Challenge In Biotechnology Research Articles

Polyhydroxyalkanoate (PHA) Biopolyesters - Emerging and Major Products of Industrial Biotechnology

Abstract Background: Industrial Biotechnology (“White Biotechnology”) is the large-scale production of materials and chemicals using renewable raw materials along with biocatalysts like enzymes derived from microorganisms or by using microorganisms themselves (“whole cell biocatalysis”). While the production of ethanol has existed for several millennia and can be considered a product of Industrial Biotechnology, the application of complex and engineered biocatalysts to produce industrial scale products with acceptable economics is only a few decades old. Bioethanol as fuel, lactic acid as food and PolyHydroxyAlkanoates (PHA) as a processible material are some examples of products derived from Industrial Biotechnology. Purpose and Scope: Industrial Biotechnology is the sector of biotechnology that holds the most promise in reducing our dependence on fossil fuels and mitigating environmental degradation caused by pollution, since all products that are made today from fossil carbon feedstocks could be manufactured using Industrial Biotechnology – renewable carbon feedstocks and biocatalysts. To match the economics of fossil-based bulk products, Industrial Biotechnology-based processes must be sufficiently robust. This aspect continues to evolve with increased technological capabilities to engineer biocatalysts (including microorganisms) and the decreasing relative price difference between renewable and fossil carbon feedstocks. While there have been major successes in manufacturing products from Industrial Biotechnology, challenges exist, although its promise is real. Here, PHA biopolymers are a class of product that is fulfilling this promise. Summary and Conclusion: The authors illustrate the benefits and challenges of Industrial Biotechnology, the circularity and sustainability of such processes, its role in reducing supply chain issues, and alleviating societal problems like poverty and hunger. With increasing awareness among the general public and policy makers of the dangers posed by climate change, pollution and persistent societal issues, Industrial Biotechnology holds the promise of solving these major problems and is poised for a transformative upswing in the manufacture of bulk chemicals and materials from renewable feedstocks and biocatalysts.

The living interface between synthetic biology and biomaterial design.

Recent far-reaching advances in synthetic biology have yielded exciting tools for the creation of new materials. Conversely, advances in the fundamental understanding of soft-condensed matter, polymers and biomaterials offer new avenues to extend the reach of synthetic biology. The broad and exciting range of possible applications have substantial implications to address grand challenges in health, biotechnology and sustainability. Despite the potentially transformative impact that lies at the interface of synthetic biology and biomaterials, the two fields have, so far, progressed mostly separately. This Perspective provides a review of recent key advances in these two fields, and a roadmap for collaboration at the interface between the two communities. We highlight the near-term applications of this interface to the development of hierarchically structured biomaterials, from bioinspired building blocks to 'living' materials that sense and respond based on the reciprocal interactions between materials and embedded cells.

THE ROLE OF BIOTECHNOLOGY IN PHYTOMEDICINE

This paper is a review of the application, relevance, advances and achievements of biotechnology in phytomedicine research and development. It also gave insight on the specific role of various biotechnological tools such as micropropagation, somatic embryogenesis, callus mediated organogenesis, cryopreservation, the bioreactor system, combinatorial biosynthesis and genetic transformation in selecting, multiplying and conserving critical genotypes of medicinal plants. Also highlighted are the opportunities and challenges of biotechnology in phtytomedicine.

Enzyme Immobilization and Co-Immobilization: Main Framework, Advances and Some Applications

Enzymes are outstanding (bio)catalysts, not solely on account of their ability to increase reaction rates by up to several orders of magnitude but also for the high degree of substrate specificity, regiospecificity and stereospecificity. The use and development of enzymes as robust biocatalysts is one of the main challenges in biotechnology. However, despite the high specificities and turnover of enzymes, there are also drawbacks. At the industrial level, these drawbacks are typically overcome by resorting to immobilized enzymes to enhance stability. Immobilization of biocatalysts allows their reuse, increases stability, facilitates process control, eases product recovery, and enhances product yield and quality. This is especially important for expensive enzymes, for those obtained in low fermentation yield and with relatively low activity. This review provides an integrated perspective on (multi)enzyme immobilization that abridges a critical evaluation of immobilization methods and carriers, biocatalyst metrics, impact of key carrier features on biocatalyst performance, trends towards miniaturization and detailed illustrative examples that are representative of biocatalytic applications promoting sustainability.

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Biotransformation of Tetracyclines by Fungi: Challenges and Future Research Perspectives.

Tetracycline antibiotics are used worldwide in human and veterinary medicine. On the basis of low metabolization and through organic fertilizers, tetracyclines enter the environment in a biologically active form. This can have toxic effects on microbial communities and promote the selection of resistant strains. The use of fungi could be a promising approach to deactivate tetracyclines by degradation or derivatization as a result of their particular enzyme endowment. Here, we highlight the current analytical and biotechnological challenges associated with the bioconversion of tetracyclines by fungi and propose research approaches to advance the technology for wastewater and manure treatment.

White biotechnology and the production of bio-products

White biotechnology uses enzymes and microorganisms to produce value-added chemicals from renewable sources. White biotechnology provides valuable components for the food, pharmaceutical, agricultural sectors as well as other industries. Metabolic diversity in fungi, yeast, and bacteria can be exploited to produce food additives and other industrial products. This is an interesting topic for those interested in screening and metabolic testing of microorganisms, industrial biotechnology, fermentation technology, and the biological products research community. The use of microbial-derived compounds has a long history in the food industry, and compounds such as flavorings, essential amino acids, poly-unsaturated fatty acids, organic acids, gelling, etc. can be obtained from microbial sources. Also, the role of microbes in human health and well-being cannot be ignored. Microbes produce primary metabolites such as vitamins, nucleotides, and amino acids, as well as secondary metabolites. These secondary metabolites are used to make many drugs. In agriculture, microbes are also used to make fertilizers and biological pesticides. This paper reviews the types of bio-products obtained through biotechnology and the barriers and challenges of white biotechnology.

Bioethics and Human Rights: The Genomic Editing Technique Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR-CAS9) and the Contemporary Biotechnological Challenges

This article deals with biotechnological advances, especially in the field of editing and genetic engineering. In contemporary times, the possibility of manipulating the human genetic heritage and creating perfect organisms is no longer a simple theoretical hypothesis, notably from the development of the genomic editing technique Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR-CAS9), a system created from immunological mechanisms of bacteria that allows the modification of the human genome. In this sense, supported by the matrix of global bioethics, it aims to investigate the immediate and mediate implications of human genetic editing in the contemporary world, through an exploratory methodology and literature review, highlighting divergent positions in Brazil and abroad. In the end, it is possible to conclude that the establishment of limits to biotechnological advances is a measure that is imposed, in respect of the determining border between techno science’s and human rights, not disregarding, however, the benefits of biotechnology, but considering ethical, legal, humanitarian and social criteria.

Production and Properties of Microbial Polyhydroxyalkanoates Synthesized from Hydrolysates of Jerusalem Artichoke Tubers and Vegetative Biomass.

One of the major challenges in PHA biotechnology is optimization of biotechnological processes of the entire synthesis, mainly by using new inexpensive carbon substrates. A promising substrate for PHA synthesis may be the sugars extracted from the Jerusalem artichoke. In the present study, hydrolysates of Jerusalem artichoke (JA) tubers and vegetative biomass were produced and used as carbon substrate for PHA synthesis. The hydrolysis procedure (the combination of aqueous extraction and acid hydrolysis, process temperature and duration) influenced the content of reducing substances (RS), monosaccharide contents, and the fructose/glucose ratio. All types of hydrolysates tested as substrates for cultivation of three strains—C. necator B-10646 and R. eutropha B 5786 and B 8562—were suitable for PHA synthesis, producing different biomass concentrations and polymer contents. The most productive process, conducted in 12-L fermenters, was achieved on hydrolysates of JA tubers (X = 66.9 g/L, 82% PHA) and vegetative biomass (55.1 g/L and 62% PHA) produced by aqueous extraction of sugars at 80 °C followed by acid hydrolysis at 60 °C, using the most productive strain, C. necator B-10646. The effects of JA hydrolysates on physicochemical properties of PHAs were studied for the first time. P(3HB) specimens synthesized from the JA hydrolysates, regardless of the source (tubers or vegetative biomass), hydrolysis conditions, and PHA producing strain employed, exhibited the 100–120 °C difference between the Tmelt and Tdegr, prevailing of the crystalline phase over the amorphous one (Cx between 69 and 75%), and variations in weight average molecular weight (409–480) kDa. Supplementation of the culture medium of C. necator B-10646 grown on JA hydrolysates with potassium valerate and ε-caprolactone resulted in the synthesis of P(3HB-co-3HV) and P(3HB-co-4HB) copolymers that had decreased degrees of crystallinity and molecular weights, which influenced the porosity and surface roughness of polymer films prepared from them. The study shows that JA hydrolysates used as carbon source enabled productive synthesis of PHAs, comparable to synthesis from pure sugars. The next step is to scale up PHA synthesis from JA hydrolysates and conduct the feasibility study. The present study contributes to the solution of the critical problem of PHA biotechnology—finding widely available and inexpensive substrates.

Antibacterial Activity of Microalgae Dunaliella salina, Tetraselmis chuii and Isochrysis galbana Against Aquatic Pathogens

Recently, expanded consideration has been paid to the business and potentiality of microalgae. Some microalgae are at present being studied for their capacity to find important metabolites for the drug industry or aquacultural applications. Concerning these biotechnological challenges, there is a consistent exertion accommodated in both finding and taking advantage of new microalgal assets and fostering their putative business results or modern valorizations. The crude methanol extracts of three microalgae, Dunaliella salina, Tetraselmis chuii, and Isochrysis galbana, have been investigated for antibacterial activity using the disk diffusion method against aquatic pathogens of fish, shrimp, and shellfish e.g. Pseudomonas fluorescence and Vibrio harveyi. This research aimed to analyze the antibacterial activity of crude extract of these microalgae against aquatic pathogens. The disk diffusion method was used to investigate the antibacterial activity. The result showed that only T. chuii has the inhibition zone for both tested bacteria P. fluorescence and V. harveyi with the inhibition zone of 3.0 ± 0.6 mm on 100 µg. g–1 of extract concentration and 4.20 ± 1.1 mm against P. fluorescence on 10000 µg. g–1 of extract concentration. While D. salina and I. galbana have inhibition zone only for V. harveyi with the inhibition zone of 4.4 ± 0.6 mm and 3.2 ± 0.7 mm on 10000 µg. g–1 of extract concentration. These three microalgae may have potential use in aquatic pathogens as antimicrobial agents. It would be possible to develop biologically active compounds of microalgae as a functional feed for aquaculture.

Bacterial Outer Membrane Vesicles as Antibiotic Delivery Vehicles.

Bacterial outer membrane vesicles (OMVs) are nanometer-scale, spherical vehicles released by Gram-negative bacteria into their surroundings throughout growth. These OMVs have been demonstrated to play key roles in pathogenesis by delivering certain biomolecules to host cells, including toxins and other virulence factors. In addition, this biomolecular delivery function enables OMVs to facilitate intra-bacterial communication processes, such as quorum sensing and horizontal gene transfer. The unique ability of OMVs to deliver large biomolecules across the complex Gram-negative cell envelope has inspired the use of OMVs as antibiotic delivery vehicles to overcome transport limitations. In this review, we describe the advantages, applications, and biotechnological challenges of using OMVs as antibiotic delivery vehicles, studying both natural and engineered antibiotic applications of OMVs. We argue that OMVs hold great promise as antibiotic delivery vehicles, an urgently needed application to combat the growing threat of antibiotic resistance.

Bridging the gap between microbial limits and extremes in space: space microbial biotechnology in the next 15 years.

SummaryThe establishment of a permanent human settlement in space is one of humanity’s ambitions. To achieve this, microorganisms will be used to carry out many functions such as recycling, food and pharmaceutical production, mining and other processes. However, the physical and chemical extremes in all locations beyond Earth exceed known growth limits of microbial life. Making microbes more tolerant of a greater range of extraterrestrial extremes will not produce organisms that can grow in unmodified extraterrestrial environments since in many of them not even liquid water can exist. However, by narrowing the gap, the engineering demands on bioindustrial processes can be reduced and greater robustness can be incorporated into the biological component. I identify and describe these required microbial biotechnological modifications and speculate on long‐term possibilities such as microbial biotechnology on Saturn’s moon Titan to support a human presence in the outer Solar System and bioprocessing of asteroids. A challenge for space microbial biotechnology in the coming decades is to narrow the microbial gap by systemically identifying the genes required to do this and incorporating them into microbial systems that can be used to carry out bioindustrial processes of interest.

Fish farm effluent as a nutrient source for algae biomass cultivation

One of the challenges of microalgae biotechnology is the cost of growth media nutrients, with microalgae consuming enormous quantities of fertilisers, more than other oil crops. The traditional use of synthetic fertilisers in mass cultivation of microalgae is associated with rising prices of crude oil and competition from traditional agriculture. The fact that fish farm wastewater (FFW) nutrients are released in the form preferred by microalgae (NH3 for nitrogen and PO4-3 for phosphate), and the ability of microalgae to use nitrogen from different sources, can be exploited by using fish farm effluent rich in nutrients (nitrogen and phosphorus) in the cultivation of cheaper microalgae biomass for production of biodiesel. The cultivation of algae biomass in FFW will also serve as wastewater treatment. We reviewed the benefits and potential of fish effluent in algae cultivation for the production of biodiesel. Microalgae can utilise nutrients in FFW for different applications desirable for the production of biomass, including the accumulation of lipids, and produce a fuel with desirable properties. Also, treating wastewater and reducing demand for fresh water are advantageous. The high lipid content and comparable biodiesel properties of Chlorella sorokiniana and Scenedesmus obliquus make both species viable for FFW cultivation for biodiesel production.
 Significance:
 
 The cost associated with microalgae growth media nutrients can be saved by using fish farm wastewater, which contains nutrients (nitrogen and phosphorus) suitable for microalgae cultivation.
 Fish farm wastewater has lower nutrient concentrations when compared to standard growth media suitable for higher lipid accumulation.
 Microalgae used as a biodiesel feedstock, cultivated in fish farm wastewater, has added benefits, including wastewater treatment.

Halomonas as a chassis.

With the rapid development of systems and synthetic biology, the non-model bacteria, Halomonas spp., have been developed recently to become a cost-competitive platform for producing a variety of products including polyesters, chemicals and proteins owing to their contamination resistance and ability of high cell density growth at alkaline pH and high salt concentration. These salt-loving microbes can partially solve the challenges of current industrial biotechnology (CIB) which requires high energy-consuming sterilization to prevent contamination as CIB is based on traditional chassis, typically, Escherichia coli, Bacillus subtilis, Pseudomonas putida and Corynebacterium glutamicum. The advantages and current status of Halomonas spp. including their molecular biology and metabolic engineering approaches as well as their applications are reviewed here. Moreover, a systematic strain engineering streamline, including product-based host development, genetic parts mining, static and dynamic optimization of modularized pathways and bioprocess-inspired cell engineering are summarized. All of these developments result in the term called next-generation industrial biotechnology (NGIB). Increasing efforts are made to develop their versatile cell factories powered by synthetic biology to demonstrate a new biomanufacturing strategy under open and continuous processes with significant cost-reduction on process complexity, energy, substrates and fresh water consumption.

Improving the Thermostability of Xylanase A from Bacillus subtilis by Combining Bioinformatics and Electrostatic Interactions Optimization.

The rational improvement of the enzyme catalytic activity is one of the most significant challenges in biotechnology. Most conventional strategies used to engineer enzymes involve selecting mutations to increase their thermostability. Determining good criteria for choosing these substitutions continues to be a challenge. In this work, we combine bioinformatics, electrostatic analysis, and molecular dynamics to predict beneficial mutations that may improve the thermostability of XynA from Bacillus subtilis. First, the Tanford-Kirkwood surface accessibility method is used to characterize each ionizable residue contribution to the protein native state stability. Residues identified to be destabilizing were mutated with the corresponding residues determined by the consensus or ancestral sequences at the same locations. Five mutants (K99T/N151D, K99T, S31R, N151D, and K154A) were investigated and compared with 12 control mutants derived from experimental approaches from the literature. Molecular dynamics results show that the mutants exhibited folding temperatures in the order K99T > K99T/N151D > S31R > N151D > WT > K154A. The combined approaches employed provide an effective strategy for low-cost enzyme optimization needed for large-scale biotechnological and medical applications.

In silico Design for Systems-Based Metabolic Engineering for the Bioconversion of Valuable Compounds From Industrial By-Products.

Selecting appropriate metabolic engineering targets to build efficient cell factories maximizing the bioconversion of industrial by-products to valuable compounds taking into account time restrictions is a significant challenge in industrial biotechnology. Microbial metabolism engineering following a rational design has been widely studied. However, it is a cost-, time-, and laborious-intensive process because of the cell network complexity; thus, it is important to use tools that allow predicting gene deletions. An in silico experiment was performed to model and understand the metabolic engineering effects on the cell factory considering a second complexity level by transcriptomics data integration. In this study, a systems-based metabolic engineering target prediction was used to increase glycerol bioconversion to succinic acid based on Escherichia coli. Transcriptomics analysis suggests insights on how to increase cell glycerol utilization to further design efficient cell factories. Three E. coli models were used: a core model, a second model based on the integration of transcriptomics data obtained from growth in an optimized culture media, and a third one obtained after integration of transcriptomics data from adaptive laboratory evolution (ALE) experiments. A total of 2,402 strains were obtained with fumarase and pyruvate dehydrogenase being frequently predicted for all the models, suggesting these reactions as essential to increase succinic acid production. Finally, based on using flux balance analysis (FBA) results for all the mutants predicted, a machine learning method was developed to predict new mutants as well as to propose optimal metabolic engineering targets and mutants based on the measurement of the importance of each knockout’s (feature’s) contribution. Glycerol has become an interesting carbon source for industrial processes due to biodiesel business growth since it has shown promising results in terms of biomass/substrate yields. The combination of transcriptome, systems metabolic modeling, and machine learning analyses revealed the versatility of computational models to predict key metabolic engineering targets in a less cost-, time-, and laborious-intensive process. These data provide a platform to improve the prediction of metabolic engineering targets to design efficient cell factories. Our results may also work as a guide and platform for the selection/engineering of microorganisms for the production of interesting chemical compounds.

Predicting microbial growth dynamics in response to nutrient availability.

Developing mathematical models to accurately predict microbial growth dynamics remains a key challenge in ecology, evolution, biotechnology, and public health. To reproduce and grow, microbes need to take up essential nutrients from the environment, and mathematical models classically assume that the nutrient uptake rate is a saturating function of the nutrient concentration. In nature, microbes experience different levels of nutrient availability at all environmental scales, yet parameters shaping the nutrient uptake function are commonly estimated for a single initial nutrient concentration. This hampers the models from accurately capturing microbial dynamics when the environmental conditions change. To address this problem, we conduct growth experiments for a range of micro-organisms, including human fungal pathogens, baker’s yeast, and common coliform bacteria, and uncover the following patterns. We observed that the maximal nutrient uptake rate and biomass yield were both decreasing functions of initial nutrient concentration. While a functional form for the relationship between biomass yield and initial nutrient concentration has been previously derived from first metabolic principles, here we also derive the form of the relationship between maximal nutrient uptake rate and initial nutrient concentration. Incorporating these two functions into a model of microbial growth allows for variable growth parameters and enables us to substantially improve predictions for microbial dynamics in a range of initial nutrient concentrations, compared to keeping growth parameters fixed.

Plant-Based Alternatives to Yogurt: State-of-the-Art and Perspectives of New Biotechnological Challenges.

Due to the increasing demand for milk alternatives, related to both health and ethical needs, plant-based yogurt-like products have been widely explored in recent years. With the main goal to obtain snacks similar to the conventional yogurt in terms of textural and sensory properties and ability to host viable lactic acid bacteria for a long-time storage, several plant-derived ingredients (e.g., cereals, pseudocereals, legumes, and fruits) as well as technological solutions (e.g., enzymatic and thermal treatments) have been investigated. The central role of fermentation in yogurt-like production led to specific selections of lactic acid bacteria strains to be used as starters to guarantee optimal textural (e.g., through the synthesis of exo-polysaccharydes), nutritional (high protein digestibility and low content of anti-nutritional compounds), and functional (synthesis of bioactive compounds) features of the products. This review provides an overview of the novel insights on fermented yogurt-like products. The state-of-the-art on the use of unconventional ingredients, traditional and innovative biotechnological processes, and the effects of fermentation on the textural, nutritional, functional, and sensory features, and the shelf life are described. The supplementation of prebiotics and probiotics and the related health effects are also reviewed.

Nanoscale Construction Biotechnology for Cementitious Materials: A Prospectus

Climate change, infrastructure resilience, and resource recovery from waste have emerged as grand challenges for civil engineers in the 21st century. Wicked problems associated with these global grand challenges are necessitating innovative, multidisciplinary thinking and multiscale, integrated solutions that are spurring the development of a new field—construction biotechnology. While the field of construction biotechnology spans multiple scales, this review highlights the promise and potential of nanoscale (<100 nm) biotechnological applications to civil engineering. While the field of nanotechnology has revolutionized other industries, applications of nanotechnology in civil engineering have remained limited due to techno-economic and environmental barriers. Biological production of functional nanoparticles (NPs), however, offers new economical routes to develop resilient, high-performance cementitious materials while simultaneously addressing critical needs related to wastewater and air-pollution treatment. Recent research has elucidated that biological production of NPs exhibit preferred—and genetically controllable—morphological characteristics that could tailor the structure-property relationships of civil engineering materials. The natural ability as well as the programmability via synthetic biology allow microorganisms to immobilize heavy metals (e.g., Hg, Cr, Zn, Cd, Cu, Ag) and interact with greenhouse gases and volatile organic compounds, therefore affording civil engineers a grand opportunity to treat wastewater and recover rare earth elements, and remove air pollutions. In addition to featuring state-of-the-art research in the field, this review summarizes the opportunities and challenges of nanoscale biotechnology and proposes a roadmap of research for civil engineers of the 21st century.

High-yield production of major T-cell ESAT6-CFP10 fusion antigen of M. tuberculosis complex employing codon-optimized synthetic gene

Translation engineering and bioinformatics have accelerated the rate at which gene sequences can be improved to generate multi-epitope proteins. Strong antigenic proteins for tuberculosis diagnosis include individual ESAT6 and CFP10 proteins or derived peptides. Obtention of heterologous multi-component antigens in E. coli without forming inclusion bodies remain a biotechnological challenge. The gene sequence for ESAT6-CFP10 fusion antigen was optimized by codon bias adjust for high-level expression as a soluble protein. The obtained fusion protein of 23.7 kDa was observed by SDS-PAGE and Western blot analysis after Ni-affinity chromatography and the yield of expressed soluble protein reached a concentration of approximately 67 mg/L in shake flask culture after IPTG induction. Antigenicity was evaluated at 4 μg/mL in whole blood cultures from bovines, and protein stimuli were assessed using a specific in vitro IFN-γ release assay. The hybrid protein was able to stimulate T-cell specific responses of bovine TB suspects. The results indicate that improved E. coli codon usage is a good and cost-effective strategy to potentialize large scale production of multi-epitope proteins with sustained antigenic properties for diagnostic purposes.