Plant Micropropagation and Temporary Immersion Systems.

Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.

Bioremediation strategies against pesticides: An overview of current knowledge and innovations.

Pesticides pose significant risks to both human health, such as cancer, neurological disorders, and endocrine disruption, and ecosystems, through the destruction of beneficial insects, contamination of soil and water, and impact on non-target species. In the face of escalating pesticide pollution, there is an urgent need for multifaceted approaches to address the issue. Bioremediation emerges as a potent tool in the environmental pollution mitigation arsenal. Ideally aiming for the complete decomposition of pesticides into harmless molecules, bioremediation encompasses diverse approaches - from bioabsorption, bioadsorption, and biotransformation using enzymes and nanoenzymes to comprehensive degradation facilitated by microorganisms such as bacteria, fungi, macro- and microalgae, or phytoremediation. Exploring nature's biodiversity offers a promising avenue to find solutions to this pressing human-induced problem. The acceleration of biodegradation necessitates identifying and developing efficient organisms, achieved through bioprospection and targeted modifications. Specific strategies to enhance process efficiency and throughput include optimizing biomass production, strategic inoculation in diverse environments, and employing bioreactor systems for processing heavily contaminated waters or soils. This comprehensive review presents various bioremediation approaches, emphasizing the importance of microorganisms' exploration and new technologies development, including current innovations and patents to effectively combat pesticide pollution. Furthermore, challenges regarding the effective implementation of these technologies are also addressed.

Production of biologically active human basic fibroblast growth factor (hFGFb) using Nicotiana tabacum transplastomic plants.

MAIN CONCLUSION: We generated transplastomic tobacco lines that stably express a human Basic Fibroblast Growth Factor (hFGFb) in their chloroplasts stroma and purified a biologically active recombinant hFGFb. MAIN: The use of plants as biofactories presents as an attractive technology with the potential to efficiently produce high-value human recombinant proteins in a cost-effective manner. Plastid genome transformation stands out for its possibility to accumulate recombinant proteins at elevated levels. Of particular interest are recombinant growth factors, given their applications in animal cell culture and regenerative medicine. In this study, we produced recombinant human Fibroblast Growth Factor (rhFGFb), a crucial protein required for animal cell culture, in tobacco chloroplasts. We successfully generated two independent transplastomic lines that are homoplasmic and accumulate rhFGFb in their leaves. Furthermore, the produced rhFGFb demonstrated its biological activity by inducing proliferation in HEK293T cell lines. These results collectively underscore plastid genome transformation as a promising plant-based bioreactor for rhFGFb production.

Types of Temporary Immersion Systems Used in Commercial Plant Micropropagation.

Technological innovation in the design and manufacture of temporary immersion systems (TIS) has increased in the past decade. Innovations have involved the size, fitting, and replacement of components, as well as manufacturing materials. Air replacement by compressor has also been substituted by air replacement by preset tilting/rotation of culture bottles. This design modification aims to increase the biological yield (number of shoots) produced in these bottles and reduce manufacturing costs. However, the operative principle has remained unchanged through time: promote an environment where explant immersions in the culture medium are programmable. The changes in the TIS design involve advantages and disadvantages, generating the efficiency of one type over another. However, validation to identify the most effective type of TIS should be carried out for each plant species. This chapter lists the different types of temporary immersion available on the market, emphasizing the advantages and disadvantages of each when used for plant micropropagation.

Temporary Immersion Systems in Plant Micropropagation.

Temporary immersion systems (TIS) are technological tools that support plant micropropagation. Given their high efficiency in the in vitro propagation of shoots, a current goal is to update the protocols addressing micropropagation in semisolid culture systems to protocols involving TIS. To this end, different parameters have been evaluated, including TIS types and designs, immersion times, immersion frequencies, and volume of medium per explant, among other characteristics. This has resulted in the improved production of propagules of plants of economic interest and the production of physiologically upgraded plants with high percent survival during acclimatization. TIS are specialized culture flasks that provide countless advantages during the commercial micropropagation of plants.

Conclusions and Perspectives on Plant Micropropagation in Temporary Immersion Systems.

In vitro propagation protocols that include temporary immersion systems are available for the most economically important plant species. However, these have not been established yet for multiple species. Having protocols validated by the scientific community guarantees the success of the mass production of commercial propagules. Besides, adequate TIS parameters should be established for each plant species to improve the efficiency of micropropagation processes. This book compiles basic and applied aspects of temporal immersion systems used for in vitro plant micropropagation, along with several detailed protocols already established, which may be used as a guide by those interested in this technique, including laboratory technicians, scientists, and other professionals.

Secondary Embryogenesis of Linaloe in Temporary Immersion Bioreactor-Type RITA®.

The linaloe [Bursera linanoe (La Llave) Rzed, Calderon and Medina] is an endemic species of Mexico, representative of the low deciduous forest of the states of Guerrero, Puebla, Morelos, and Oaxaca, and has been of great economic importance for the people, mainly for the artisanal use of its aromatic wood that is used to make boxes, trunks, and furniture that are manufactured in Olinala, Guerrero, Mexico; and industrial, thanks to the fine aroma of its essential oil (linalool), which is used in the manufacture of perfumes and pharmaceuticals. Overexploitation has endangered the species in recent years, and propagation by seed and/or cuttings has produced very poor results compared to those obtained with other recalcitrant Bursera species. The protection of endangered species makes urgent the need to propose new alternatives for its propagation. Somatic embryogenesis is a reliable and feasible technique, including induction, maintenance, multiplication, and maturation of embryos, often in semisolid culture media; however, the recent use of liquid media has allowed semi-automation in temporary immersion bioreactors, for example, the RITA® system, which favors both the multiplication rate and the final conversion to seedlings.

Sustainable production of bioemulsifiers, a critical overview from microorganisms to promising applications.

Microbial bioemulsifiers are molecules of amphiphilic nature and high molecular weight that are efficient in emulsifying two immiscible phases such as water and oil. These molecules are less effective in reducing surface tension and are synthesized by bacteria, yeast and filamentous fungi. Unlike synthetic emulsifiers, microbial bioemulsifiers have unique advantages such as biocompatibility, non-toxicity, biodegradability, efficiency at low concentrations and high selectivity under different conditions of pH, temperature and salinity. The adoption of microbial bioemulsifiers as alternatives to their synthetic counterparts has been growing in ongoing research. This article analyzes the production of microbial-based emulsifiers, the raw materials and fermentation processes used, as well as the scale-up and commercial applications of some of these biomolecules. The current trend of incorporating natural compounds into industrial formulations indicates that the search for new bioemulsifiers will continue to increase, with emphasis on performance improvement and economically viable processes.

Indicators for the sustainability assessment of MBR technologies for wastewater reuse in Chile: The good, the bad, and the ugly.

While Chile faces a mega-drought, wastewater reuse emerges as an alternative solution. In this study we develop a set of indicators for the comprehensive sustainability assessment for the application of advanced wastewater treatment technologies (e.g., MBRs) in a wastewater reuse project in Chile. The methodology is based on the Integrative Concept of Sustainable Development (ICoS) framework. A critical analysis of the set of indicators is presented in terms of the benefits (The Good), the difficulties (The Bad), and the barriers (the Ugly) for their development and potential application. The characterization of the environmental benefits constitutes the useful aspects (e.g., recovery of nutrients, energy, and water). Difficulties include economic aspects (e.g., continuous monitoring of emerging contaminants) and public acceptance. Political and administrative aspects were found to be the main barrier, including water rights in Chile and the absence of a clear regulatory framework for wastewater reuse. To our knowledge, this study is the first to present a detailed methodology for developing indicators for membrane-based water reuse projects in Chile. The steps to develop the indicators are: •Identification of the study zone or case study, characterization of treatment technology.•Identification and formulation of indicators for the specific case study, based on the ICoS framework.•Verification of the relevance of indicators for the case study according to data availability and expert reviews.

Biofouling in Membrane Bioreactors-Mitigation and Current Status: a Review.

Biological fouling as termed biofouling is caused by varied living organisms and is difficult to eliminate from the environment thus becoming a major issue during membrane bioreactors. Biofouling in membrane bioreactors (MBRs) is a crucial problem in increasing liquid pressure due to reduced pore diameter, clogging of the membrane pores, and alteration of the chemical composition of the water which greatly limits the growth of MBRs. Thus, membrane biofouling and/or microbial biofilms is a hot research topic to improve the market competitiveness of the MBR technology. Though several antibiofouling strategies (addition of bioflocculant or sponge into MBRs) came to light, biological approaches are sustainable and more practicable. Among the biological approaches, quorum sensing-based biofouling control (so-called quorum quenching) is an interesting and promising tool in combating biofouling issues in the MBRs. Several review articles have been published in the area of membrane biofouling and mitigation approaches. However, there is no single source of information about biofouling and/or biofilm formation in different environmental settings and respective problems, antibiofilm strategies and current status, quorum quenching, and its futurity. Thus, the objectives of the present review were to provide latest insights on mechanism of membrane biofouling, quorum sensing molecules, biofilm-associated problems in different environmental setting and antibiofilm strategies, special emphasis on quorum quenching, and its futurity in the biofilm/biofouling control. We believe that these insights greatly help in the better understanding of biofouling and aid in the development of sustainable antibiofouling strategies.

Immobilization of the metagenomic lipase, LipG9, on porous pellets of poly-hydroxybutyrate produced by the double emulsion solvent evaporation technique.

This work aimed to produce porous poly-hydroxybutyrate (PHB) pellets in order to evaluate the pellets as a support for immobilization of the metagenomic lipase, LipG9. Four types of pelletized PHB particles with different morphological characteristics were obtained using the double emulsion and solvent evaporation technique (DESE). The micropores of these PHB pellets had similar average diameters (about 3 nm), but the pellets had different specific surface areas: 11.7 m2 g-1 for the PHB powder, 8.4 m2  g-1 for the control pellets (Ø < 0.5 mm, produced without the pore forming agent), 10.0 m2  g-1 for the small pellets (Ø < 0.5 mm), 9.5 m2  g-1 for the medium pellets (0.5 < Ø < 0.8 mm) and 8.4 m2  g-1 for the large pellets (Ø > 1.4 mm). Purified LipG9 was immobilized by adsorption on these pellets, and the results were compared with those obtained with PHB powder. The highest immobilization yield (83%) was obtained for the medium PHB pellets, followed by large (76%) and small (55%) PHB pellets. The activity of LipG9 immobilized on the pellets, for the synthesis of ethyl oleate in n-hexane, was highest for the medium pellets (22 U g-1 ). The immobilization yield was high for PHB powder (99%) but the esterification activity was slightly lower (20 U g-1 ). These results show that pelletized PHB beads can be used for the immobilization of lipases, with the advantage that pelletized PHB will perform better than PHB powder in large-scale enzyme bioreactors.

Flow cytometry: a tool for understanding the behaviour of polyhydroxyalkanoate accumulators.

The use of mixed microbial cultures (MMCs) is seen as an attractive strategy for polyhydroxyalkanoate (PHA) production. In order to optimize the MMC-PHA production process, tools are required to improve our understanding of the physiological state of the PHA-storing microorganisms within the MMC. In the present study, we explored the use of flow cytometry to analyse the metabolic state and polyhydroxybutyrate (PHB) content of the microorganisms from an MMC-PHA production process. A sequencing batch reactor under a feast and famine regime was used to enrich an MMC with PHB-storing microorganisms. Interestingly, once the PHB-storing microorganisms are selected, the level of PHB accumulation depends largely on the metabolic state of these microorganisms and not exclusively on the consortium composition. These results demonstrate that flow cytometry is a powerful tool to help to understand the PHA storage response of an MMC-PHA production process. KEY POINTS: • Flow cytometry allows to measure PHB content and metabolic activity over time. • Microorganisms showing high PHB content also have high metabolic activity. • PHB producers with low metabolic activity show low PHB content.

Comparison of two acidophilic sulfidogenic consortia for the treatment of acidic mine water.

Sulfate-reducing bioreactors are a biotechnological alternative for the treatment of acid mine drainage (AMD). In this study, two separate bioreactors with pH and temperature-controlled (Bio I and II) were operated with two different acidophilic microbial consortia to determine their efficiencies in sulfate removal from a synthetic acidic mine water. The bioreactors were operated for 302 days in continuous flow mode under the same parameters: fed with a sulfate solution of ∼30 mM with a pH of 2.5, the temperature at 30°C, stirred gently at 40 rpm and using a continuous stream of nitrogen to help remove the H2S produced in the bioreactor. The glycerol consumption, acetate production, and sulfate removal were monitored throughout the course of the experiment. The community composition and potential metabolic functional groups were analyzed via 16S rRNA partial gene sequencing. Bio I consortium reduced the sulfate, achieving a range of sulfate concentration from 4.7 to 19 mM in the effluent liquor. The removal of sulfate in Bio II was between 5.6 and 18 mM. Both bioreactors' communities showed the presence of the genus De sulfosporosinus as the main sulfate-reducing bacteria (SRB). Despite differences in microbial composition, both bioreactors have similar potential metabolism, with a higher percentage of microorganisms that can use sulfate in respiration. Overall, both bioreactors showed similar performance in treating acidic mine water containing mostly sulfate using two different acidophilic sulfidogenic consortia obtained from different global locations.

Comparison of Different Semi-Automated Bioreactors for In Vitro Propagation of Taro (Colocasia esculenta L. Schott).

Taro is important for its nutritional content, medicinal use, and bioethanol production. The aim of the present study was to compare different semi-automated bioreactors (SABs) during in vitro multiplication of C. esculenta. The SABs used were temporary immersion bioreactors (TIBs), SETIS™ bioreactors and ebb-and-flow bioreactors; semi-solid culture medium was used as a control treatment. At 30 d of culture, different developmental variables, determination of chlorophyll, stomatal content, and survival percentage during acclimatization were evaluated. SABs increased the shoot multiplication rate relative to the semi-solid medium; however, the SETIS™ bioreactor showed the highest shoot production, with 36 shoots per explant, and the highest chlorophyll content. The stomatal index was higher in the semi-solid medium compared to the SABs, while the percentage of closed stomata was higher in the SABs than in the semi-solid culture medium. The survival rate during acclimatization showed no differences among the culture systems assessed, obtaining survival rates higher than 99%. In conclusion, the SETIS™ bioreactor showed the highest multiplication rate; however, other bioreactor alternatives are available for semi-automation and cost reduction for micropropagation of C. esculenta.

A survey on experiences in leachate treatment: Common practices, differences worldwide and future perspectives.

The necessity for landfill leachate treatment is a requisite to reduce the environmental impact related to municipal solid waste landfills and different aspects must be considered while deciding for an appropriate treatment process. For example, it was demonstrated that the landfill leachate stabilization in tropical regions is achieved right after its first year of operation, requiring technologies capable of treating leachates of a higher recalcitrant character if compared to those leachates from temperate regions and same landfill age. In view of its complexity and variability, stand-alone processes (either biological or physicochemical) are often ineffective in attaining the threshold values for its discharge in receiving bodies. Due to that fact, full-scale facilities have adopted integrated routes, harvesting the benefits of both biological and physicochemical processes. The implementation of membrane bioreactors followed by polishing membrane separation process (nanofiltration and reverse osmosis) seems to be a trend in leachate treatment by full-scale treatment plants. This technology is widely employed in China, European countries, and tropical countries as Brazil, generally with a treatment cost lower than the costs related to its disposal in domestic effluent collection systems. From the technologies already employed by full-scale facilities, four integrated routes were proposed for a sensitive analysis considering the treatment of a landfill leachate of different physicochemical characteristics. From all routes, those employing the membrane separation process as a polishing step had a better efficacy in attaining the threshold values for leachate disposal, being that an interesting alternative for leachate polishing by full-scale facilities.

Use of equivalent circuit analysis and Cole-Cole model in evaluation of bioreactor operating conditions for biomass monitoring by impedance spectroscopy.

The most important parameter in bioprocesses is biomass, where not only the quantity produced in a culture but also the behavior that is presented are important concerns. It is clear that conditions of operation in a bioreactor affect biomass production, but how operation conditions affect the measurement of biomass on-line is of special interest. We studied the effect of bioreactor operating condition variations on model parameters using impedance spectroscopy for biomass monitoring. The model parameters analyzed were capacitance, resistance, alpha (α), conductivity delta (∆σ) and critical frequency (fc). These model parameters were obtained by fitting data from impedance measurements to an equivalent circuit model and Cole-Cole conductivity model. The effect of operating conditions on the medium with no cells was estimated by the percentage of change in each model parameter. The operating conditions with the most significant percentage of change were determined, by comparing to the percentage of change of the same model parameters obtained, during a fermentation of Bacillus thuringiensis as a cellular model. Equivalent circuit parameters were mainly affected by variation in pH, temperature and aeration, whereas Cole-Cole parameters were affected by variation in agitation, aeration, temperature and pH. Therefore, any variation in these operating conditions (within the test interval) during a fermentation may generate changes in monitoring parameters, which will not be a direct consequence of any change in the properties of the biomass.

Influence of yeast inoculation on the quality of fermented coffee (Coffea arabica var. Mundo Novo) processed by natural and pulped natural processes.

Starter cultures during fermentation of Coffea arabica var. Mundo Novo processed in open stainless-steel vessels by natural and pulped natural methods were studied. The yeasts Meyerozyma caribbica (CCMA 0198), Saccharomyces cerevisiae (CCMA 0543), Candida parapsilosis (CCMA 0544), and Torulaspora delbrueckii (CCMA 0684) were inoculated separately in two different coffee processes: natural and pulped natural. The qPCR (real-time quantitative polymerase chain reaction) was used as a culture-independent method to monitor the inoculum's permanence. Changes in microbial metabolites (organic acids and volatile) production were evaluated by high-performance liquid chromatography (HPLC) and gas chromatograph-mass spectrometry (GC-MS), respectively. The sensory analysis was assessed in roasted beans. The fermentation lasted 27 h, and the coffee temperature ranged from 16.5 to 24.0 °C. The starter culture population was dominant throughout fermentation. S. cerevisiae (CCMA 0543) and T. delbrueckii (CCMA 0684) presented a higher population in natural processing. However, in pulped natural processing, M. caribbica (CCMA 0198) and C. parapsilosis (CCMA 0544) were the dominant populations. Citric, malic, and succinic acids were naturally present in coffee. Lactic, isobutyric, and isovaleric acids were detected at the end of the fermentation in different treatments. Lactic acid was detected in samples at the end of fermentation in Control and CCMA 0198 treatment. NAT coffee inoculated with CCMA 0684 presented isobutyric acid and isovaleric acid concentrations. Volatile compounds, such as 2,6-diethylpyrazine was detected in treatments inoculated with yeasts, but not in Controls. 2-acetoxymethylfuran was only detected in samples inoculated with CCMA 0198 from both NAT and PN methods. Samples fermented with S. cerevisiae (CCMA 0543) presented the highest sensorial scores in both processing (84.75 and 84.92). The inoculated coffee beans showed higher scores of sweetness, long aftertaste, and greater complexity. The starter cultures influenced the sensorial profiles through the synthesis of specific volatile constituents. However, considering all parameters analyzed, S. cerevisiae (CCMA 0543) would be the most suitable yeast for the var. Mundo Novo processed by both fermentation methods.

Novel stainless steel tanks enhances coffee fermentation quality.

Fermenting in bioreactors can improve coffee quality, standardize the fermentation process, and generate specialty coffees. This work aimed to evaluate novel stainless steel bioreactors with inoculated and non-inoculated coffees processed via natural and pulped natural. Yeast and bacteria populations were evaluated and grown on Yeast Extract Peptone Glucose; De Man, Rogosa, and Sharpe; and Nutrient agar media. Volatile compounds from roasted beans were analyzed in a Gas Chromatography-Mass Spectrometry equipment, and the sensory perception was evaluated through a cup test. The mesophilic bacteria population was statistically significant in pulped natural coffee compared to yeast and lactic acid bacteria. Furans had the highest concentration among the chemical groups. Beverage inoculated with CCMA 0535 presented the highest SCA score. Prune, peach, and floral attributes were only perceived in Nat CCMA 0535. The sensory perception indicated that the inoculated yeasts modified the flavor attributes, enhanced the quality, and increase their SCA scores.

A metagenomic assessment of microbial communities in anaerobic bioreactors and sediments: Taxonomic and functional relationships.

The present study used metagenomic sequencing, metagenome assembly and physical-chemical analysis to describe taxonomically and functionally 3 anaerobic bioreactors treating manure (LI), brewery (BR) and cornmeal (CO) wastes, and an anaerobic estuarine sediment (ES). Proteobacteria, Firmicutes, Euryarchaeota and Bacteroidetes were the most abundant Phyla in all metagenomes. A bacteria/archaea ratio of 3.4 was found in the industrial full-scale anaerobic bioreactors BR and CO, while ratios greater than 10 were found for LI and ES. Canonical correspondence analysis showed that environmental variables such as chemical oxygen demand, lipid content, and ammonium nitrogen influenced the ordination of taxonomic groups. Mesotoga prima was linked to high-temperature conditions, particularly in the BR bioreactor, along with the presence of heat shock proteins genes. Likewise, the hydrogenotrophic methanogen, Methanoregula formicica, was associated with high ammonium concentration in LI bioreactor. The interactions of microbes with specific methanogenic pathways were identified using Clusters of Orthologous Groups (COG) functions, while metagenome-assembled genomes (MAGs) further confirmed relationships between taxa and functions. Our results provide valuable information to understand microbial processes in anaerobic environments.

Novel external-loop-airlift milliliter scale bioreactors for cell growth studies: Low cost design, CFD analysis and experimental characterization.

Many researchers have limited access to fully equipped laboratory-scale batch bioreactors and chemostats due to their relatively high cost. This becomes particularly prohibitive when multiple replicas of the same experiment are required, but not enough bioreactors are available to operate simultaneously. Additionally, experiments using shaken flasks are common but show significant limitations in terms of maintaining homogeneous conditions in liquid cultures or installing instrumentation for monitoring. Here, we proposed to tackle this significant hurdle by providing a route to make available the manufacture of low-cost, milliliter-scale bioreactors. This approach seems plausible for enabling proof-of-concept experiments before moving to a larger scale without significant investments. The conceptually designed systems were based on external-loop bioreactors due to their flexibility, simplicity, and ease of assembling and testing. Designs were initially evaluated in silico with the aid of COMSOL Multiphysics. The successfully evaluated systems were then constructed via additive manufacturing and assembled for hydrodynamics testing via tracer methods. This was enabled by a newly home-made optical absorbance sensor (OAS) for in-line and real-time measurements. Both the in silico and experimental results indicated close to ideal mixing conditions and low shear stress. Cell growth curves were prepared by culturing Escherichia coli and following its cell density in real-time. Our cell growth rate and maximum cell density were similar to those previously obtained in closely related systems. Therefore, the proposed bioreactors are an affordable alternative for batch and continuous cell growth studies rapidly and inexpensively.