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.

Impact of Uniaxial Static Strain on Myoblast Differentiation in Collagen-Coated PCL Microfilament Scaffolds: Role of Onset Time of Mechanical Stimulation.

Tissue engineering endeavors to create in vitro constructs that replicate the properties of native tissue, such as skeletal muscle. This study investigated the use of mechanical stimulation to promote myogenic differentiation and enhance the functionality of bioengineered tissues. Specifically, it aimed to facilitate the differentiation of myoblasts within a three-dimensional scaffold using a defined pattern of mechanical stimulation. C2C12 cells were cultured on a collagen-coated PCL microfilament scaffold and subjected to 24 h of uniaxial static strain using a biomechanical stimulation system. Two onset times of stimulation, 72 h and 120 h post-seeding, were evaluated. Cell proliferation, myogenic marker expression, and alterations in cell morphology and orientation were assessed. Results indicate that static strain on the scaffold promoted myoblast differentiation, evidenced by morphological and molecular changes. Notably, strain initiated at 72 h induced an early differentiation stage marked by MyoD expression, whereas stimulation beginning at 120 h led to a mid-stage differentiation characterized by the co-expression of MyoD and Myogenin, culminating in myotube formation. These results highlight the critical influence of myoblast maturity at the time of strain application on the differentiation outcome. This study provides insights that could guide the optimization of mechanical stimulation protocols in tissue engineering applications.

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.

Bioconversion of L-Tyrosine into p-Coumaric Acid by Tyrosine Ammonia-Lyase Heterologue of Rhodobacter sphaeroides Produced in Pseudomonas putida KT2440.

p-Coumaric acid (p-CA) is a valuable compound with applications in food additives, cosmetics, and pharmaceuticals. However, traditional production methods are often inefficient and unsustainable. This study focuses on enhancing p-CA production efficiency through the heterologous expression of tyrosine ammonia-lyase (TAL) from Rhodobacter sphaeroides in Pseudomonas putida KT2440. TAL catalyzes the conversion of L-tyrosine into p-CA and ammonia. We engineered P. putida KT2440 to express TAL in a fed-batch fermentation system. Our results demonstrate the following: (i) successful integration of the TAL gene into P. putida KT2440 and (ii) efficient bioconversion of L-tyrosine into p-CA (1381 mg/L) by implementing a pH shift from 7.0 to 8.5 during fed-batch fermentation. This approach highlights the viability of P. putida KT2440 as a host for TAL expression and the successful coupling of fermentation with the pH-shift-mediated bioconversion of L-tyrosine. Our findings underscore the potential of genetically modified P. putida for sustainable p-CA production and encourage further research to optimize bioconversion steps and fermentation conditions.

Advances in submerged liquid fermentation and formulation of entomopathogenic fungi.

Entomopathogenic fungi (EPF) can be defined as beneficial multifunctional eukaryotic microorganisms that display pivotal ecological services in pest management, with some species possessing the special ability to establish mutualistic relationships with plants. Mass production of these fungi is critical to support affordable widespread commercialization and worldwide field application. Among the mass production methods explored mainly by industry, submerged liquid fermentation is a robust and versatile technology that allows the formation of different types of propagules designated for various applications in pest control. Many hypocrealean EPF are easily culturable on artificial substrates by producing single-celled structures (hyphal bodies, blastospores, and submerged conidia) or multicellular structures (mycelium and microsclerotia). Less frequently, some EPF may form environmentally resistant chlamydospores, but these structures have almost always been overlooked. A continued research pipeline encompassing screening fungal strains, media optimization, and proper formulation techniques aligned with the understanding of molecular cues involved in the formation and storage stability of these propagules is imperative to unlock the full potential and to fine-tune the development of robust and effective biocontrol agents against arthropod pests and vectors of diseases. Finally, we envision a bright future for the submerged liquid fermentation technology to supplement or replace the traditional solid substrate fermentation method for the mass production of many important EPF. KEY POINTS: • Submerged liquid fermentation (SLF) allows precise control of nutritional and environmental factors • SLF provides a scalable, robust, and cost-effective platform for mycopesticide production • Enhancing formulation, shelf life, and field efficacy of submerged propagules remain crucial • Understanding the molecular mechanisms behind submerged propagule formation is key to advancing SLF technology.

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.

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 complex bioprocess learning through simulation technology and hybrid teaching: A case study in university education.

The utilization of computer simulators in university education is progressively being embraced to offer students a practical exposure to industrial bioprocesses. Bioreactor computer simulators hold various advantages over conventional laboratory experiments, such as cost-effectiveness and enhanced safety. The research objective is to assess the effectiveness of integrating bioreactor computer simulators into hybrid teaching to promote active and collaborative learning experiences and evaluate their impact on student participation and understanding. A hybrid strategy combining synchronous, face-to-face, and online teaching has been implemented to enhance the teaching-learning processes in the Industrial Bioprocesses course for Biochemistry students. The simulation software BIOSTAT®T Yeast was used. This software models the production of ethanol with Saccharomyces cerevisiae through batch cultivation and the determination of the kLa value of a bioreactor. In the first simulation activity, students analyzed the software response based on parameter values input by the instructor, while in the second simulation activity, students autonomously used the computer simulator under the primary oversight of the instructor. The survey results indicate that the pedagogical innovation was positively received and significantly motivating for the students. Comparing student satisfaction surveys between the two simulation activities suggests that fostering student autonomy and engagement through simulation technology can improve satisfaction and learning outcomes in bioprocess education.

Tertiary treatment of dairy wastewater applying a microalga-fungus consortium.

This paper aimed to apply filamentous fungi (Penicillium oxalicum and Cunninghamella echinulata), the microalga Tetradesmus obliquus and their co-culture in advanced treatment (tertiary treatment) of cheese whey. The bioremediation process was carried out in agitated flasks and bubble column bioreactors with different concentrations of chemical oxygen demand (COD) (223-1663 mg L-1), total nitrogen (TN) (13-61 mg L-1), and total phosphorus (TP) (3-26 mg L-1). The results obtained in shaken flasks showed a superiority of the consortium compared to the systems with separated species. In this sense, the treatment was carried out in a bubble column reactor, and the consortium formed by the microalga and the fungus C. echinulata showed a greater efficiency (at a light intensity of 100 µmol m-2 s-1), promoting by the symbiosis to reach removal efficiencies of up to 93.7, 78.8 and 93.4% for COD, TN and TP, respectively; meeting Brazilian and European standards for discharge into water bodies. In addition, no pH adjustment was required during the co-culture treatment, demonstrating the buffering effect of using these two types of microorganisms. Therefore, the use of the consortium formed by T. obliquus and C. echinulata as a remediator was highly promising to promote the advanced treatment of cheese whey.

Dairy wastewater needs a polishing treatment stage after secondary treatmentThe microalga-fungus consortium met legislation requirementsCOD, nitrogen and phosphorus were efficiently removed by the consortiumNo pH control was applied during the biological treatment by the consortium.

Effect of impeller type on cellular morphology and production of clavulanic acid by Streptomyces clavuligerus.

It is essential to evaluate the effects of operating conditions in submerged cultures of filamentous microorganisms. In particular, the impeller type influences the flow pattern, power consumption, and energy dissipation, leading to differences in the hydrodynamic environment that affect the morphology of the microorganism. This work investigated the effect of different impeller types, namely the Rushton turbine (RT-RT) and Elephant Ear impellers in up-pumping (EEUP) and down-pumping (EEDP) modes, on cellular morphology and clavulanic acid (CA) production by Streptomyces clavuligerus in a stirred-tank bioreactor. At 800 rpm and 0.5 vvm, the cultivations performed using RT-RT and EEUP impellers provided higher shear conditions and oxygen transfer rates than those observed with EEDP. These conditions resulted in higher clavulanic acid production using RT-RT (380.7 mg/L) and EEUP (453.3 mg/L) impellers, compared to EEDP (196.6 mg/L). Although the maximum CA concentration exhibited the same order of magnitude for RT-RT and EEUP impellers, the latter presented 40% of the specific power consumption (4.9 kW/m3) compared to the classical RT-RT (12.0 kW/m3). The specific energy for CA production ( E CA ), defined as the energy cost to produce 1 mg of CA, was 3.5 times lower using the EEUP impeller (1.91 kJ/mgCA) when compared to RT-RT (5.91 kJ/mgCA). Besides, the specific energy for O2 transfer ( E O 2 ), the energy required to transfer 1 mmol of O2, was 2.3 times lower comparing the EEUP impeller (3.28 kJ/mmolO2) to RT-RT (7.65 kJ/mmolO2). The results demonstrated the importance of choosing the most suitable impeller configuration in conventional bioreactors to manufacture bioproducts.

Global insight into the occurrence, treatment technologies and ecological risk of emerging contaminants in sanitary sewers: Effects of the SARS-CoV-2 coronavirus pandemic.

The SARS-CoV-2 pandemic caused changes in the consumption of prescribed/non-prescribed drugs and the population's habits, influencing the detection and concentration of emerging contaminants (ECs) in sanitary sewage and harming environmental and health risks. Therefore, the present work sought to discuss current literature data on the effects of the "COVID-19 pandemic factor" on the quality of raw sewage produced over a five-year period (2018-2019: pre-pandemic; 2020-2022: during the pandemic) and biological, physical, chemical and hybrid treatment technologies, influencing factors in the removal of ECs and potential ecological risks (RQs). Seven hundred thirty-one publications correlating sewage and COVID-19 were identified: 184 pre-pandemic and 547 during the pandemic. Eight classes and 37 ECs were detected in sewage between 2018 and 2022, with the "COVID-19 pandemic factor" promoting an increase in estrogens (+31,775 %), antibiotics (+19,544 %), antiepileptics and antipsychotics (+722 %), pesticides (+200 %), analgesics, anti-inflammatories and anticoagulants (+173 %), and stimulant medications (+157 %) in sanitary sewage. Among the treatment systems, aerated reactors integrated into biomembranes removed >90 % of cephalexin, clarithromycin, ibuprofen, estrone, and 17ß-estradiol. The absorption, adsorption, and biodegradation mechanisms of planted wetland systems contributed to better cost-benefit in reducing the polluting load of sewage ECs in the COVID-19 pandemic, individually or integrated into the WWTP. The COVID-19 pandemic factor increased the potential ecological risks (RQs) for aquatic organisms by 40 %, with emphasis on clarithromycin and sulfamethoxazole, which changed from negligible risk and low risk to (very) high risk and caffeine with RQ > 2500. Therefore, it is possible to suggest that the COVID-19 pandemic intensified physiological, metabolic, and physical changes to different organisms in aquatic biota by ECs during 2020 and 2022.

BioMINT: A Temporary Immersion System for Agave Micropropagation.

Agaves are cultivated in Mexico as a source of industrial products such as fibers, nutritional supplements, and alcoholic beverages. Due to the demand for plant material, its long-life cycle, and the need to avoid predation on its natural populations, in vitro micropropagation represents a good option for agaves. Plant tissue culture has been successfully used to micropropagate selected elite individuals from plants of various Agave species of economic interest. However, it is necessary to implement systems that lower production costs without losing the quality of the plantlets obtained. This chapter describes the BioMINT™ bioreactor as an alternative for the micropropagation of agaves in the different stages of the micropropagation process.

In Vitro Multiplication of Agave (A. marmorata and A. potatorum) by Temporary Immersion in SETIS™ Bioreactor.

In Mexico, wild agaves are important for the production of alcoholic beverages known as mezcal and pulque. However, the propagation of agave seeds and pups is not enough to satisfy the national demand. Temporary immersion systems represent an agave micropropagation alternative that reduces the labor force, increases development, and improves the quality of seedlings. The use of the SETIS™ bioreactor in A. marmorata and A. potatorum improves the multiplication rate and allows rooting. Additionally, this bioreactor reduces the culture time, labor force, and reagents needed while maintaining high survival rates during the acclimatization phase.

Alstroemeria Micropropagation in a RITA® Temporary Immersion System.

The use of temporary immersion systems (TIS) for plant micropropagation is an efficient technique for plant production, and we have applied it for the production of alstroemerias. This method involves the cultivation of explants such as rhizomes and axillary buds in a nutrient medium to stimulate shoot growth. TIS offer advantages such as accelerated multiplication processes, uniform production, and cost reduction. This process has shown promise in meeting the growing demand for alstroemeria plants in the market. This chapter describes a specific protocol for temporary immersion bioreactor micropropagation of the "Albatroz" cultivar, with the potential for large-scale automation.

Micropropagation of Encyclia cordigera (Kunth) Dressler in Ebb-and-Flow Bioreactor.

The use of new technologies for micropropagation such as temporary immersion systems (TISs) is important, because it reduces costs by 40% lowering labor, agar and containers. TISs are containers designed for large-scale, semiautomatic production of plants in a liquid medium, which has been used in propagation of commercial orchids. This tool has high potential for application in micropropagation of medicinal and endangered orchids for conservation and commercial purposes. In this chapter, we describe a detailed protocol for propagation and development of Encyclia cordigera to be used in research projects for small-scale production. This protocol comprises all steps from explant preparation to the establishment orchids plantlets.

Biooxidation of hydrogen sulfide to sulfur by moderate thermophilic acidophilic bacteria.

The copper industry utilizes significant amounts of sulfuric acid in its processes, generating sulfate as waste. While sulfate-reducing bacteria can remove sulfate, it produces hydrogen sulfide (H2S) as a byproduct. This study examined the capability of a consortium consisting of Sulfobacillus thermosulfidooxidans and Sulfobacillus acidophilus to partially oxidize H2S to S° at a temperature of 45 °C. A fixed-bed bioreactor, with glass rings as support material and sodium thiosulfate as a model electron donor, was inoculated with the consortium. Formation of biofilms was crucial to maintain the bioreactor's steady state, despite high flow rates. Afterward, the electron donor was changed to H2S. When the bioreactor was operated continuously and with high aeration, H2S was fully oxidized to SO42-. However, under conditions of low aeration and at a concentration of 0.26 g/L of H2S, the consortium was able to oxidize H2S to S° with a 13% yield. S° was discovered attached to the glass rings and jarosite. The results indicate that the consortium could oxidize H2S to S° with a 13% yield under low aeration and at a concentration of 0.26 g/L of H2S. The findings highlight the capability of a Sulfobacillus consortium to convert H2S into S°, providing a potential solution for addressing environmental and safety issues associated with sulfate waste generated by the mining industry.

Sf9 Cell Metabolism Throughout the Recombinant Baculovirus and Rabies Virus-Like Particles Production in Two Culture Systems.

This work aimed to assess the Sf9 cell metabolism during growth, and infection steps with recombinant baculovirus bearing rabies virus proteins, to finally obtain rabies VLP in two culture systems: Schott flask (SF) and stirred tank reactor (STR). Eight assays were performed in SF and STR (four assays in each system) using serum-free SF900 III culture medium. Two non-infection growth kinetics assays and six recombinant baculovirus infection assays. The infection runs were carried out at 0.1 pfu/cell multiplicity of infection (MOI) for single baculovirus bearing rabies glycoprotein (BVG) and matrix protein (BVM) and a coinfection with both baculoviruses at MOI of 3 and 2 pfu/cell for BVG and BVM, respectively. The SF assays were done in triplicate. The glucose, glutamine, glutamate, lactate, and ammonium uptake or release specific rates were quantified over the exponential growth phase and infection stage. The highest uptake specific rate was observed for glucose (42.5 × 10-12 mmol cell/h) in SF and for glutamine (30.8 × 10-12 mmol/cell/h) in STR, in the exponential growth phases. A wave pattern was observed for assessed analytes throughout the infection phase and the glucose had the highest wave amplitude within the 10-10 mmol cell/h order. This alternative uptake and release behavior is in harmony with the lytic cycle of baculovirus in insect cells. The virus propagation and VLP generation were not limited by glucose, glutamine, and glutamate, neither by the toxicity of lactate nor ammonium under the conditions appraised in this work. The findings from this work can be useful to set baculovirus infection processes at high cell density to improve rabies VLP yield, purity, and productivity.