Microbial Conversion Processes Research Articles

Metagenomic analysis reveals the responses of microbial communities and nitrogen metabolic pathways to polystyrene micro(nano)plastics in activated sludge systems.

Microplastics (MPs) and nanoplastics (NPs) are prevalent in sewage and pose a potential threat to nitrogen biotransformation in wastewater treatment systems. However, investigations on how MPs and NPs affect the microbial nitrogen conversion and metabolism of the activated sludge are still scanty. Herein, the responses of microbiomes and functional genes to polystyrene MPs and NPs in activated sludge systems were investigated by metagenomic analysis. Results indicated that 1mg/L MPs and NPs had marginal impacts on the nitrogen removal performance of the activated sludge systems, whereas high concentrations of MPs and NPs (20 and 100mg/L) decreased the total nitrogen removal efficiency (13.4%-30.6%) by suppressing the nitrogen transformation processes. Excessive reactive oxygen species induced by MPs and NPs caused cytotoxicity, as evidenced by impaired cytomembranes and decreased bioactivity. Metagenomic analysis revealed that MPs and NPs diminished the abundance of denitrifiers (e.g. Mesorhizobium, Rhodobacter and Thauera), and concurrently reduced the abundance of functional genes (e.g. napA, napB and nirS) encoding for key enzymes involved in the nitrogen transformations, as well as the genes (e.g. mdh) related to the electron donor production, thereby declining the nitrogen removal efficiency. Network analysis further clarified the attenuate association between denitrifiers and denitrification-related genes in the plastic-exposed systems, elucidating that MPs and NPs restrained the nitrogen removal by inhibiting the contributions of microorganisms to nitrogen transformation processes. This study provides vital insights into the responses of the microbial community structure and nitrogen conversion processes to micro(nano)plastics disturbance in activated sludge systems.

Enhancing tolerance of Kluyveromyces marxianus to lignocellulose-derived inhibitors and its ethanol production from corn cob via overexpression of a nitroreductase gene

Despite its potential as a sustainable feedstock for producing renewable fuels and chemicals, lignocellulosic biomass (LCB) remains largely untapped due to problems associated with its deconstruction and the toxicity of its sugar-rich hydrolysates in microbial conversion processes. Using genetic modification to facilitate inhibitor tolerance, the present study aimed to construct a robust yeast cell factory for cellulosic ethanol bioproduction from non-detoxified LCB. To this effect, two putative nitroreductase genes (KmHBN1 and KmFRM2) hypothesized to be possible mediators of oxidative stress response were separately overexpressed and disrupted in Kluyveromyces marxianus. In contrast to KmFRM2 modified strains (disrupted or overexpressed) with no significant change, the inhibitor tolerance to phenols, furfural, and acetic acid cocktail was improved in YKmHBN1-URA3 strain overexpressing KmHBN1. Although the recombinant KmHBN1 and KmFRM2 had nitroreductase activity, they did not degrade or convert inhibitors. The change of intracellular reactive oxygen species level (ROS) of KmHBN1 disrupted or overexpressed strains indicated that KmHBN1 affected intracellular ROS elimination. Cells with disrupted KmHBN1 failed to respond to ROS created during inhibitor metabolism, and this disruption also led to reduced expression of respiratory chain genes which translates to a decreased inhibitor tolerance. The overexpression of KmHBN1 not only enhanced the ethanol production from glucose in the presence of inhibitors but also improved ethanol productivity (18.75%) from simultaneously-saccharified and co-fermented inhibitor-ladened corn cob. These findings unlock a new pathway for the creation of robust yeast strains to aid in the biorefinery conversion of lignocellulosic feedstock.

The influence of the configurations of multiple-impeller on canrenone bioconversion using resting cells of Aspergillus ochraceus

Abstract 11 α-Hydroxycanrenone is a key intermediate in the synthesis of eplernone which is a drug that protects the cardiovascular system. It can be obtained by microbial transformation of canrenone using Aspergillus ochraceus. The impeller configuration has a great impact on the microbial transformation efficiency. In this study, three kinds of multiple-impeller including six-blade Rushton turbine (lower) and six-blade Rushton turbine (upper) (RT + RT), six-blade Rushton turbine (lower) and six-arrow blade turbine (upper) (RT + ABT), six-blade Rushton turbine impeller (lower) and six-blade Chemineer CD6 impeller (upper) (RT + CD6) were employed to carry out the microbial conversion process, which was investigated by experiments and computational fluid dynamic (CFD) simulations. The CFD simulation was performed only for the hydrodynamic part of the bioreactor in this article. The results showed that RT + CD6 gave better conversion ratio compared to the other two multiple impellers. It had higher axial flow and better air volume fraction distribution which was benefit for the biotransformation process. A certain amount of cell content should be guaranteed in order to obtain a good substrate conversion (45 % approximately). The final conversion ratio of canrenone was proportional to the content of mycelium at the late stage of conversion, while the content of mycelium at the early stage had a subtle effect. Besides, A. ochraceus resting cells could tolerate the maximum and average shear strain rate in the order of 2598 s−1 and 52 s−1, respectively. The research results provided a guide for the selection of impeller for the biotransformation of canrenone in biopharmaceutical industry.

Unraveling the mechanism of furfural tolerance in engineered Pseudomonas putida by genomics.

As a dehydration product of pentoses in hemicellulose sugar streams derived from lignocellulosic biomass, furfural is a prevalent inhibitor in the efficient microbial conversion process. To solve this obstacle, exploiting a biorefinery strain with remarkable furfural tolerance capability is essential. Pseudomonas putida KT2440 (P. putida) has served as a valuable bacterial chassis for biomass biorefinery. Here, a high-concentration furfural-tolerant P. putida strain was developed via adaptive laboratory evolution (ALE). The ALE resulted in a previously engineered P. putida strain with substantially increased furfural tolerance as compared to wild-type. Whole-genome sequencing of the adapted strains and reverse engineering validation of key targets revealed for the first time that several genes and their mutations, especially for PP_RS19785 and PP_RS18130 [encoding ATP-binding cassette (ABC) transporters] as well as PP_RS20740 (encoding a hypothetical protein), play pivotal roles in the furfural tolerance and conversion of this bacterium. Finally, strains overexpressing these three striking mutations grew well in highly toxic lignocellulosic hydrolysate, with cell biomass around 9-, 3.6-, and two-fold improvement over the control strain, respectively. To our knowledge, this study first unravels the furan aldehydes tolerance mechanism of industrial workhorse P. putida, which provides a new foundation for engineering strains to enhance furfural tolerance and further facilitate the valorization of lignocellulosic biomass.

Unraveling the potential of non-conventional yeasts in biotechnology.

ABSTRACTCost-effective microbial conversion processes of renewable feedstock into biofuels and biochemicals are of utmost importance for the establishment of a robust bioeconomy. Conventional baker's yeast Saccharomyces cerevisiae, widely employed in biotechnology for decades, lacks many of the desired traits for such bioprocesses like utilization of complex carbon sources or low tolerance towards challenging conditions. Many non-conventional yeasts (NCY) present these capabilities, and they are therefore forecasted to play key roles in future biotechnological production processes. For successful implementation of NCY in biotechnology, several challenges including generation of alternative carbon sources, development of tailored NCY and optimization of the fermentation conditions are crucial for maximizing bioproduct yields and titers. Addressing these challenges requires a multidisciplinary approach that is facilitated through the ‘YEAST4BIO’ COST action. YEAST4BIO fosters integrative investigations aimed at filling knowledge gaps and excelling research and innovation, which can improve biotechnological conversion processes from renewable resources to mitigate climate change and boost transition towards a circular bioeconomy. In this perspective, the main challenges and research efforts within YEAST4BIO are discussed, highlighting the importance of collaboration and knowledge exchange for progression in this research field.

Reactor Designs and Configurations for Biological and Bioelectrochemical C1 Gas Conversion: A Review.

Microbial C1 gas conversion technologies have developed into a potentially promising technology for converting waste gases (CO2, CO) into chemicals, fuels, and other materials. However, the mass transfer constraint of these poorly soluble substrates to microorganisms is an important challenge to maximize the efficiencies of the processes. These technologies have attracted significant scientific interest in recent years, and many reactor designs have been explored. Syngas fermentation and hydrogenotrophic methanation use molecular hydrogen as an electron donor. Furthermore, the sequestration of CO2 and the generation of valuable chemicals through the application of a biocathode in bioelectrochemical cells have been evaluated for their great potential to contribute to sustainability. Through a process termed microbial chain elongation, the product portfolio from C1 gas conversion may be expanded further by carefully driving microorganisms to perform acetogenesis, solventogenesis, and reverse β-oxidation. The purpose of this review is to provide an overview of the various kinds of bioreactors that are employed in these microbial C1 conversion processes.

Exceptionally rich keratinolytic enzyme profile found in the rare actinomycetes Amycolatopsis keratiniphila D2T.

The non-spore forming Gram-positive actinomycetes Amycolatopsis keratiniphila subsp. keratiniphila D2T (DSM 44,409) has a high potential for keratin valorization as demonstrated by a novel biotechnological microbial conversion process consisting of a bacterial growth phase and a keratinolytic phase, respectively. Compared to the most gifted keratinolytic Bacillus species, a very large number of 621 putative proteases are encoded by the genome of Amycolatopsis keratiniphila subsp. keratiniphila D2T, as predicted by using Peptide Pattern Recognition (PPR) analysis. Proteome analysis by using LC-MS/MS on aliquots of the supernatant of A. keratiniphila subsp. keratiniphila D2T culture on slaughterhouse pig bristle meal, removed at 24, 48, 96 and 120h of growth, identified 43 proteases. This was supplemented by proteome analysis of specific fractions after enrichment of the supernatant by anion exchange chromatography leading to identification of 50 proteases. Overall 57 different proteases were identified corresponding to 30% of the 186 proteins identified from the culture supernatant and distributed as 17 metalloproteases from 11 families, including an M36 protease, 38 serine proteases from 4 families, and 13 proteolytic enzymes from other families. Notably, M36 keratinolytic proteases are prominent in fungi, but seem not to have been discovered in bacteria previously. Two S01 family peptidases, named T- and C-like proteases, prominent in the culture supernatant, were purified and shown to possess a high azo-keratin/azo-casein hydrolytic activity ratio. The C-like protease revealed excellent thermostability, giving promise for successful applications in biorefinery processes. Notably, the bacterium seems not to secrete enzymes for cleavage of disulfides in the keratinous substrates. KEY POINTS: • A. keratiniphila subsp. keratiniphila D2T is predicted to encode 621 proteases. • This actinomyceteefficiently converts bristle meal to a protein hydrolysate. • Proteome analysis identified 57 proteases in its secretome.

A framework for the development of Pedagogical Process Simulators (P2Si) using explanatory models and gamification

Process simulators are powerful supporting tools employed at all educational levels of engineering education. Meanwhile, digitalization is obliging the educational systems to adapt. In this new era marked by the Industry 4.0 movement, the formulation and implementation of models are fundamental for process digitalization; therefore, students and new trainees must be familiar with process models, simulators and programming. In this work, a computer-aided framework for the development of process simulators is proposed (P2Si). This framework integrates the following aspects: (i) the use, reuse, and explanation of process models; (ii) a tailored learning design; (iii) game elements; and, (iv) the use of students as co-designers through participatory design to improve the human-computer interaction. Through the application of a series of hierarchical steps, the framework’s workflow aims to arrive at a promising candidate to facilitate the teaching of processes. Furthermore, a proof of concept is developed for the teaching and training of undergraduate students, where the case of an aerobic microbial conversion process at industrial scale is explored. By applying this framework, we obtained the conceptual design of a pedagogical process simulator, which was then implemented as a prototype software platform, BioVL (www.biovl.com), for further development and testing. The main output of this work is the conceptual design of pedagogical process simulators integrating gamification and the use of process models as educational tools. In conclusion, the proposed framework has shown its merit when designing a computer-aided tool suited to support the transition towards the increased industrial digitalization as well as to address current educational challenges.

Direct and Indirect Effects of Increased CO2 Partial Pressure on the Bioenergetics of Syntrophic Propionate and Butyrate Conversion.

Simultaneous digestion and in situ biogas upgrading in high-pressure bioreactors will result in elevated CO2 partial pressure (pCO2). With the concomitant increase in dissolved CO2, microbial conversion processes may be affected beyond the impact of increased acidity. Elevated pCO2 was reported to affect the kinetics and thermodynamics of biochemical conversions because CO2 is an intermediate and end-product of the digestion process and modifies the carbonate equilibrium. Our results showed that increasing pCO2 from 0.3 to 8 bar in lab-scale batch reactors decreased the maximum substrate utilization rate (rsmax) for both syntrophic propionate and butyrate oxidation. These kinetic limitations are linked to an increased overall Gibbs free energy change (ΔGOverall) and a potential biochemical energy redistribution among syntrophic partners, which showed interdependence with hydrogen partial pressure (pH2). The bioenergetics analysis identified a moderate, direct impact of elevated pCO2 on propionate oxidation and a pH-mediated effect on butyrate oxidation. These constraints, combined with physiological limitations on growth exerted by increased acidity and inhibition due to higher concentrations of undissociated volatile fatty acids, help to explain the observed phenomena. Overall, this investigation sheds light on the role of elevated pCO2 in delicate biochemical syntrophic conversions by connecting kinetic, bioenergetic, and physiological effects.

Flexibility assessment of a biorefinery distillation train: Optimal design under uncertain conditions

Multicomponent mixtures can be separated into their single components by mean of different distillation system configurations. The typical distillation train design procedure consists of the assessment of the optimal columns configuration according to the economic and operational aspects. However, this optimal design is strictly related to the operating conditions, i.e. perturbations, when present, can seriously compromise the operation profitability. In these cases a flexibility analysis could be of critical importance to assess the operating conditions range of better performance for different system configurations. This is the typical case of biorefineries where the floating nature of the feedstock causes composition disturbances downstream the fermenter across the year’s seasons. A brand new ABE/W mixture separation case study has been set up; this mixture derives from an upstream microbial conversion process and the successful recovery of at least biobutanol and acetone is crucial for the return on the investments. This paper then compares the possible distillation train configurations from a flexibility point of view. The analysis is focused in particular in highlighting the differences, if present, between the economic optimal solution and flexibility optimal configuration that could not be the same, causing this way a very profitable design to be much less performant under perturbated conditions. Furthermore, a detailed analysis correlating the complex thermodynamics to the operation under uncertain conditions is thoroughly discussed. The proposed design procedure allowed to highlight the differences between weak and strong flexibility constraints and resulted in a dedicated “additional costs vs. flexibility” trend useful to improve the decision making.

Catalyst: Is the Amino Acid a New Frontier for Biorefineries?

Prof. Ning Yan obtained his BS and PhD degrees from Peking University in 2004 and 2009, respectively. Thereafter, he worked as a Marie-Curie research fellow at Ecole Polytechnique Federale de Lausanne, Switzerland. He joined the National University of Singapore (NUS) as an assistant professor, established the Lab of Green Catalysis in 2012, and was promoted to the position of associate professor in 2018. Yunzhu Wang received her BE degree from NUS in 2014. She joined Prof. Yan’s group in the same year and is currently pursuing her PhD. Her research interests include nanoscale metal catalysts and their applications in producing renewable nitrogen-containing chemicals.

Towards biobased industry: acetate as a promising feedstock to enhance the potential of microbial cell factories.

A broad range of different chemical and pharmaceutical compounds have been produced in microbial cell factories. To compete with traditional crude oil based production processes, the use of complex alternative raw materials such as lignocellulosic biomass, waste streams and utilization of CO2 in gas fermentations has been suggested. All of these streams contain acetate, a cheap and potentially interesting carbon source for microbial production processes. Acetate (co-)utilization remains challenging, which is the reason for extensive research on the use of acetate for the production of value-added compounds. For industrial implementation of microbial conversion processes using acetate as a feedstock gaining a deeper insight into acetate metabolism of microorganisms is essential. Systems level analyses and manipulation of potential host organisms should be applied to achieve full utilization of this prospective substrate.

Syngas Fermentation: A Microbial Conversion Process of Gaseous Substrates to Various Products

Biomass and other carbonaceous materials can be gasified to produce syngas with high concentrations of CO and H2. Feedstock materials include wood, dedicated energy crops, grain wastes, manufacturing or municipal wastes, natural gas, petroleum and chemical wastes, lignin, coal and tires. Syngas fermentation converts CO and H2 to alcohols and organic acids and uses concepts applicable in fermentation of gas phase substrates. The growth of chemoautotrophic microbes produces a wide range of chemicals from the enzyme platform of native organisms. In this review paper, the Wood–Ljungdahl biochemical pathway used by chemoautotrophs is described including balanced reactions, reaction sites physically located within the cell and cell mechanisms for energy conservation that govern production. Important concepts discussed include gas solubility, mass transfer, thermodynamics of enzyme-catalyzed reactions, electrochemistry and cellular electron carriers and fermentation kinetics. Potential applications of these concepts include acid and alcohol production, hydrogen generation and conversion of methane to liquids or hydrogen.

Conversion and assimilation of furfural and 5-(hydroxymethyl)furfural by Pseudomonas putida KT2440

The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

Advancing understanding of microbial bioenergy conversion processes by activity-based protein profiling.

The development of renewable biofuels is a global priority, but success will require novel technologies that greatly improve our understanding of microbial systems biology. An approach with great promise in enabling functional characterization of microbes is activity-based protein profiling (ABPP), which employs chemical probes to directly measure enzyme function in discrete enzyme classes in vivo and/or in vitro, thereby facilitating the rapid discovery of new biocatalysts and enabling much improved biofuel production platforms. We review general design strategies in ABPP, and highlight recent advances that are or could be pivotal to biofuels processes including applications of ABPP to cellulosic bioethanol, biodiesel, and phototrophic production of hydrocarbons. We also examine the key challenges and opportunities of ABPP in renewable biofuels research. The integration of ABPP with molecular and systems biology approaches will shed new insight on the catalytic and regulatory mechanisms of functional enzymes and their synergistic effects in the field of biofuels production.

Heterologous expression of xylanase enzymes in lipogenic yeast Yarrowia lipolytica.

To develop a direct microbial sugar conversion platform for the production of lipids, drop-in fuels and chemicals from cellulosic biomass substrate, we chose Yarrowia lipolytica as a viable demonstration strain. Y. lipolytica is known to accumulate lipids intracellularly and is capable of metabolizing sugars to produce lipids; however, it lacks the lignocellulose-degrading enzymes needed to break down biomass directly. While research is continuing on the development of a Y. lipolytica strain able to degrade cellulose, in this study, we present successful expression of several xylanases in Y. lipolytica. The XynII and XlnD expressing Yarrowia strains exhibited an ability to grow on xylan mineral plates. This was shown by Congo Red staining of halo zones on xylan mineral plates. Enzymatic activity tests further demonstrated active expression of XynII and XlnD in Y. lipolytica. Furthermore, synergistic action in converting xylan to xylose was observed when XlnD acted in concert with XynII. The successful expression of these xylanases in Yarrowia further advances us toward our goal to develop a direct microbial conversion process using this organism.

Production of long-chain hydroxy fatty acids by microbial conversion

Hydroxy fatty acids (HFAs) are very important chemicals for versatile applications in biodegradable polymer materials and cosmetic and pharmaceutical industries. They are difficult to be synthesized via chemical routes due to the inertness of the fatty acyl chain. In contrast, these fatty acids make up a major class of natural products widespread among bacteria, yeasts, and fungi. A number of microorganisms capable of producing HFAs from fatty acids or vegetable oils have been reported. Therefore, HFAs could be produced by biotechnological strategies, especially by microbial conversion processes. Microorganisms could oxidize fatty acids either at the terminal carbon or inside the acyl chain to produce various HFAs, including α-HFAs, β-HFAs, mid-position HFAs, ω-HFAs, di-HFAs, and tri-HFAs. The enzymes and their encoded genes responsible for the hydroxylation of the carbon chain have been identified and characterized during the past few years. The involved microbes and catalytic mechanisms for the production of different types of HFAs are systematically demonstrated in this review. It provides a better view of HFA biosynthesis and lays the foundation for further industrial production.

Microbial production of bulk chemicals: development of anaerobic processes

Innovative fermentation processes are necessary for the cost-effective production of bulk chemicals from renewable resources. Current microbial processes are either anaerobic processes, with high yield and productivity, or less-efficient aerobic processes. Oxygen utilization plays an important role in energy generation and redox metabolism that is necessary for product formation. The aerobic productivity, however, is relatively low because of rate-limiting volumetric oxygen transfer; whereas the product yield in the presence of oxygen is generally low because part of the substrate is completely oxidized to CO₂. Hence, new microbial conversion processes for the production of bulk chemicals should be anaerobic. In this opinion article, we describe different scenarios for the development of highly efficient microbial conversion processes for the anaerobic production of bulk chemicals.

地下常在微生物を利用した二酸化炭素のメタン変換システム構築

地下常在微生物を利用した二酸化炭素のメタン変換システム構築

Enhanced production of 2,3-butanediol from glycerol by forced pH fluctuations

The glycerol fermentation by Klebsiella pneumoniae occurs by receiving more than five liquid products-organic acids, diols, and ethanol. Aiming to direct the glycerol conversion towards predominant production of 2,3-butanediol (2,3-BD), the main influencing parameters (the aeration and the pH) were investigated during fed-batch processes. The regime of intensive aeration (2.2 vvm air supply) was evaluated as most favorable for 2,3-BD synthesis and ensured the decrease of all other metabolites. Thus, without pH control, 52.5 g/l 2,3-BD were produced, as the carbon conversion of glycerol into 2,3-BD reached 60.6%. Additional enhancement in 2,3-BD production (by significant increase of glycerol utilization) was achieved by the development of a new method of "forced pH fluctuations". It was realized by consecutive raisings of pH using definite DeltapH value, at exact time intervals, allowing multiple variations. Thus, the optimal conditions for maximal glycerol consumption were defined, and 70 g/l 2,3-BD were produced, which is the highest amount obtained from glycerol as a sole carbon source until now. The forced pH fluctuations emphasized pH as a governing factor in microbial conversion processes.