Lysine Fermentation Research Articles

Artificial neural network and genetic algorithm coupled fermentation kinetics to regulate L-lysine fermentation

Fermentation plays a pivotal role in the industrialization of bioproducts, yet there is a substantial lag in the fermentation process regulation. Here, an artificial neural network (ANN) and genetic algorithm (GA) coupled with fermentation kinetics were employed to establish an innovative lysine fermentation control. Firstly, the strategy of coupling GA with ANN was established. Secondly, specific lysine formation rate (qp), specific substrate consumption rate (qs), and specific cell growth rate (μ) were predicted and optimized by ANN-GA. The optimal ANN model adopts a three-layer feed-forward back-propagation structure (4:10:1). The optimal fermentation control parameters are obtained through GA. Finally, when the carbon to nitrogen ratio, residual sugar concentration, ammonia nitrogen concentration, and dissolved oxygen were [2.5, 4.5], [6.5, 9.5] g·L−1, [1.0, 2.0] g·L−1 and [20, 30] %, respectively, the lysine concentration reaches its peak at 213.0 ± 5.10 g·L−1. The novel control strategy holds significant potential for optimizing the fermentation of other bioproducts.

Valorization of paper industry rejects by combined thermo-chemical pretreatment and biological conversion to L-lysine

Paper and pulp industry is one of the biggest industries that generate waste material and waste water. Conversion of the industrial waste to renewable products through green process is one of the recent trends for the production of renewable fuels and other industrially important bioproducts like bioethanol, biobutanol and bioplastics. In pulp and paper industry, the main process for the production of cellulose pulp is Kraft process and various wastes are generated during this process. These wastes mainly contain the plant biomass which consists carbohydrates like cellulose, hemicellulose and other components of the wood. The present work focuses on the potential of utilizing paper industry rejects for the production of amino acids. The paper rejects were pretreated using mild acids and hydrolyzed enzymatically using cellulases. The effect of the acid pretreatment and hydrolysis on cellulose fiber structure were studied. The pretreatment and hydrolysis yielded 37% sugar from the paper rejects and the glucose obtained was used for subsequent lysine fermentation. Maximum sugar yield was observed when the pretreatment was carried out in a laboratory autoclave at 121 ºC for 30 min at 15 lb pressure. When the pretreatment is carried out at higher temperature charring occurred which resulted in a reduction in glucose yield. The study was further outstretch by appraising the fermentability of using the paper reject hydrolyzateblended with CGXII synthetic medium in 1:1 ratio for L-lysine production using a strain of Corynebacterium glutamicum DM1729 which resulted in the production of 11.5 mg/ml lysine compared to 15.1 mg/ml obtained from pure synthetic medium. • Paper industry rejects as a suitable substrate for the production of value added products. • Acid pretreated hydrolyzate suitable for lysine production. • Corynebacterium glutamicum DM1729 produced 11.5 mg/ml of lysine.

Soft-sensor development for monitoring the lysine fermentation process

Monitoring cell growth and target production in working fermentors is important for stabilizing high level production. In this study, we developed a novel soft sensor for estimating the concentration of a target product (lysine), substrate (sucrose), and bacterial cell in commercially working fermentors using machine learning combined with available on-line process data. The lysine concentration was accurately estimated in both linear and nonlinear models; however, the nonlinear models were also suitable for estimating the concentrations of sucrose and bacterial cells. Data enhancement by time interpolation improved the model prediction accuracy and eliminated unnecessary fluctuations. Furthermore, the soft sensor developed based on the dataset of the same process parameters in multiple fermentor tanks successfully estimated the fermentation behavior of each tank. Machine learning-based soft sensors may represent a novel monitoring system for digital transformation in the field of biotechnological processes.

Integration of constraint-based modeling with fecal metabolomics reveals large deleterious effects of Fusobacterium spp. on community butyrate production

ABSTRACT Characterizing the metabolic functions of the gut microbiome in health and disease is pivotal for translating alterations in microbial composition into clinical insights. Two major analysis paradigms have been used to explore the metabolic functions of the microbiome but not systematically integrated with each other: statistical screening approaches, such as metabolome-microbiome association studies, and computational approaches, such as constraint-based metabolic modeling. To combine the strengths of the two analysis paradigms, we herein introduce a set of theoretical concepts allowing for the population statistical treatment of constraint-based microbial community models. To demonstrate the utility of the theoretical framework, we applied it to a public metagenomic dataset consisting of 365 colorectal cancer (CRC) cases and 251 healthy controls, shining a light on the metabolic role of Fusobacterium spp. in CRC. We found that (1) glutarate production capability was significantly enriched in CRC microbiomes and mechanistically linked to lysine fermentation in Fusobacterium spp., (2) acetate and butyrate production potentials were lowered in CRC, and (3) Fusobacterium spp. presence had large negative ecological effects on community butyrate production in CRC cases and healthy controls. Validating the model predictions against fecal metabolomics, the in silico frameworks correctly predicted in vivo species metabolite correlations with high accuracy. In conclusion, highlighting the value of combining statistical association studies with in silico modeling, this study provides insights into the metabolic role of Fusobacterium spp. in the gut, while providing a proof of concept for the validity of constraint-based microbial community modeling.

Data science-based modeling of the lysine fermentation process

Mathematical modeling of the fermentation process is useful for understanding the influence of operating parameters on target production and control performance, depending on the situation, to stabilize the target production at a high-level. However, the previous approaches using physical modeling methods and traditional knowledge-based methods are difficult to apply on working fermentors at a commercial plant scale because they have unknown and unmeasured parameters involved in target production. This study focused on developing an ensemble learning model that can predict the amino acid fermentation process behavior based on observation values, which can be obtained from fermentation tanks and future control input. The results revealed the influence of each control input on lysine production during the culturing period. Furthermore, high-order stability, which achieved the target trajectory for lysine production, was realized using dynamic fermentation controls. Additionally, this study demonstrates that the fermentation behavior on a commercial plant scale is reproduced using the ensemble device. The ensemble learning model will provide novel control system with data-science based model of Industry 4.0 in the field of biotechnological processes.

Phytoremediation of acid mine drainage using by-product of lysine fermentation.

The main point of this research is to assess the applicability of condensed molasses soluble (CMS), which is an organic by-product of lysine fermentation, as an environmentally friendly complexing agent in rhizofiltration of heavy metal contaminated acid mine drainage (AMD). First, the ecotoxicological properties (growth inhibition, seed germination) of CMS were examined with often applied indicator plant species such as duckweed (Lemna minor) and lettuce (Lactuca sativa) so as to define the possible applicable CMS concentration. Then the heavy metal accumulation and translocation properties of root accumulator plant species, i.e. common reed (Phragmites australis) and sedge (Carex flacca), were studied to optimize CMS concentration for rhizofiltration. Due to the CMS application, significant increase in bioaccumulation was detected in the case of every examined heavy metal (As, Cd, Cu, Pb and Zn) at the end of the experiment. Results also showed that CMS increased the heavy metal concentration in shoots, but did not affect the root accumulation characteristics of the plants. Furthermore, CMS treated plants accumulated heavy metals at higher rates in their roots compared to control. The results suggest that CMS is a viable additive and a complexing agent to aid rhizofiltration of heavy metal contaminated AMD.

Short communication: Lysine retained among 2 lipid-coated lysine products after exposure to alfalfa or corn silage with different amounts of acidity

We conducted 2 experiments to determine lysine loss from 2 lipid-coated lysine products after mixing with silage. In our first experiment, we mixed 2 lipid-coated lysine products, crystalline lysine or crystalline lysine and amounts of lipid identical to amounts included in lipid-coated lysine products, with alfalfa or corn silage that had 2 different amounts of acidity. Lysine appeared to disassociate from lipid-coated lysine products in a nonlinear manner after mixing with either alfalfa or corn silage at different amounts of acidity. Additionally, silage source and acidity affected amounts of lysine released from lipid-coated lysine products after mixing. In a corresponding experiment, in vitro estimates of lysine available to ruminal microbiota after mixing with alfalfa or corn silage at different amounts of acidity were measured by ammonia release. In vitro measures were conducted with or without monensin to allow estimates of effects of monensin on amounts of lysine released from the 2 lipid-coated lysine products. It is unclear whether in vitro estimates of lysine fermentation from lipid-coated lysine are truly reflective of ruminal degradation of lysine from lipid-coated lysine because amounts of time needed to measure differences between different lysine sources were greater than typical estimates of mean ruminal particulate retention time. Nonetheless, monensin apparently reduced ammonia release from lysine, but ammonia release from lipid-coated lysine did not differ from crystalline lysine. Clearly, methods of manufacture together with physical and chemical characteristics of diet can affect amounts of lysine provided from lipid-coated lysine products to ruminants.

Computational minimization of the specific energy demand of large-scale aerobic fermentation processes based on small-scale data

During the assessment of the potential profitability and feasibility of new production routes, aerobic fermentation can turn out to be a major factor contributing to the operating expenditure of the overall process. The accurate evaluation of its energy demand is, therefore, of prime importance. To this end, a tool was developed based on established and generally accepted correlations considering relevant parameters and constraints for bioreactor operation. While the assessment is performed for large-scale reactors (10–100m3), it is based on data from small-scale experiments using the oxygen transfer rate as a scale-up criterion. Among the evaluated constraints, the flooding-loading transition of the bioreactor proved to be a crucial criterion for culture volumes of more than 20m3. Minimum energy demand for cooling, agitation, and aeration could thus be achieved at low agitation and high aeration intensity. At moderate oxygen transfer requirements, bioreactor pressurization was not found to increase energy efficiency of aerobic processes. It was, however, shown to be indispensable for processes with high oxygen demands and elevated foam formation. The tool was applied to assess itaconic acid as well as lysine fermentation processes. Due to considerable differences in oxygen demand, average power consumption was found to be at 0.51kWm−3 (itaconic acid) and 2.61kWm−3 (lysine), thus arguing against the utilization of general rule of thumb values. The established tool, therefore, provides an efficient means to specify estimates of the energy demand in consideration of the respective process.

The metabolism and biotechnological application of betaine in microorganism.

Glycine betaine (betaine) is widely distributed in nature and can be found in many microorganisms, including bacteria, archaea, and fungi. Due to its particular functions, many microorganisms utilize betaine as a functional chemical and have evolved different metabolic pathways for the biosynthesis and catabolism of betaine. As in animals and plants, the principle role of betaine is to protect microbial cells against drought, osmotic stress, and temperature stress. In addition, the role of betaine in methyl group metabolism has been observed in a variety of microorganisms. Recent studies have shown that betaine supplementation can improve the performance of microbial strains used for the fermentation of lactate, ethanol, lysine, pyruvate, and vitamin B12, during which betaine can act as stress protectant or methyl donor for the biosynthesis of structurally complex compounds. In this review, we summarize the transport, synthesis, catabolism, and functions of betaine in microorganisms and discuss potential engineering strategies that employ betaine as a methyl donor for the biosynthesis of complex secondary metabolites such as a variety of vitamins, coenzymes, and antibiotics. In conclusion, the biocompatibility, C/N ratio, abundance, and comprehensive metabolic information of betaine collectively indicate that this molecule has great potential for broad applications in microbial biotechnology.

Co-expression of endoglucanase and β-glucosidase in Corynebacterium glutamicum DM1729 towards direct lysine fermentation from cellulose

The aim of the present study is the development of a consolidated bioprocess for the production of lysine with recombinant Corynebacterium glutamicum DM1729 strains expressing endoglucanase and β-glucosidase genes. Here, the endoglucanase genes from Xanthomonas campestris XCC3521 and XCC2387 and betaglucosidase gene from Saccharophagus degradans Sde1394 were cloned in C. glutamicum DM1729 and expressed either extracellularly or on cell surface. The highest β-glucosidase activity of 9±0.5U/OD600 of 1 and endoglucanase activity of 5.5±0.8U was obtained in C. glutamicum DM 1729 (pVWEx1-TATXCC2387) (pEKEx3-PorC-Sde1394) when cellobiose (20g/L) alone or in combination with carboxymethyl cellulose (20g/L) was used as the carbon sources respectively. The overall efforts resulted in a lysine titre of 5.9±0.5mM. The ability of the constructs to utilize carboxymethyl cellulose and cellobiose for growth and amino acid production proves the concept of utilization of C. glutamicum as a biocatalyst in the lignocellulosic biorefinery.

Lysine Fermentation: History and Genome Breeding.

Lysine fermentation by Corynebacterium glutamicum was developed in 1958 by Kyowa Hakko Kogyo Co. Ltd. (current Kyowa Hakko Bio Co. Ltd.) and is the second oldest amino acid fermentation process after glutamate fermentation. The fundamental mechanism of lysine production, discovered in the early stages of the process's history, gave birth to the concept known as "metabolic regulatory fermentation," which is now widely applied to metabolite production. After the development of rational metabolic engineering, research on lysine production first highlighted the need for engineering of the central metabolism from the viewpoints of precursor supply and NADPH regeneration. Furthermore, the existence of active export systems for amino acids was first demonstrated for lysine in C. glutamicum, and this discovery has resulted in the current recognition of such exporters as an important consideration in metabolite production. Lysine fermentation is also notable as the first process to which genomics was successfully applied to improve amino acid production. The first global "genome breeding" strategy was developed using a lysine producer as a model; this has since led to new lysine producers that are more efficient than classical industrial producers. These advances in strain development technology, combined with recent systems-level approaches, have almost achieved the optimization of entire cellular systems as cell factories for lysine production. In parallel, the continuous improvement of the process has resulted not only in fermentation processes with reduced load on downstream processing but also in commercialization of various product forms according to their intended uses. Nowadays lysine fermentation underpins a giant lysine demand of more than 2 million metric tons per year.

A Novel Acyl-CoA Beta-Transaminase Characterized from a Metagenome

BackgroundBacteria are key components in all ecosystems. However, our knowledge of bacterial metabolism is based solely on the study of cultivated organisms which represent just a tiny fraction of microbial diversity. To access new enzymatic reactions and new or alternative pathways, we investigated bacterial metabolism through analyses of uncultivated bacterial consortia.Methodology/Principal FindingsWe applied the gene context approach to assembled sequences of the metagenome of the anaerobic digester of a municipal wastewater treatment plant, and identified a new gene which may participate in an alternative pathway of lysine fermentation.ConclusionsWe characterized a novel, unique aminotransferase that acts exclusively on Coenzyme A (CoA) esters, and proposed a variant route for lysine fermentation. Results suggest that most of the lysine fermenting organisms use this new pathway in the digester. Its presence in organisms representative of two distinct bacterial divisions indicate that it may also be present in other organisms.

3-Keto-5-aminohexanoate Cleavage Enzyme: A COMMON FOLD FOR AN UNCOMMON CLAISEN-TYPE CONDENSATION

The exponential increase in genome sequencing output has led to the accumulation of thousands of predicted genes lacking a proper functional annotation. Among this mass of hypothetical proteins, enzymes catalyzing new reactions or using novel ways to catalyze already known reactions might still wait to be identified. Here, we provide a structural and biochemical characterization of the 3-keto-5-aminohexanoate cleavage enzyme (Kce), an enzymatic activity long known as being involved in the anaerobic fermentation of lysine but whose catalytic mechanism has remained elusive so far. Although the enzyme shows the ubiquitous triose phosphate isomerase (TIM) barrel fold and a Zn(2+) cation reminiscent of metal-dependent class II aldolases, our results based on a combination of x-ray snapshots and molecular modeling point to an unprecedented mechanism that proceeds through deprotonation of the 3-keto-5-aminohexanoate substrate, nucleophilic addition onto an incoming acetyl-CoA, intramolecular transfer of the CoA moiety, and final retro-Claisen reaction leading to acetoacetate and 3-aminobutyryl-CoA. This model also accounts for earlier observations showing the origin of carbon atoms in the products, as well as the absence of detection of any covalent acyl-enzyme intermediate. Kce is the first representative of a large family of prokaryotic hypothetical proteins, currently annotated as the "domain of unknown function" DUF849.

Mechanism of Concerted Inhibition of α2β2-type Hetero-oligomeric Aspartate Kinase from Corynebacterium glutamicum

Aspartate kinase (AK) is the first and committed enzyme of the biosynthetic pathway producing aspartate family amino acids, lysine, threonine, and methionine. AK from Corynebacterium glutamicum (CgAK), a bacterium used for industrial fermentation of amino acids, including glutamate and lysine, is inhibited by lysine and threonine in a concerted manner. To elucidate the mechanism of this unique regulation in CgAK, we determined the crystal structures in several forms: an inhibitory form complexed with both lysine and threonine, an active form complexed with only threonine, and a feedback inhibition-resistant mutant (S301F) complexed with both lysine and threonine. CgAK has a characteristic alpha(2)beta(2)-type heterotetrameric structure made up of two alpha subunits and two beta subunits. Comparison of the crystal structures between inhibitory and active forms revealed that binding inhibitors causes a conformational change to a closed inhibitory form, and the interaction between the catalytic domain in the alpha subunit and beta subunit (regulatory subunit) is a key event for stabilizing the inhibitory form. This study shows not only the first crystal structures of alpha(2)beta(2)-type AK but also the mechanism of concerted inhibition in CgAK.

Comprehensive proteome profiling of the Fe(III)‐reducing myxobacterium Anaeromyxobacter dehalogenans 2CP‐C during growth with fumarate and ferric citrate

Anaeromyxobacter dehalogenans is a microaerophilic member of the delta-proteobacteria which is able to utilize a wide range of electron acceptors, including halogenated phenols, U(VI), Fe(III), nitrate, nitrite, oxygen and fumarate. To date, the knowledge regarding general metabolic activities of this ecologically relevant bacterium is limited. Here, we present a first systematic 2-D reference map of the soluble A. dehalogenans proteome in order to provide a sound basis for further proteomic studies as well as to gain first global insights into the metabolic activities of this bacterium. Using a combination of 2-DE and MALDI-TOF-MS, a total of 720 proteins spots were identified, representing 559 unique protein species. Using the proteome data, altogether 50 metabolic pathways were found to be expressed during growth with fumarate as primary electron acceptor. An analysis of the pathways revealed an extensive display of enzymes involved in the catabolism and anabolism of a variety of amino acids, including the unexpected fermentation of lysine to butyrate. Moreover, using the reference gel as basis, a semi-quantitative analysis of protein expression changes of A. dehalogenans during growth with ferric citrate as electron acceptor was conducted. The adaptation to Fe(III) reducing conditions involved the expression changes of a total of 239 proteins. The results suggest that the adaptation to Fe(III) reductive conditions involves an increase in metabolic flux through the tricarboxylic acid cycle, which is fueled by an increased catabolism of amino acids.

Lysine degradation by ruminal Fusobacteriumnecrophorum (2010)

Three experiments were conducted to characterize lysine fermentation by Fusobacterium necrophorum, a ruminal bacterium that is known to degrade amino acids. In Experiment 1, 7 strains of Fusobacterium necrophorum were inoculated into media containing lysine (50 mM), lactate (50 mM), or lysine plus lactate (50 mM each) as the major energy substrate to evaluate growth and ammonia production. All strains grew with lysine, lactate, or lactate plus lysine as the primary substrate. When grown with lysine, all strains produced ammonia as an end product, even if lactate was also present. Smaller concentrations of ammonia for medium containing lactate plus lysine when compared with lysine alone indicate that the Fusobacterium strains used lactate as a growth substrate that stimulated utilization of ammonia. In Experiment 2, the 2 strains tested were able to degrade extensively both lysine and glutamic acid. Some evidence was detected for partial utilization for growth of histidine, methionine, and tryptophan by strain A21. In Experiment 3, the minimum inhibitory concentration (MIC) of the antibiotic tylosin was 25 μg/mL when Fusobacterium necrophorum strains A21 and B35 were grown in either lysine or lactate-enriched medium. The MIC of monensin was 6.25 and 3.9 μg/mL for strains A21 and B35, respectively, when grown in lysine-enriched medium, but > 50 and 10.9 μg/mL when the strains were grown in lactate-enriched medium. These findings may lead to ways that ruminal lysine degradation may be controlled.; Dairy Day, 2010, Kansas State University, Manhattan, KS, 2010; Dairy Research, 2010 is known as Dairy Day, 2010

NEURAL NETWORK SOFTWARE SENSORS DESIGN FOR LYSINE FERMENTATION PROCESS

The main goal of this article is to study the possibility for neural network software sensors design for Lysine fermentation process on the basis of only one on-line measurable process variable, in this case—the dissolved oxygen concentration. Software sensors for biomass, lysine, glucose, and ammonium concentrations were designed. It is shown that past values of the dissolved oxygen must also be included in order to get successful results, or to enhance the quality of the estimation. The applicability of the sensors in the framework of a closed loop control system was also investigated.

Selection of lactic acid bacteria for acidification of brown juice (grass juice), with the aim of making a durable substrate for L‐lysine fermentation

AbstractBACKGROUND: Brown juice is a waste product from the green crop‐drying industry. Heating and pressing of green crops prior to drying produces a juice rich in nutrients, which can be converted to a stable universal fermentation medium by lowering its pH to 4.0–4.5 via lactic acid fermentation. The aim of this study was to select a strain of lactic acid bacteria for industrial acidification of brown juice.RESULTS: Several strains were tested in fermentation experiments and Lactobacillus salivarius ssp. salivarius DSM 20555 was found to be the best choice. It showed high growth rates at temperatures ranging from 40 to 48 °C, with an optimum temperature of 46 °C (µmax = 2.2 h−1), and maintained a high productivity at pH 4.0 in continuous fermentation. The highest productivity of 7.23 g L−1 h−1 was found at a dilution rate of 1.0 h−1.CONCLUSION: With today's increased focus on utilisation of residues from food and agro‐industry, coupled with restrictions on their use as fertiliser for farmlands, lactic acid fermentation could play a significant role in conservation of these nutrient‐rich liquids. This study shows that Lb. salivarius ssp. salivarius DSM 20555 is a very promising micro‐organism for use in such a process. Copyright © 2008 Society of Chemical Industry

Biosorption of Reactive black 5 by Corynebacterium glutamicum biomass immobilized in alginate and polysulfone matrices

Corynebacterium glutamicum, a lysine fermentation industry waste, showed promise for the removal of Reactive black 5 (RB5). Due to practical difficulties in solid-liquid separation, the free biomass was immobilized in two polymer matrices: calcium alginate and polysulfone. Initially, the optimization of biomass loading in polymeric beads and bead dosage were examined. Of the different combinations examined, 4% (with bead dosage of 2 g per 40 ml) and 14% (with bead dosage of 1 g per 40 ml) in the case of alginate and polysulfone beads, respectively, were identified as the optimal conditions. According to the Langmuir model, at pH 1, the maximum RB5 uptakes of 352, 282 and 291 mg g(-1) were observed for free, alginate and polysulfone-immobilized biomass, respectively. According to the Weber-Morris model, intraparticle diffusion was found to be the potential rate limiting step for the immobilized beads. Regeneration experiments, with 0.01 M NaOH and Na(2)CO(3) as eluents, revealed that polysulfone beads exhibited invariable RB5 uptake capacity and very high mechanical stability even at the end of twentieth cycle, confirming the technical feasibility of the biosorption process for industrial applications.

Identification of the Last Unknown Genes in the Fermentation Pathway of Lysine

Although the proteins of the lysine fermentation pathway were biochemically characterized more than thirty years ago, the genes encoding the proteins that catalyze three steps of this pathway are still unknown. We combined gene context, similarity of enzymatic mechanisms, and molecular weight comparisons with known proteins to select candidate genes for these three orphan proteins. We used a wastewater metagenomic collection of sequences to find and characterize the missing genes of the lysine fermentation pathway. After recombinant protein production and purification following cloning in Escherichia coli, we demonstrated that these genes (named kdd, kce, and kal) encode a l-erythro-3,5-diaminohexanoate dehydrogenase, a 3-keto-5-aminohexanoate cleavage enzyme, and a 3-aminobutyryl-CoA ammonia lyase, respectively. Because all of the genes of the pathway are now identified, we used this breakthrough to detect lysine-fermenting bacteria in sequenced genomes. We identified twelve bacteria that possess these genes and thus are expected to ferment lysine, and their gene organization is discussed.