillus oryzae fungal biomass grown on organic residue derived-VFAs as a promising ruminant feed supplement.

BACKGROUND: As demand for high quality animal feed continues to raise, it becomes increasingly important to minimize the environmental impact of feed production. An appealing sustainable approach to provide feed fractions is to use organic residues from agro-food industry. In this regard, volatile fatty acids (VFAs) such as acetic, propionic and butyric acids, derived from bioconversion of organic residues can be used as precursors for production of microbial protein with ruminant feed inclusion potential. This study aims to investigate the in vitro digestibility of the Aspergillus oryzae edible fungal biomass cultivated on VFAs-derived from anaerobic digestion of residues. The produced fungal protein biomass, along with hay clover silage and rapeseed meal were subjected to various in vitro assays using two-stage Tilley and Terry (TT), gas, and bag methods to evaluate and compare its digestibility for application in ruminant feed. RESULTS: The produced fungal biomass contained a higher crude protein (CP) (41%-49%) and rather similar neutral detergent fiber (NDF) (41%-56%) compared to rapeseed meal. The rumen in vitro dry matter digestibility (IVDMD) of the fungal biomass in the TT method ranged from 82% to 88% (statistically similar to that of the gas method (72% to 85%)). The IVDMD of fungal biomass were up to 26% and 40% greater than that of hay clover silage and rapeseed meal, respectively. The type of substrate and bag method had pronounced effect on the fermentation products (ammonium-N (NH4+-N), total gas and VFAs). Fungal biomass digestion resulted in the highest release of NH4+-N (340-540 mg/L) and the ratio of acetate to propionate ratio (3.5) among subjected substrates. CONCLUSION: The results indicate that gas method can be used as a reliable predictor for IVDMD as well as fermentation products. Furthermore, the high IVDMD and fermentation product observed for Aspergillus oryzae fungal biomass digestion, suggest that the supplementation of fungal biomass will contribute to improving the rumen digestion by providing necessary nitrogen and energy to the ruminant and microbiota.

Application of Fungal Biomass for the Development of New Polylactic Acid-Based Biocomposites.

Fungal biomass (FB), a by-product of the fermentation processes produced in large volumes, is a promising biomaterial that can be incorporated into poly(lactic acid) (PLA) to develop enhanced biocomposites that fully comply with the biobased circular economy concept. The PLA/FB composites, with the addition of triethyl citrate (TEC) as a biobased plasticizer, were fabricated by a microcompounder at 150 °C followed by injection molding. The effects of FB (10 and 20 wt %) and TEC (5, 10, and 15 wt %) contents on the mechanical, thermal and surface properties of the biocomposites were analyzed by several techniques. The PLA/FB/TEC composites showed a rough surface in their fracture section. A progressive decrease in tensile strength and Young's modulus was observed with increasing FB and TEC, while elongation at break and impact strength started to increase. The neat PLA and biocomposite containing 10% FB and 15% TEC exhibited the lowest (3.84%) and highest (224%) elongation at break, respectively. For all blends containing FB, the glass transition, crystallization and melting temperatures were shifted toward lower values compared to the neat PLA. The incorporation of FB to PLA thus offers the possibility to overcome one of the main drawbacks of PLA, which is brittleness.

Factors influencing pressure-driven membrane-assisted volatile fatty acids recovery and purification-A review.

Volatile fatty acids (VFAs) are building block chemicals that can be produced through bioconversion of organic waste streams via anaerobic digestion as intermediate products. Purified VFAs are applicable in a wide range of industrial applications such as food, textiles, cosmetics, pharmaceuticals etc. production. The present review focuses on VFAs recovery methods and technologies such as adsorption, distillation, extraction, gas stripping, esterification and membrane based techniques etc., while presenting a discussion of their pros and cons. Moreover, a great attention has been given to the recovery of VFAs through membrane filtration as a promising sustainable clarification, fractionation and concentration approach. In this regard, a thorough overview of factors affecting membrane filtration performance for VFAs recovery has been presented. Filtration techniques such as nanofiltration and reverse osmosis have shown to be capable of recovering over 90% of VFAs content from organic effluent steams, proving the direct effect of membrane materials/surface chemistry, pore size and solution pH in recovery success level. Overall, this review presents a new insight into challenges and potentials of membrane filtration for VFAs recovery based on the effects of factors such as operational parameters, membrane properties and effluent characteristics.

Potential of food waste-derived volatile fatty acids as alternative carbon source for denitrifying moving bed biofilm reactors.

Fossil-based materials such as methanol are frequently used in the denitrification process of advanced biological wastewater treatment as external carbon source. Volatile fatty acids (VFAs) produced by anaerobic digestion of food waste, are sustainable compounds with the potential to act as carbon sources for denitrification, reducing carbon footprint and material costs. In this study, the effectiveness of food waste-derived VFAs (AD-VFA) was investigated in the post-denitrification process in comparison with synthetic VFA and methanol as carbon sources. Acetic acid had the highest rate of disappearance among single tested VFAs with a denitrification rate of 0.44 g NOx-N removed/m2/day, indicating a preferential utilization pattern. While AD-VFA had a denitrification rate of 0.61 mg NOx-N removed/m2/day, sVFA had a rate of 0.57 mg NOx-N removed/m2/day, indicating that impurities in AD-VFA did not play substantial role in denitrification. AD-VFA proved to be promising carbon source alternative for denitrification in wastewater treatment plants.

Double-stage membrane-assisted anaerobic digestion process intensification for production and recovery of volatile fatty acids from food waste.

The potential of organic waste streams (i.e., food waste) for the sustainable production of precursor chemicals such as volatile fatty acids (VFAs) using anaerobic digestion (AD) has received significant attention in the present days. AD-derived VFAs have great market appeal if the challenges with their recovery and purification from the complex AD effluent is overcome. In this study, a microfiltration immersed membrane bioreactor (MBR) was used for the production of VFAs from food waste and simultaneously in-situ recovery of VFAs. The MBR set-up was applied for 98 days, with a maximum yield of 0.2 gVFA/gVSadded at an organic loading rate (OLR) of 4 g VS/L/d. The recovered permeate was then subjected to further purification using a side stream ultrafiltration unit. It was found that the removal rates of total solids (TS), total suspended solids (TSS), dissolved solids (DS), volatile solids (VS) and volatile suspended solids (VSS) were above 70-80% in both membranes (10 kDa and 50 kDa), and Phosphorus (P), Total Kjeldahl Nitrogen (TKN), chemical oxygen demand (COD), and NH4+-N were also removed partially. Particularly, VFAs concentration (above 6 g/L) was higher for 10 kDa at pH 5.4 in ultrafiltered solution and permeate flux decline was higher for 10 kDa at pH 5.4. These results are also supported by the measurement of UV-Vis spectra of the solution and visual appearance, providing a promising approach towards building a VFAs-based platform.

Waste-derived volatile fatty acid production and ammonium removal from it by ion exchange process with natural zeolite.

Volatile fatty acids (VFAs) produced during anaerobic digestion (AD) of organic waste are a promising alternative carbon source for various biological processes; however, their applications are limited due to the presence of impurities such as ammonium (NH4+). This study investigates the potential for removal of ammonium using a naturally occurring zeolite (clinoptilolite) from chicken manure (CKM) derived VFA effluent recovered from an anaerobic membrane bioreactor (MBR). Experiments were conducted for both synthetic and actual VFA (AD-VFA) solutions, and the effects of different parameters were investigated with batch and continuous studies. It was observed that the Langmuir-type isotherm provided the best fit to the equilibrium data in the isotherm investigations carried out with the AD-VFA solution. The maximum adsorption capacity (qm) was found as 15.7 mg NH4+/g clinoptilolite. The effect of some operational parameters on process performance such as pH, initial NH4+ loading and potassium ion (K+) concentration was investigated. The pH had a negligible effect on ammonium removal for a pH range of 3-7, while the removal efficiency of ammonium decreased with the increase of initial NH4+ loading and K+ concentration. At the optimum conditions determined in batch experiments, the ammonium removal from synthetic and AD-VFA solutions were compared and average ammonium removal efficiencies of 93 and 94% were found in 12 h equilibrium time for synthetic and AD-VFA solutions, respectively. Overall findings indicated that clinoptilolite has excellent potential for ion exchange when combined with biological processes such as acidogenic fermentation of VFAs to purify the solution from high-ammonium content.

A Glimpse of the World of Volatile Fatty Acids Production and Application: A review.

Sustainable provision of chemicals and materials is undoubtedly a defining factor in guaranteeing economic, environmental, and social stability of future societies. Among the most sought-after chemical building blocks are volatile fatty acids (VFAs). VFAs such as acetic, propionic, and butyric acids have numerous industrial applications supporting from food and pharmaceuticals industries to wastewater treatment. The fact that VFAs can be produced synthetically from petrochemical derivatives and also through biological routes, for example, anaerobic digestion of organic mixed waste highlights their provision flexibility and sustainability. In this regard, this review presents a detailed overview of the applications associated with petrochemically and biologically generated VFAs, individually or in mixture, in industrial and laboratory scale, conventional and novel applications.