Field Of Industrial Biotechnology Research Articles

Production of Cellulolytic Enzymes by Paecilomyces TP08 Using Grape Stalk and Wheat Bran as Substrates in Solid State Culture

Objective: The objective of this study is to investigate the use of grape stems and wheat bran as substrates in solid state fermentation, for the production of cellulolytic enzymes using the fungus Paecilomyces TP08 sp. Theoretical Framework: In grape production, driven by increased consumption and exports, by-products represent 30% of the total volume of grapes processed for wine, requiring efficient management to ensure sustainability. Method: The methodology adopted for this research involves solid-state cultivation, with a substrate made up of 50% grape stems and 50% wheat bran, with samples taken every 24 hours. Each sample taken went through a filtration process, followed by enzyme activity analysis using the DNS method. Results and Discussion: The results obtained showed that the cultivation with the association of wheat bran and grape stalks presented good total cellulase activity during the process, value of 0.846 U/g in 144h, followed by the activity of 0.663U/g observed in 24h. Research Implications: The practical and theoretical implications of this research are discussed, providing insights into how the results can be applied or influence practices in the field of industrial biotechnology. These implications could cover the food, textile and pharmaceutical industries. Originality/Value: This study contributes to the literature by associating grape stalks with wheat bran for the production of fungal enzymes. The relevance and value of this research are evidenced by the use of an innovative substrate (grape stalks) associated with a conventional substrate (wheat bran) for induction of the microorganism little used in the literature for enzyme production.

BioDT: An Integrated Digital-Twin-Based Framework for Intelligent Biomanufacturing

The field of industrial biotechnology has shown an increasing interest in adopting digital twins for improving process productivity and management efficiency. Despite its potential benefits, digital-twin-based biomanufacturing has not been fully implemented. As a preliminary undertaking, we developed an open-source digital twin framework for cell culture. The core models of the digital twin were coded in C++ and compiled as a reusable Python library. A web-based, cloud-native HMI application that links the physical and virtual systems was developed. A microbioreactor digital twin system was implemented using the framework as a proof of concept. The system features a 3D-printed rocking platform that is customized to fit T25 flasks, enabling automated rocking rate and angle control and in-place optical cell density measurement. The digital twin was validated using Chinese Hamster Ovary (CHO) cells and was found to be able to predict the changes in cell density, glucose consumption, lactic acid production, and oxygen uptake rate (OUR). Finally, we performed a case study to demonstrate the system’s practical applicability in Advanced Process Control (APC) by constructing real-time glucose and lactic acid soft sensors, which are in turn used to alert the operator for manual media change or for automated feeding.

Diversity of culturable thermophilic bacteria from Tata Pani hotspring of Kotli Azad Jammu and Kashmir.

Hot water springs are unique areas populated by mesophiles, thermotolerant and hyperthermophiles. They are the source of diversity of thermophiles, mainly belonging to archaea and bacteria domains. The diversity of thermophiles gives an outline of the huge biological potential that can be exploited for industrial applications.To this end, this study was aimed to isolate and characterise the unexplored thermophilic microorganisms from hot water spring in Tatapani, Tehsil & District Kotli AJK, Pakistan. Around 10 bacterial isolates were identified using morphological, biochemical, physiological and molecular attributes. Sequencing of the 16S rDNA gene of the isolates followed by BLAST search revealed that the strain MBT008 has 100% similarity with Anoxybacilluskamchatkensis. MBT012 showed 99.57% similarity with A.mongoliensis, MBT014 was affiliated with A.tengchongensis with 99.43% similarity, MBT009 showed 99.83% homology with A.gonensis and MBT018, 98.70% similarity with A.karvacharensis. The presence of all this microbial diversity in one common source is of immense importance related to envioronmental and industrial aspects in general and extraction of thermostable enzymes from these thermophiles specifically opens new horizons in the field of industrial biotechnology. These thermophiles are revealing new capabilities and are being manipulated by biotechnologists in utilizing them in different unique ways.

Review on Thermozymes Produced by Thermophilic Fungi: A Gold Mine for Industrial Applications

Thermophilic fungi have often been proposed as magnificent sources, since, they grow in extremely harsh temperatures, ranging between 20-50 °C. They have ability to produce industrially germane thermo-resistant enzymes that can endure the austere environment of industrialized procedures, and have outspread mercantile acceptability in food, paper, feed, pulp and also in textile industries. The isolates of numerous thermophilic fungi are sources of biotechnologically valuable enzymes like pectinases, laccases, xylanases, phytases, proteases and cellulases etc., which display high levels of temperature tolerances up to a maximum range of 72 °C. Despite lesser-known knowledge about them, they have already aided humans in many ways and still have lot more to give. In the subsequent years, the utilization of thermostable enzymes is anticipated to escalate exponentially due to their role in biofuels. In this review I describe about their isolation from their respective habitat, production of thermostable enzymes and their applications in industry, which helped scientists to break through the field of Industrial Biotechnology.

Preface to the special issue on the 10th anniversary of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences

Industrial biotechnology offers great promises to the development of a green, low-carbon and sustainable economy. The Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, which was formally established ten years ago, is an important powerhouse in the field of industrial biotechnology. To date, the Institute has developed a scientific and technological innovation chain, covering from basic research in life sciences to industrial application of biotechnology. On the occasion of the Institute's tenth anniversary, we publish this special issue to review the progress and trends of industrial biotechnology from six aspects, namely expert perspectives, development strategy, cutting-edge research, enabling technology, core technology innovation, and industrialization promotion. This will facilitate the construction of a green and low-carbon circular economy system, and promote the high-quality development of bioeconomy.

Type I fimbriae subunit fimA enhances Escherichia coli biofilm formation but affects L-threonine carbon distribution.

The biofilm (BF) provides favorable growth conditions to cells, which has been exploited in the field of industrial biotechnology. Based on our previous research works on type I fimbriae for the biosynthesis of L-threonine (LT) in Escherichia coli, in this study, a fimA-overexpressing strain was engineered, which improved BF formation under industrial fermentation conditions. The morphological observation and characterization of BF formation were conducted to verify the function of the subunit FimA. However, it was not suitable for repeated-batch immobilized fermentation as the LT titer was not elevated significantly. The underlying molecular mechanisms of BF formation and the LT carbon flux were explored by transcriptomic analysis. The results showed that fimA regulated E. coli BF formation but affected LT carbon distribution. This study will stimulate thoughts about how the fimbriae gene regulated biofilms and amino acid excretion and will bring some consideration and provide a reference for the development of BF-based biomanufacturing processes in E. coli.

Engineering thermotolerant Yarrowia lipolytica for sustainable biosynthesis of mannitol and fructooligosaccharides

Yarrowia lipolytica is an important industrial microbe and a commonly used synthetic chassis in synthetic biology. In recent years, it has become increasingly popular in the field of industrial biotechnology to synthesize lipophilic molecules, such as lycopene, β-carotene, docosahexaenoic acid (DHA) and arachidonic acid. It is also a novel chassis for synthesizing erythritol and D-mannitol. Chemical synthesis is currently the most common method for producing mannitol, which has disadvantages, including multi-steps and great risks (under high pressure and high temperature). Based on the unique properties of Y. lipolytica to synthesize erythritol and mannitol simultaneously, this study overexpressed its heat shock protein Hsp90 gene to enhance its heat tolerance and ferment glucose at a higher temperature (34–35 °C) to realize the shift from mainly synthesizing erythritol to mannitol. The relative expression level of genes in the mannitol and erythritol synthesis pathway was detected by qPCR. The shift was achieved, according to preliminary findings, by raising the expression level of genes in the mannitol synthesis route and decreasing the expression level of genes in the erythritol synthesis pathway. To diversify the sugar alcohol profile, fructosyltransferase was displayed on the cell surface. The yeast cells, after mannitol synthesis, can also be used as a whole cell catalyst to catalyze the synthesis of fructooligosaccharides (FOS) from sucrose.

Development and evaluation of culture media based on extracts of the cyanobacterium Arthrospira platensis.

Continuous advances in the fields of industrial biotechnology and pharmacy require the development of new formulations of culture media based on new nutrient sources. These new sources must be sustainable, high yielding, and non-animal-based, with minimal environmental impact. Thus, culture media prepared from cyanobacterial extracts can be an interesting alternative to the current formulations. In this study, we prepared various minimal formulations of culture media using the extracts of Arthrospira platensis, and analyzed the efficiency of these formulations, based on their effect on the production of biomass and molecules of industrial interest, using different types of bacteria. All media formulations prepared in this study showed better performance than conventional media, including those based on animal ingredients. Thus, based on their versatility and high-yielding capacity, we conclude that culture media prepared from cyanobacterial extracts are a good alternative to conventional media for meeting the current demands of the cosmetic and pharmaceutical industries.

The soil bacterium, Corynebacterium glutamicum, from biosynthesis of value-added products to bioremediation: A master of many trades

Ever since its discovery in 1957, Corynebacterium glutamicum has become a well-established industrial strain and is known for its massive capability of producing various amino acids (like L-lysine and L-glutamate) and other value-added chemicals. With the rising demand for these bio-based products, the revelation of the whole genome sequences of the wild type strains, and the astounding advancements made in the fields of metabolic engineering and systems biology, our perspective of C. glutamicum has been revolutionized and has expanded our understanding of its strain development. With these advancements, a new era for C. glutamicum supremacy in the field of industrial biotechnology began. This led to remarkable progress in the enhancement of tailor-made over-producing strains and further development of the substrate spectrum of the bacterium, to easily accessible, economical, and renewable resources. C. glutamicum has also been metabolically engineered and used in the degradation/assimilation of highly toxic and ubiquitous environmental contaminant, arsenic, present in water or soil. Here, we review the history, current knowledge, progress, achievements, and future trends relating to the versatile metabolic factory, C. glutamicum. This review paper is devoted to C. glutamicum which is one of the leading industrial microbes, and one of the most promising and versatile candidates to be developed. It can be used not only as a platform microorganism to produce different value-added chemicals and recombinant proteins, but also as a tool for bioremediation, allowing to enhance specific properties, for example in situ bioremediation.

Microfluidics in Biotechnology: Overview and Status Quo.

Microfluidics has emerged as a powerful tool, enabling biotechnological processes to be performed on a microscale where certain physical processes (such as laminar flow, surface-to-volume ratio, and surface interactions) become dominant factors. At the same time, volumes and assay times are also reduced in microscale - which can substantially lower experimental costs. A decade ago, most microfluidic systems were only used for proof-of-concept studies; today, a wide array of microfluidic systems have been deployed to tackle various biotechnological research questions - especially regarding the analysis, screening, and understanding of cellular systems. Examples cover all biotechnological areas, from diagnostic applications in the field of medical biotechnology to the screening of potentially useful cells in the field of industrial biotechnology. As part of this review, we provide a brief introduction to microfluidics technology (including the vision of Lab-on-a-chip (LOC) systems) and survey some of the most notable applications of microfluidic technology in biotechnology to date.

Increased carvone production in Escherichia coli by balancing limonene conversion enzyme expression via targeted quantification concatamer proteome analysis

(−)-Carvone is a monoterpenoid with a spearmint flavor. A sustainable biotechnological production process for (−)-carvone is desirable. Although all enzymes in (−)-carvone biosynthesis have been functionally expressed in Escherichia coli independently, the yield was low in previous studies. When cytochrome P450 limonene-6-hydroxylase (P450)/cytochrome P450 reductase (CPR) and carveol dehydrogenase (CDH) were expressed in a single strain, by-product formation (dihydrocarveol and dihydrocarvone) was detected. We hypothesized that P450 and CDH expression levels differ in E. coli. Thus, two strains independently expressing P450/CPR and CDH were mixed with different ratios, confirming increased carvone production and decreased by-product formation when CDH input was reduced. The optimum ratio of enzyme expression to maximize (−)-carvone production was determined using the proteome analysis quantification concatamer (QconCAT) method. Thereafter, a single strain expressing both P450/CPR and CDH was constructed to imitate the optimum expression ratio. The upgraded strain showed a 15-fold improvement compared to the initial strain, showing a 44 ± 6.3 mg/L (−)-carvone production from 100 mg/L (−)-limonene. Our study showed the usefulness of the QconCAT proteome analysis method for strain development in the industrial biotechnology field.

Complete Genome Sequence of Cupriavidus sp. Strain EM10, Isolated from Sewage Sludge.

ABSTRACTCupriavidus species have been known as versatile microorganisms in the field of industrial biotechnology. Cupriavidus sp. strain EM10 was isolated from sewage sludges. Here, we report the complete genome sequence of this bacterium, which contains 6,658,510 bp (GC content, 65.12%) and 6,248 genes.

Ustilaginaceae Biocatalyst for Co-Metabolism of CO2-Derived Substrates toward Carbon-Neutral Itaconate Production.

The family Ustilaginaceae (belonging to the smut fungi) are known for their plant pathogenicity. Despite the fact that these plant diseases cause agricultural yield reduction, smut fungi attracted special attention in the field of industrial biotechnology. Ustilaginaceae show a versatile product spectrum such as organic acids (e.g., itaconate, malate, succinate), polyols (e.g., erythritol, mannitol), and extracellular glycolipids, which are considered value-added chemicals with potential applications in the pharmaceutical, food, and chemical industries. This study focused on itaconate as a platform chemical for the production of resins, plastics, adhesives, and biofuels. During this work, 72 different Ustilaginaceae strains from 36 species were investigated for their ability to (co-) consume the CO2-derived substrates acetate and formate, potentially contributing toward a carbon-neutral itaconate production. The fungal growth and product spectrum with special interest in itaconate was characterized. Ustilago maydis MB215 and Ustilago rabenhorstiana NBRC 8995 were identified as promising candidates for acetate metabolization whereas Ustilago cynodontis NBRC 7530 was identified as a potential production host using formate as a co-substrate enhancing the itaconate production. Selected strains with the best itaconate production were characterized in more detail in controlled-batch bioreactor experiments confirming the co-substrate utilization. Thus, a proof-of-principle study was performed resulting in the identification and characterization of three promising Ustilaginaceae biocatalyst candidates for carbon-neutral itaconate production contributing to the biotechnological relevance of Ustilaginaceae.

Aspergillus: A Powerful Protein Production Platform

Aspergilli have been widely used in the production of organic acids, enzymes, and secondary metabolites for almost a century. Today, several GRAS (generally recognized as safe) Aspergillus species hold a central role in the field of industrial biotechnology with multiple profitable applications. Since the 1990s, research has focused on the use of Aspergillus species in the development of cell factories for the production of recombinant proteins mainly due to their natively high secretion capacity. Advances in the Aspergillus-specific molecular toolkit and combination of several engineering strategies (e.g., protease-deficient strains and fusions to carrier proteins) resulted in strains able to generate high titers of recombinant fungal proteins. However, the production of non-fungal proteins appears to still be inefficient due to bottlenecks in fungal expression and secretion machinery. After a brief overview of the different heterologous expression systems currently available, this review focuses on the filamentous fungi belonging to the genus Aspergillus and their use in recombinant protein production. We describe key steps in protein synthesis and secretion that may limit production efficiency in Aspergillus systems and present genetic engineering approaches and bioprocessing strategies that have been adopted in order to improve recombinant protein titers and expand the potential of Aspergilli as competitive production platforms.

Safe-by-Design: Stakeholders' Perceptions and Expectations of How to Deal with Uncertain Risks of Emerging Biotechnologies in the Netherlands.

Advanced gene editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas have increased the pace of developments in the field of industrial biotechnology. Such techniques imply new possibilities when working with living organisms, possibly leading to uncertain risks. In the Netherlands, current policy fails to address these uncertain risks because risk classification is determined process‐wise (i.e., genetically modified organism [GMO] and non‐GMO), there is a strong focus on quantifiable risks, and the linearity within current governance (science–policy–society) hinders iterative communication between stakeholders, leaving limited room to anticipate uncertainties at an early stage of development. A suggested concept to overcome these shortcomings is the Safe‐by‐Design (SbD) approach, which, theoretically, allows stakeholders to iteratively incorporate safety measures throughout a technology's development process, creating a dynamic environment for the anticipation of uncertain risks. Although this concept originates from chemical engineering and is already widely applied in nanotechnology, for the field of biotechnology, there is no agreed upon definition yet. To explore the possibilities of SbD for future governance of biotechnology, we should gain insight in how various stakeholders perceive notions of risk, safety, and inherent safety, and what this implies for the applicability of SbD for risk governance concerning industrial biotechnology. Our empirical research reveals three main themes: (1) diverging expectations with regard to safety and risks, and to establish an acceptable level of risk; (2) different applications of SbD and inherent safety, namely, product‐ and process‐wise; and (3) unclarity in allocating responsibilities to stakeholders in the development process of a biotechnology and within society.

Vital parameters for biomass, lipid, and carotenoid production of thraustochytrids

Thraustochytrids have recently gained attention owing to their widespread applications in biodiesel production, biomedicine, and nutraceutical industries. They have a promising capability to produce high amounts of polyunsaturated fatty acids (PUFAs), including health-benefiting docosahexaenoic acid (DHA) and saturated fatty acid (SFA) which are renewable sources of biofuels. Since biofuels are an economical and renewable alternative source of energy to fossil fuels, lipid biofactories have become the centre of attention in the field of industrial biotechnology. Although thraustochytrids are a valuable source of bioenergy and biomedical products, their optimal use has some challenges and limitations to overcome. The originality of the current review lies in the evaluation of relevant literature on the upstream and downstream processing of thraustochytrids to provide background knowledge for future researchers to enhance the growth rate, product synthesis, and scale-up to an industrial level of thraustochytrids.

Sustainability and Life Cycle Assessment in Industrial Biotechnology: A Review of Current Approaches and Future Needs.

The development and implementation of industrial biotechnology (IB) is associated with high expectations for reductions of environmental impacts and risks, particularly in terms of climate change and fossil resource depletion, positive socioeconomic effects, hopes for new competitive products and processes, and development in rural areas. However, not all products and processes are really advantageous with regard to sustainability criteria, and not all are economically successful and accepted by stakeholders. Sustainability and life cycle assessment can play an important role to assess IB products and processes, often accompanying development processes from the early stages onwards. Such assessments can identify key factors regarding sustainability criteria, enable a determination of both product and process performance, or aid in prospectively estimating such performance and its consequences. Thus, development processes, investment decisions, policymaking, and the communication with stakeholders can be supported. This contribution reviews the field of sustainability and life cycle assessment in IB. We explore relevant literature from a methodical and application perspective and categorise suitable methodologies, methods, and tools. We characterise IB from an assessment perspective and indicate challenges, discuss approaches to address these, and identify possible fields of future research. Thus, students, researchers, and practitioners in the field of IB will obtain an up-to-date overview, references to relevant fields of literature, and guidance for own studies in this important and fast-emerging topic.

The Sustainability and Life Cycle Assessments of Industrial Biotechnology: An Introduction.

Industrial biotechnology (IB) uses biological and biochemical processes in industrial production and is often regarded as an emerging key technology revolutionizing the production of many products while protecting resources and the environment and fostering economic development. This contribution describes the background and sketches the content of the volume 'Sustainability and Life Cycle Assessment of Industrial Biotechnology' in the Springer series 'Advances in Biochemical Engineering/Biotechnology'. The field of IB is introduced from different perspectives (milestones in IB history, economics of biotechnology industry, environmental and social as well as ethical issues and impacts, green chemistry) and in several applications fields (production of chemicals, geobiotechnology in mining).

Primary and Secondary Metabolic Effects of a Key Gene Deletion (ΔYPL062W) in Metabolically Engineered Terpenoid-Producing Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an established cell factory for production of terpenoid pharmaceuticals and chemicals. Numerous studies have demonstrated that deletion or overexpression of off-pathway genes in yeast can improve terpenoid production. The deletion of YPL062W in S. cerevisiae, in particular, has benefitted carotenoid production by channeling carbon toward carotenoid precursors acetyl coenzyme A (acetyl-CoA) and mevalonate. The genetic function of YPL062W and the molecular mechanisms for these benefits are unknown. In this study, we systematically examined this gene deletion to uncover the gene function and its molecular mechanism. RNA sequencing (RNA-seq) analysis uncovered that YPL062W deletion upregulated the pyruvate dehydrogenase bypass, the mevalonate pathway, heterologous expression of galactose (GAL) promoter-regulated genes, energy metabolism, and membrane composition synthesis. Bioinformatics analysis and serial promoter deletion assay revealed that YPL062W functions as a core promoter for ALD6 and that the expression level of ALD6 is negatively correlated to terpenoid productivity. We demonstrate that ΔYPL062W increases the production of all major terpenoid classes (C10, C15, C20, C30, and C40). Our study not only elucidated the biological function of YPL062W but also provided a detailed methodology for understanding the mechanistic aspects of strain improvement.IMPORTANCE Although computational and reverse metabolic engineering approaches often lead to improved gene deletion mutants for cell factory engineering, the systems level effects of such gene deletions on the production phenotypes have not been extensively studied. Understanding the genetic and molecular function of such gene alterations on production strains will minimize the risk inherent in the development of large-scale fermentation processes, which is a daunting challenge in the field of industrial biotechnology. Therefore, we established a detailed experimental and systems biology approach to uncover the molecular mechanisms of YPL062W deletion in S. cerevisiae, which is shown to improve the production of all terpenoid classes. This study redefines the genetic function of YPL062W, demonstrates a strong correlation between YPL062W and terpenoid production, and provides a useful modification for the creation of terpenoid production platform strains. Further, this study underscores the benefits of detailed and systematic characterization of the metabolic effects of genetic alterations on engineered biosynthetic factories.

Social Life Cycle Assessment for Industrial Biotechnology.

This chapter focuses on social assessment methods (known as SLCA) applied to industrial biotechnology (IB), which are part of the "life cycle" approach. IB is a heading for a set of different technologies. The first section presents a review of the literature to provide an analysis of the results and limitations of IB SLCA studies, with the main focus on the biofuel industries. Often conducted via a social performance analysis based on CSR (corporate social responsibility) criteria, most studies provide little new information. Nevertheless, there are some studies on the change caused by the emergence of an IB. These studies use national accounts input-output tables, which allow us to predict impacts. The second section suggests rules to follow in order to achieve a "good" SLCA in the field of IB, in other words, to be able to anticipate the main known impacts or at least to carry out the assessment in a rigorous and transparent fashion. The conclusion focuses on the prospects and challenges of IB SLCA, in a world which is experiencing immense upheaval. Graphical Abstract.