Pilot scale production of high-content mycoprotein using Rhizopus microsporus var. oligosporus by submerged fermentation and agro-industrial by-products.

While mycoprotein has gained traction as a human food source, its potential as a nutrient for animals remains largely unexplored. The mycoprotein-producing Rhizopus microsporus var. oligosporus, a fungus traditionally used for human food in Indonesia, is promising. It could revolutionise animal nutrition once it is Generally Recognized as Safe (GRAS) and is a biosafety level 1 (BSL1) organism. To enhance sustainably, we propose using sugar cane molasses (SM) and corn steep liquor (CSL) as nutrient sources. Also, we investigated the growth of R. microsporus var. oligosporus in five 14 L external-loop airlift bioreactors using CSL as the sole nutrient source. After 96 h of fermentation, at 25 °C and 0.5 vvm, the mycelium produced had an average biomass yield of 38.34 g L-1, with 70.18 % (m v-1) crude protein (mycoprotein). This bioprocess, which is scalable and economically viable, produces high amounts of mycoprotein for animal feed using CSL, a cost-effective agro-industrial by-product, providing a practical solution to the growing demand for animal protein.

Can natural zeolite improve the removal of micropollutants in a nitrifying sequencing batch reactor? Insights on bioreactor performance, kinetics, and microbial community using Ibuprofen and Diclofenac as model micropollutants.

This study presents the effect of natural zeolite (NZ) on a nitrifying sequencing batch reactor for removing ibuprofen (IBP) and diclofenac (DFC) in the long term, including kinetics and microbial community. The research was conducted in two 2 L liquid-volume bioreactors, one with 5 g/L of NZ. Nitrogen load rates ranging between 5.8 and 8.5 mg N/L h were studied. Bioreactors were operated for 217 days, with IBP and DFC concentrations ranging between 20 and 2000 µg/L. The results showed that using NZ in a nitrifying SBR only improves IBP removal at low concentrations (40 µg/L). IBP and DFC do not affect the nitrification efficiency or kinetic of ammonia removal. In the presence of IBP and DFC, NZ also favored a higher relative abundance in the genus Nitrosomonas and the Bradyrhizobiaceae family (responsible for nitrite-oxidizing activity), allowing higher IBP degradations at low IBP concentrations. Finally, IBP and DFC stimulated heterotrophic nitrification.

Bioethanol production by immobilized co-culture of Saccharomyces cerevisiae and Scheffersomyces stipitis in a novel continuous 3D printing microbioreactor.

Biorefineries require low-cost production processes, low waste generation and equipment that can be used not only for a single process, but for the manufacture of several products. In this context, in this research a continuous 3D printing microbioreactor coupled to an Arduino-controlled automatic feeding system was developed for the intensification of the ethanol production process from xylose/xylulose (3:1), using a new biocatalyst containing the co-culture of Scheffersomyces stipitis and Saccharomyces cerevisiae (50/50). Initially, batch fermentations of monocultures of S. cerevisiae and S. stipitis and co-culture were carried out. Subsequently, the immobilized co-culture was used as a biocatalyst in continuous fermentations using the developed microreactor. Fermentations carried out in the microbioreactor presented a 2-fold increase in the ethanol concentration and a 3-fold increase in productivity when compared to monocultures. The microbioreactor developed proved to be efficient and can be extended for other bioproducts production. This approach proved to be a promising alternative for the use of the hemicellulose fraction of biomasses without the need to use modified strains.

Innovative sustainable bioreactor-in-a-granule formulation of Trichoderma asperelloides.

The advancement of fungal biocontrol agents depends on replacing cereal grains with low-cost agro-industrial byproducts for their economical mass production and development of stable formulations. We propose an innovative approach to develop a rice flour-based formulation of the beneficial biocontrol agent Trichoderma asperelloides CMAA1584 designed to simulate a micro-bioreactor within the concept of full biorefinery process, affording in situ conidiation, extended shelf-life, and effective control of Sclerotinia sclerotiorum, a devastating pathogen of several dicot agricultural crops worldwide. Rice flour is an inexpensive and underexplored byproduct derived from broken rice after milling, capable of sustaining high yields of conidial production through our optimized fermentation-formulation route. Conidial yield was mainly influenced by nitrogen content (0.1% w/w) added to the rice meal coupled with the fermentor type. Hydrolyzed yeast was the best nitrogen source yielding 2.6 × 109 colony-forming units (CFU)/g within 14 days. Subsequently, GControl, GLecithin, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru formulations were obtained by extrusion followed by air-drying and further assessed for their potential to induce secondary sporulation in situ, storage stability, and efficacy against Sclerotinia. GControl, GBreak-Thru, GBentonite, and GOrganic compost+Break-Thru stood out with the highest number of CFU after sporulation upon re-hydration on water-agar medium. Shelf-life of formulations GControl and GBentonite remained consistent for > 3 months at ambient temperature, while in GBentonite and GOrganic compost+Break-Thru formulations remained viable for 24 months during refrigerated storage. Formulations exhibited similar efficacy in suppressing the myceliogenic germination of Sclerotinia irrespective of their concentration tested (5 × 104 to 5 × 106 CFU/g of soil), resulting in 79.2 to 93.7% relative inhibition. Noteworthily, all 24-month-old formulations kept under cold storage successfully suppressed sclerotia. This work provides an environmentally friendly bioprocess method using rice flour as the main feedstock to develop waste-free granular formulations of Trichoderma conidia that are effective in suppressing Sclerotinia while also improving biopesticide shelf-life. KEY POINTS: • Innovative "bioreactor-in-a-granule" system for T. asperelloides is devised. • Dry granules of aerial conidia remain highly viable for 24 months at 4 °C. • Effective control of white-mold sclerotia via soil application of Trichoderma-based granules.

Kinetics of Riboflavin Production by Hyphopichia wangnamkhiaoensis under Varying Nutritional Conditions.

Riboflavin, an essential vitamin for humans, is extensively used in various industries, with its global demand being met through fermentative processes. Hyphopichia wangnamkhiaoensis is a novel dimorphic yeast species capable of producing riboflavin. However, the nutritional factors affecting riboflavin production in this yeast species remain unknown. Therefore, we conducted a kinetic study on the effects of various nutritional factors-carbon and energy sources, nitrogen sources, vitamins, and amino acids-on batch riboflavin production by H. wangnamkhiaoensis. Batch experiments were performed in a bubble column bioreactor to evaluate cell growth, substrate consumption, and riboflavin production. The highest riboflavin production was obtained when the yeast growth medium was supplemented with glucose, ammonium sulfate, biotin, and glycine. Using these chemical components, along with the mineral salts from Castañeda-Agullo's culture medium, we formulated a novel, low-cost, and effective culture medium (the RGE medium) for riboflavin production by H. wangnamkhiaoensis. This medium resulted in the highest levels of riboflavin production and volumetric productivity, reaching 16.68 mg/L and 0.713 mg/L·h, respectively, within 21 h of incubation. These findings suggest that H. wangnamkhiaoensis, with its shorter incubation time, could improve the efficiency and cost-effectiveness of industrial riboflavin production, paving the way for more sustainable production methods.

Oxygen Transfer Effect on the Growth of Limosilactobacillus reuteri ATCC 53608 and on Its Metabolic Capacity.

Limosilactobacillus reuteri is a probiotic microorganism used in the treatment of gastrointestinal disorders. The effect of oxygen transfer on cultures of L. reuteri ATCC 53608 at shake flask and stirred tank bioreactor scales was studied, using MRS and molasses-based media. At shake flask scale, in MRS medium, a maximum bacterial concentration of 2.01 ± 0.02 g L-1 was obtained; the oxygen transfer coefficient was 2.01 ± 0.04 h-1. Similarly, in a 7.5 L bioreactor, in MRS, a maximum bacterial concentration of 2.46 ± 0.16 g L-1 was achieved (kLa = 2.64 ± 0.06 h-1). In contrast, using a molasses-based medium, bacterial concentration reached 3.13 ± 0.17 g L-1 in the 7.5 L bioreactor. A progressive reduction in lactic acid concentration and yield was observed as the oxygen transfer coefficient increased, at shake flask scale. Also, the oxygen transfer coefficient strongly affected the growth of L. reuteri in shake flask and bioreactor and allowed us to successfully scale up L. reuteri culture, producing similar maximum bacterial concentrations in both scales (2.01 g L-1 and 2.46 g L-1 in MRS). This is the first study on oxygen transfer coefficients in L. reuteri, and it is a valuable contribution to the field as it provides important insights about how this organism tolerates oxygen and adapts its metabolism for larger biomass production.

Co-digestion and co-treatment of sewage and organic waste in mainstream anaerobic reactors: operational insights and future perspectives.

The global shift towards sustainable waste management has led to an intensified exploration of co-digestion and co-treatment of sewage and organic waste using anaerobic reactors. This review advocates for an integrated approach where organic waste is treated along with the sewage stream, as a promising solution to collect, treat, and dispose of organic waste, thereby reducing the environmental and economic burden on municipalities. Various efforts, ranging from laboratory to full-scale studies, have been undertaken to assess the feasibility and impacts of co-digestion or co-management of sewage and organic waste, using technologies such as up-flow anaerobic sludge blankets or anaerobic membrane bioreactors. However, there has been no consensus on a standardized definition of co-digestion, nor a comprehensive understanding of its impacts. In this paper, we present a comprehensive review of the state-of-the-art in liquid anaerobic co-digestion systems, which typically operate at 1.1% total solids. The research aims to investigate how the integration of organic waste into mainstream anaerobic-based sewage treatment plants has the potential to enhance the sustainability of both sewage and organic waste management. In addition, utilizing the surplus capacity of existing anaerobic reactors leads to significant increases in methane production ranging from 190 to 388% (v/v). However, it should be noted that certain challenges may arise, such as the necessity for the development of tailored strategies and regulatory frameworks to enhance co-digestion practices and address the inherent challenges.

Raman laser intensity and sample clarification on biochemical monitoring over Zika-VLP upstream stages.

In the current biopharmaceutical scenario, constant bioprocess monitoring is crucial for the quality and integrity of final products. Thus, process analytical techniques, such as those based on Raman spectroscopy, have been used as multiparameter tracking methods in pharma bioprocesses, which can be combined with chemometric tools, like Partial Least Squares (PLS) and Artificial Neural Networks (ANN). In some cases, applying spectra pre-processing techniques before modeling can improve the accuracy of chemometric model fittings to observed values. One of the biological applications of these techniques could have as a target the virus-like particles (VLP), a vaccine production platform for viral diseases. A disease that has drawn attention in recent years is Zika, with large-scale production sometimes challenging without an appropriate monitoring approach. This work aimed to define global models for Zika VLP upstream production monitoring with Raman considering different laser intensities (200 mW and 495 mW), sample clarification (with or without cells), spectra pre-processing approaches, and PLS and ANN modeling techniques. Six experiments were performed in a benchtop bioreactor to collect the Raman spectral and biochemical datasets for modeling calibration. The best models generated presented a mean absolute error and mean relative error respectively of 3.46 × 105 cell/mL and 35 % for viable cell density (Xv); 4.1 % and 5 % for cell viability (CV); 0.245 g/L and 3 % for glucose (Glc); 0.006 g/L and 18 % for lactate (Lac); 0.115 g/L and 26 % for glutamine (Gln); 0.132 g/L and 18 % for glutamate (Glu); 0.0029 g/L and 3 % for ammonium (NH4+); and 0.0103 g/L and 2 % for potassium (K+). Sample without conditioning (with cells) improved the models' adequacy, except for Glutamine. ANN better predicted CV, Gln, Glu, and K+, while Xv, Glc, Lac, and NH4+ presented no statistical difference between the chemometric tools. For most of the assessed experimental parameters, there was no statistical need for spectra pre-filtering, for which the models based on the raw spectra were selected as the best ones. Laser intensity impacts quality model predictions in some parameters, Xv, Gln, and K+ had a better performance with 200 mW of intensity (for PLS, ANN, and ANN, respectively), for CV the 495 mW laser intensity was better (for PLS), and for the other biochemical variables, the use of 200 or 495 mW did not impact model fitting adequacy.

Production and concentration of keratinases and application of fermentation residual in removing hexavalent chromium.

The production of keratinases was evaluated in submerged fermentation with Aspergillus niger and by pigs' swine hair in a batch bioreactor. Experimental planning was performed to assess the interaction between different variables. The enzyme extract produced was characterized at various pH and temperatures and subjected to enzyme concentration using a biphasic aqueous system and salt/solvent precipitation techniques. In addition, the substrate's potential in reducing hexavalent chromium from synthetic potassium dichromate effluent with an initial concentration of 20 mg L-1 of chromium was evaluated. The resulting enzyme extract showed 89 ± 2 U mL-1 of keratinase. The enzyme concentration resulted in a purification factor of 1.3, while sodium chloride/acetone and ammonium sulfate/acetone resulted in a purification factor of 1.9 and 1.4, respectively. Still using the residual substrate of swine hair from the fermentation, a 94% reduction of hexavalent chromium concentration occurred after 9 h of reaction. Thus, the study proved relevant for producing keratinases, with further environmental applicability and the possibility of concentrating the extract via low-cost processes.

Cunninghamella echinulata DSM1905 biofilm-based L-asparaginase production in pneumatically-driven bioreactors.

We evaluated by comparing the performance of three pneumatically-driven bioreactors in the production of L-asparaginase (L-ASNase), an enzyme used to treat leukaemia and lymphoma. A two-step screening process was conducted to detect Cunninghamella spp. strains producing L-ASNase. Cunninghamella echinulata DSM1905 produced the highest levels of L-ASNase during screening assays. Subsequently, fermentations were performed in bubble column (BCR), airlift (ALR), and hybrid fixed-bed airlift (FB-ALR) bioreactors to determine the best upstream bioprocess. Mycelial biomass production was higher in BCR than in ALR and FB-ALR (p ≤ 0.0322). The activity of L-ASNase produced in FB-ALR, in which the fungus grew as a consistent biofilm, was significantly higher (p ≤ 0.022) than that from ALR, which was higher than that of BCR (p = 0.036). The specific activity of ALR and FB-ALR presented no differences (p = 0.073), but it was higher than that of BCR (p ≤ 0.032). In conclusion, C. echinulata DSM1905, grown under the biofilm phenotype, produced the highest levels of L-ASNase, and FB-ALR was the best upstream system for enzyme production.

Adsorption of extracellular lipase in a packed-bed reactor: an alternative immobilization approach.

In light of the growing demand for novel biocatalysts and enzyme production methods, this study aimed to evaluate the potential of Aspergillus tubingensis for producing lipase under submerged culture investigating the influence of culture time and inducer treatment. Moreover, this study also investigated conditions for the immobilization of A. tubingensis lipase by physical adsorption on styrene-divinylbenzene beads (Diaion HP-20), for these conditions to be applied to an alternative immobilization system with a packed-bed reactor. Furthermore, A. tubingensis lipase and its immobilized derivative were characterized in terms of their optimal ranges of pH and temperature. A. tubingensis was shown to be a good producer of lipase, obviating the need for inducer addition. The enzyme extract had a hydrolytic activity of 23 U mL-1 and achieved better performance in the pH range of 7.5 to 9.0 and in the temperature range of 20 to 50 °C. The proposed immobilization system was effective, yielding an immobilized derivative with enhanced hydrolytic activity (35 U g-1), optimum activity over a broader pH range (5.6 to 8.4), and increased tolerance to high temperatures (40 to 60 ℃). This research represents a first step toward lipase production from A. tubingensis under a submerged culture and the development of an alternative immobilization system with a packed-bed reactor. The proposed system holds promise for saving time and resources in future industrial applications.

Structural and functional bacterial biodiversity in a copper, zinc and nickel amended bioreactor: shotgun metagenomic study.

BACKGROUND: At lower concentrations copper (Cu), zinc (Zn) and nickel (Ni) are trace metals essential for some bacterial enzymes. At higher concentrations they might alter and inhibit microbial functioning in a bioreactor treating wastewater. We investigated the effect of incremental concentrations of Cu, Zn and Ni on the bacterial community structure and their metabolic functions by shotgun metagenomics. Metal concentrations reported in previous studies to inhibit bacterial metabolism were investigated. RESULTS: At 31.5 µM Cu, 112.4 µM Ni and 122.3 µM Zn, the most abundant bacteria were Achromobacter and Agrobacterium. When the metal concentration increased 2 or fivefold their abundance decreased and members of Delftia, Stenotrophomonas and Sphingomonas dominated. Although the heterotrophic metabolic functions based on the gene profile was not affected when the metal concentration increased, changes in the sulfur biogeochemical cycle were detected. Despite the large variations in the bacterial community structure when concentrations of Cu, Zn and Ni increased in the bioreactor, functional changes in carbon metabolism were small. CONCLUSIONS: Community richness and diversity replacement indexes decreased significantly with increased metal concentration. Delftia antagonized Pseudomonas and members of Xanthomonadaceae. The relative abundance of most bacterial genes remained unchanged despite a five-fold increase in the metal concentration, but that of some EPS genes required for exopolysaccharide synthesis, and those related to the reduction of nitrite to nitrous oxide decreased which may alter the bioreactor functioning.

Evaluation of individual and combined effect of lactic acid-consuming bacteria on mesophilic hydrogen production from lactic acid effluent from food waste treatment.

Lactic acid has been applied as a precursor for hydrogen (H2) production from substrates rich in lactic acid bacteria (LAB), focusing on microbial interactions between producing and consuming LAB tested with model substrates. Therefore, this study evaluated the effect of single and combined lactic acid-consuming bacteria on mesophilic H2 production in batch tests from lactic acid from fermented food waste (FW). Megasphaera elsdenii, Clostridium beijerinckii, and Clostridium butyricum were inoculated at different ratios (v/v). Additionally, thermal pretreated sludge (TPS) was added to the strain mixtures. The highest production was obtained with M. elsdenii, C. beijerinckii, and C. butyricum (17:66:17 ratio), obtaining 1629.0 mL/Lreactor. The optimal mixture (68:32:0 of M. elsdenii and C. beijerinckii) enriched with TPS reached 1739.3 ± 98.6 mL H2/Lreactor, consuming 98 % of lactic acid added. M. elsdenii and Clostridium strains enhance H2 production from lactic acid as they persist in a microbial community initially dominated by LAB.

Biomass Profiling in a Horizontal-Flow Anaerobic Bioreactor Used for Limonene Degradation.

This study characterized the microbial community present in the bench scale horizontal-flow anaerobic immobilized biomass bioreactor (HAIB) used in the removal of limonene, a compound present in citrus processing industries. The HAIB was filled with three support materials (coal, polyurethane foam and gravel) which were inoculated with anaerobic sludge. The limonene initial concentration on the substrate ranged from 10 mg/L to 500 mg/L. The analysis of 16S rRNA showed the presence of 22 OTUs (based on ⩾97% sequence identity), distributed in 57 genera, considering three different matrices. Higher relative abundance of phyla was observed as Synergistetes (43-57%), Proteobacteria (32-42%), Firmicutes (7-8%) and Acidobacteria (2-3%). Actinobacteria, Bacterioidetes and Chloroflexi had the lowest relative abundances between 1 and 2%. Synergistaceae family was the predominated group (47.6%-mineral coal, 55.9%-foam and 43.5%-gravel) followed by Syntrophaceae (2.4%-coal, 1.5%-foam and 2.2%-gravel), which kept a syntrophic relationship with methanogenesis (hydrogenotrophic methanogens) to maintain the anaerobic digestion. Among the Proteobacteria phylum, the Pseudomonadaceae family was predominant in the system with 12.0% on coal, 13.1% on foam, and 20.4% on gravel. The metabolic versatility of Pseudomonas sp. makes them an important bioremediation agent by being capable of metabolizing xenobiotic and chemical toxic compounds, thus having great prominence for the limonene removal in the HAIB bioreactor.

In Vitro Propagation of Threatened Agave Species Using a Two-Stage System.

Agaves are plants with multiple possibilities of use and are naturally tolerant to low water availability conditions and high temperatures. This makes them species of great interest in the context of the necessary substitution of crops due to climate change. Unfortunately, the overexploitation of wild specimens has endangered many species of the genus that have not been domesticated or cultivated intensively. In vitro mass culture and propagation techniques have emerged as a very efficient option to produce agave plants that can be used without damage to the natural populations. A protocol is presented here for the in vitro micropropagation of agaves in a two-stage process. In the first step, clusters of slightly differentiated shoots are generated from stem segments cultivated on a semisolid medium added with cytokinin. In a second step, these shoot clusters are cultured in temporary immersion bioreactors where they grow and complete their differentiation, and then the shoots are rooted and transferred to soil. This protocol has been successfully applied to several threatened species of the Agave genus.

Scale-Up of Coffea canephora Somatic Embryogenesis in Temporary Immersion System.

Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.

Production of a bacterial secretome highly efficient for the deconstruction of xylans.

Bacteria within the Paenibacillus genus are known to secrete a diverse array of enzymes capable of breaking down plant cell wall polysaccharides. We studied the extracellular xylanolytic activity of Paenibacillus xylanivorans and examined the complete range of secreted proteins when grown on carbohydrate-based carbon sources of increasing complexity, including wheat bran, sugar cane straw, beechwood xylan and sucrose, as control. Our data showed that the relative abundances of secreted proteins varied depending on the carbon source used. Extracellular enzymatic extracts from wheat bran (WB) or sugar cane straw (SCR) cultures had the highest xylanolytic activity, coincidently with the largest representation of carbohydrate active enzymes (CAZymes). Scaling-up to a benchtop bioreactor using WB resulted in a significant enhancement in productivity and in the overall volumetric extracellular xylanase activity, that was further concentrated by freeze-drying. The enzymatic extract was efficient in the deconstruction of xylans from different sources as well as sugar cane straw pretreated by alkali extrusion (SCRe), resulting in xylobiose and xylose, as primary products. The overall yield of xylose released from SCRe was improved by supplementing the enzymatic extract with a recombinant GH43 ß-xylosidase (EcXyl43) and a GH62 α-L-arabinofuranosidase (CsAbf62A), two activities that were under-represented. Overall, we showed that the extracellular enzymatic extract from P. xylanivorans, supplemented with specific enzymatic activities, is an effective approach for targeting xylan within lignocellulosic biomass.

Improved productivity and dye removal performance of Trametes versicolor pellets using rice husk as a co-substrate.

Pellet production represents a critical step for several processes requiring fungal biomass, nevertheless, its optimization is seldom reported. The use of finely ground rice husk as a microcarrier and co-substrate permitted a marked increase (≈ 2.7×) in the productivity of fungal pellet production using Trametes versicolor compared to traditional production methods. The pellets show similar structure and smaller size compared to typical sole-mycelium pellets, as well as comparable laccase activity. The efficiency of the pellets for biodegradation was confirmed by the removal of the crystal violet dye, achieving significantly faster decolorization rates compared to the traditionally produced pellets. The use of these pellets during the continuous treatment of the dye in a stirred tank bioreactor resulted in 97% decolorization operating at a hydraulic residence time of 4.5 d.

Enhancing Selenium Accumulation in Rhodotorula mucilaginosa Strain 6S Using a Proteomic Approach for Aquafeed Development.

It is known that selenium (Se) is an essential trace element, important for the growth and other biological functions of fish. One of its most important functions is to contribute to the preservation of certain biological components, such as DNA, proteins, and lipids, providing protection against free radicals resulting from normal metabolism. The objective of this study was to evaluate and optimize selenium accumulation in the native yeast Rhodotorula mucilaginosa 6S. Sodium selenite was evaluated at different concentrations (5-10-15-20-30-40 mg/L). Similarly, the effects of different concentrations of nitrogen sources and pH on cell growth and selenium accumulation in the yeast were analyzed. Subsequently, the best cultivation conditions were scaled up to a 2 L reactor with constant aeration, and the proteome of the yeast cultured with and without sodium selenite was evaluated. The optimal conditions for biomass generation and selenium accumulation were found with ammonium chloride and pH 5.5. Incorporating sodium selenite (30 mg/L) during the exponential phase in the bioreactor after 72 h of cultivation resulted in 10 g/L of biomass, with 0.25 mg total Se/g biomass, composed of 25% proteins, 15% lipids, and 0.850 mg total carotenoids/g biomass. The analysis of the proteomes associated with yeast cultivation with and without selenium revealed a total of 1871 proteins. The results obtained showed that the dynamic changes in the proteome, in response to selenium in the experimental medium, are directly related to catalytic activity and oxidoreductase activity in the yeast. R. mucilaginosa 6S could be an alternative for the generation of selenium-rich biomass with a composition of other nutritional compounds also of interest in aquaculture, such as proteins, lipids, and pigments.

Methylotrophic methanogenesis induced by ammonia nitrogen in an anaerobic digestion system.

OBJECTIVES: This lab-scale study aimed to investigate the effect of total ammonia nitrogen (TAN) stress on the methanogenic activity and the taxonomic and functional profiles of the microbial community of anaerobic sludge (AS) from a full-scale bioreactor. METHODS: The AS was subjected to a stepwise increase in TAN every 14 days at concentrations of 1, 2, 2.5, 3, 3.5, and 4 g TAN/L (Acclimated-AS or AAS). This acclimation stage was followed by an ammonia stress stage (4 g/L). A blank-AS (BAS) was maintained without TAN during the acclimation stage. In the second stress stage (ST), the BAS was divided into two new treatments: a control (BAS') and one that received a shock load of TAN of 4 g/L (SBAS'). Methane production was measured, and a metagenomic analysis was conducted to describe the microbial community. RESULTS: A decrease in the relative abundance of Methanothrix soehngenii of 16 % was related to a decrease of 23 % in the methanogenic capacity of AAS when comparing with the final stage of BAS. However, recovery was observed at 3.5 g TAN/L, and a shift to methylotrophic metabolism occurred, indicated by a 4-fold increase in abundance of Methanosarcina mazei. The functional analysis of sludge metagenomes indicated that no statistical differences (p > 0.05, RM ANOVA) were found in the relative abundance of methanogenic genes that initiate acetoclastic and hydrogenotrophic pathways (acetyl-CoA synthetase, ACSS; acetate kinase, ackA; phosphate acetyltransferase, pta; and formylmethanofuran dehydrogenase subunit A, fwdA) into the BAS and AAS during the acclimation phase. The same was observed between groups of genes associated with methanogenesis from methylated compounds. In contrast, statistical differences (p < 0.05, one-way ANOVA) in the relative abundance of these genes were recorded during ST. The functional profiles of the genes involved in acetoclastic, hydrogenotrophic, and methylotrophic methanogenic pathways were brought to light for acclimatation and stress experimental stages. CONCLUSIONS: TAN inhibited methanogenic activity and acetoclastic metabolism. The gradual acclimatization to TAN leads to metabolic and taxonomic changes that allow for the subsequent recovery of methanogenic functionality. The study highlights the importance of adequate management of anaerobic bioprocesses with high nitrogen loads to maintain the methanogenic functionality of the microbial community.