Search Results (572)

In recent years, inulin enzymatic hydrolysis has become a very promising alternative for producing fructose on a large scale. Genetically modified chicory was used to extract inulin of industrial quality. By using an adequate kinetic model from the literature, this study aimed to determine the optimal operating alternatives of a batch (BR) or fed-batch (FBR) reactor used for the hydrolysis of inulin to fructose. The operation of the FBR with a constant or variable/dynamic feeding was compared to that of the BR to determine which best maximizes reactor production while minimizing enzyme consumption. Multi-objective optimal solutions were also investigated by using the Pareto-optimal front technique. Our in-silico analysis reveals that, for this enzymatic process, the best alternative is the FBR operated with a constant control variable but using the set-point given by the (breakpoint) of the Pareto optimal front under the imposed technological constraints. This set point reported the best performances, regarding all the considered opposite economic objectives. Also, the FBR with a constant, but NLP optimal feeding, reported fairly good performances. Full article

Due to the barriers imposed by the restriction–modification (RM) system, Nisin-producing industrial strains of Lactococcus lactis often encounter low transformation efficiency, which seriously hinders the widespread application of genetic engineering in non-model L. lactis. Herein, we present a novel pre-modification strategy (PMS) coupled with optimized plasmid delivery systems designed to systematically evade RM barriers and substantially improve Nisin biosynthesis in L. lactis. Through the use of engineered Escherichia coli strains with methylation profiles specifically optimized for L. lactis C20, we have effectively evaded RM barriers, thereby facilitating the efficient introduction of large Nisin biosynthetic gene clusters into L. lactis. The PMS tools, which significantly improve the transformation efficiency (~10³ transformants per microgram of DNA), have been further improved in combination with a Rolling Circle Amplification, resulting in a higher enhancement in transformation efficiency (~10⁴ transformants per microgram of DNA). Using this strategy, large Nisin biosynthetic gene clusters and the expression regulation of all genes within the cluster were introduced and analyzed in L. lactis, leading to a highest Nisin titer of 11,052.9 IU/mL through a fed-batch fermentation in a 5 L bioreactor. This is the first systematic report on the expression regulation and application of a complete Nisin biosynthesis gene cluster in L. lactis. Taken together, our studies provide a versatile and efficient strategy for systematic evasion and enhancement of RM barriers and Nisin biosynthesis, thereby paving the way for genetic modification and metabolic engineering in L. lactis. Full article

In the context of the surfactant-enhanced remediation of polluted sites, this work focuses on the development of non-ionic alkylpolyglucosidic (APG) surfactant formulations with different additives for the solubilization and mobilization of strongly adsorbed organic pollutants. The study involved three consecutive phases to evaluate the effect of the additives on surface behavior and the potential improvement in alkylpolyglycoside surfactant’s capability to solubilize toluene and perchloroethylene (PCE), selected as reference contaminants. After a chemical–physical characterization phase, the APG-based formulations were first used in a batch configuration test, in which the formulations’ solubilization ability was indirectly assessed by observing the effect on pollutants’ adsorption. Lastly, a continuous configuration column experiment was performed to simulate the flushing process of a synthetic matrix previously contaminated with strongly adsorbed toluene or PCE. The results showed that the presence of additives firstly reduced the ability of the surfactant to form micelles, increasing the CMC, but at the same time improved the ability to reduce surface tension. Moreover, the addition of the additives overall resulted in a significant improvement in adsorbed pollutant removal in a minimal volume of fed solution, reaching 96% and 99% efficiencies for toluene and PCE, respectively, compared with 76% and 92%, for toluene and PCE, respectively, in the presence of free-additive APG surfactant. Full article

Using a laboratory-scale system, consisting of a primary disinfection tank (PDT) and three intermittently mixed reactors (R1–R3) in series, bulk water and biofilm contributions to chlorine decay were quantified. The reactors (surface-to-volume ratio: 23.7 m⁻¹; retention time in each reactor: 42.6 ± 1.18 h) were fed with plant-filtered water (PFW). Secondary disinfection was carried out in R1. Free chlorine concentration decreased with travel time (R1: 1.2 mg/L; R2: 0.6 mg/L; and R3: 0.12 mg/L). The bacterial number (ATP) decreased from 67 pg/mL in PFW and remained at ~2–3 pg/mL in R1 and R2 but increased back to 68 pg/mL in R3. First-order chlorine decay rate coefficients decreased from R1 to R2, as expected, but increased by five-fold from R2 to R3. The increased bacterial number (ATP) in R3 and batch chlorine decay tests confirmed that bulk water (soluble compounds, microbes, and sediments) contributed approximately 40% of the decay, and the biofilm contributed 60% in R3. When ATP levels in the reactors were combined with literature data, the bacterial number increased significantly when free chlorine decreased below 0.2 mg/L, but data between 0.2 and 0.5 mg/L are limited. More investigation is needed in the future for chlorine < 0.5 mg/L regarding bacterial regrowth and its effect on bulk water chlorine decay. Full article

The production of biodiesel from single-cell oils (SCOs) utilizing industrial wastes as feedstock presents an economically viable approach. To date, studies have rarely reported the utilization of vinasse combined with industrial glycerol for the production of SCO. This study aimed to assess the performance of a Rhodotorula toruloides strain in vinasse from ethanol distilleries supplemented with pure/raw glycerol as an affordable carbon feedstock for SCO production. Several critical factors, including the C/N ratio, the impact of impurities in the crude glycerol, the proper nitrogen source, and the effects of the vinasse compositions, were evaluated. The results showed that the incorporation of urea and raw glycerol increased the lipid content to 51.8 ± 1.6% and the lipid productivity to 0.034 ± 0.001 g L⁻¹h⁻¹. Elevated biomass (42.5 g L⁻¹) and lipid (11.0 g L⁻¹) concentrations indicated that impurities in the raw glycerol positively affected the growth and lipid accumulation of this strain. Notably, supplementing raw glycerol to the vinasse led to a 16.1% increase in biomass concentration and a 25.7% rise in lipid content, significantly enhancing lipid productivity by 59.6%. The fatty acid profile predominantly featured unsaturated fatty acids (96.8%), including high percentages of stearic acid (41.8 ± 2.6%), palmitic acid (21.8 ± 1.5%), and oleic acid (18.3 ± 1.4%), aligning with the standards for vegetable-oil-based biodiesel manufacture. Fed-batch strategies using pulse-feeding turned out to be less effective than the constant-flow feeding strategy with vinasse supplemented with raw glycerol, which achieved a higher lipid productivity of 0.30 g L⁻¹h⁻¹. Full article

Traditional algorithms and single predictive models often face challenges such as limited prediction accuracy and insufficient modeling capabilities for complex time-series data in fault prediction tasks. To address these issues, this paper proposes a combined prediction model based on an improved temporal convolutional network (TCN) and bidirectional long short-term memory (BiLSTM), referred to as the TCN-BiLSTM model. This model aims to enhance the reliability and accuracy of time-series fault prediction. It is designed to handle continuous processes but can also be applied to batch and hybrid processes due to its flexible architecture. First, preprocessed industrial operation data are fed into the model, and hyperparameter optimization is conducted using the Optuna framework to improve training efficiency and generalization capability. Then, the model employs an improved TCN layer and a BiLSTM layer for feature extraction and learning. The TCN layer incorporates batch normalization, an optimized activation function (Leaky ReLU), and a dropout mechanism to enhance its ability to capture multi-scale temporal features. The BiLSTM layer further leverages its bidirectional learning mechanism to model the long-term dependencies in the data, enabling effective predictions of complex fault patterns. Finally, the model outputs the prediction results after iterative optimization. To evaluate the performance of the proposed model, simulation experiments were conducted to compare the TCN-BiLSTM model with mainstream prediction methods such as CNN, RNN, BiLSTM, and A-BiLSTM. The experimental results indicate that the TCN-BiLSTM model outperforms the comparison models in terms of prediction accuracy during both the modeling and forecasting stages, providing a feasible solution for time-series fault prediction. Full article

Direct-fed microbials (DFMs) have shown the potential to improve livestock performance and overall health. Extensive research has been conducted to identify new DFMs and understand their mechanisms of action in the gut. Bacillus species are multifunctional spore-forming bacteria that exhibit resilience to harsh conditions, making them ideal candidates for applications in the feed industry and livestock production. This study investigates the mode of action of B. licheniformis and B. subtilis in the rumen using diverse in vitro techniques. Our results revealed that both strains germinated and grew in sterile rumen and intestinal contents from dairy cows and bulls. Gas composition analysis of in vitro cultures in a medium containing 40% rumen fluid demonstrated that germination of B. licheniformis and B. subtilis strains reduced oxygen levels, promoting an anaerobic environment favorable to rumen microbes. Enzymatic activity assays showed that B. licheniformis released sugars from complex substrates and purified polysaccharides in filtered rumen content. Additionally, the combination of B. licheniformis and B. subtilis survived and grew in the presence of a commercial monensin dose in rumen fluid media. The effects of B. licheniformis and B. subtilis on rumen fermentation activity and microbiota were studied using an in vitro batch fermentation assay. In fermenters that received a combination of B. licheniformis and B. subtilis, less CO₂ was produced while dry matter degradation and CH₄ production was comparable to the control condition, indicating better efficiency of dry matter utilization by the microbiota. The investigation of microbiota composition between supplemented and control fermenters showed no significant effect on alpha and beta diversity. However, the differential analysis highlighted changes in several taxa between the two conditions. Altogether, our data suggests that the administration of these strains of Bacillus could have a beneficial impact on rumen function, and consequently, on health and performance of ruminants. Full article

Background: In biotechnology, B. subtilis is established for heterologous protein production. In addition, the species provides a variety of bioactive metabolites, including the non-ribosomally produced surfactin lipopeptide. However, to control the formation of the target product-forming enzyme, different expression systems could be introduced, including the principle of genetic code expansion by the incorporation of externally supplied non-canonical amino acids. Methods: Integration of an amber stop codon into the srfA operon and additional chromosomal integration of an aminoacyl-tRNA synthetase/tRNA mutant pair from Methanococcus jannaschii enabled site-directed incorporation of the non-canonical amino acid O-methyl-L-tyrosine (OMeY). In different fed-batch bioreactor approaches, OMeY-associated surfactin production was quantified by high-performance thin-layer chromatography (HPTLC). Physiological adaptations of the B. subtilis production strain were analyzed by mass spectrometric proteomics. Results: Using a surfactin-forming B. subtilis production strain, which enables high cell density fermentation processes, the principle of genetic code expansion was introduced. Accordingly, the biosynthesis of the surfactin-forming non-ribosomal peptide synthetase (NRPS) was linked to the addition of the non-canonical amino acid OMeY. In OMeY-associated fed-batch bioreactor fermentation processes, a maximum surfactin titre of 10.8 g/L was achieved. In addition, the effect of surfactin induction was investigated by mass spectrometric proteome analyses. Among other things, adaptations in the B. subtilis motility towards a more sessile state and increased abundances of surfactin precursor-producing enzymes were detected. Conclusions: The principle of genetic code expansion enabled a precise control of the surfactin bioproduction as a representative of bioactive secondary metabolites in B. subtilis. This allowed the establishment of inducer-associated regulation at the post-transcriptional level with simultaneous use of the native promoter system. In this way, inductor-dependent control of the production of the target metabolite-forming enzyme could be achieved. Full article

This paper presents a new method of batch-to-batch optimization control for a fed-batch fermentation process. A recursively updated extreme learning machine (ELM) neural network model is used to model a fed-batch fermentation process. ELM models have advantages over other neural networks in that they can be trained very fast and have good generalization performance. However, the ELM model loses its predictive abilities in the presence of batch-to-batch process variations or disturbances, which lead to a process–model mismatch. The recursive least squares (RLS) technique takes the model prediction error from the previous batch and uses it to update the model parameters for the next batch. This improves the performance of the model and helps it to respond to any changes in process conditions or disturbances. The updated model is used in an optimization control procedure, which generates an improved control profile for the next batch. The update of the RLS model enables the optimization control strategy to maintain a high final product quality in the presence of disturbances. The proposed batch-to-batch optimization control method is demonstrated on a simulated fed-batch fermentation process. Full article

Succinic acid is applied in many chemical industries in which it can be produced through microbial fermentation using lignocellulosic biomasses. Mixed-waste office paper (MWOP) containing lignocellulosic fibers is enormously generated globally. MWOP is recycled into toilet paper and cardboard, but the recovery process is costly. The reuse of MWOP to alternatively produce succinic acid is highly attractive. In this study, pretreatment of MWOPs with 1% (v/v) H₂SO₄ at 121 °C for 20 min was found to be optimal. The optimal conditions for the enzymatic hydrolysis of H₂SO₄-pretreated MWOP (AP-MWOP) were at 50 °C, with cellulase loading at 80 PCU/g AP-MWOP. This resulted in the highest glucose (22.46 ± 0.15 g/L) and xylose (5.11 ± 0.32 g/L). Succinic acid production via separate hydrolysis and fermentation (SHF) by Escherichia coli KJ122 reached 28.19 ± 0.98 g/L (productivity of 1.17 ± 0.04 g/L/h). For simultaneous saccharification and fermentation (SSF), succinic acid was produced at 24.58 ± 2.32 g/L (productivity of 0.82 ± 0.07 g/L/h). Finally, succinic acid at 51.38 ± 4.05 g/L with yield and productivity of 0.75 ± 0.05 g/g and 1.07 ± 0.08 g/L/h was achieved via fed-batch pre-saccharified SSF. This study not only offers means to reuse MWOP for producing succinic acid but also provides insights for exploiting other wastes to high-value succinic acid, supporting environmental sustainability and zero-waste society. Full article

Background: The Dutch branch organization for pet products promised the public that it will improve the quality of raw meat-based diets (RMBDs) for dogs after several diagnoses of tuberculosis, brucellosis, and hyperthyroidism in dogs fed RMBDs. Objective: The objective of this study was to re-evaluate the risk factors of commercially available raw meat diets for dogs in The Netherlands. Methods: Seven commercial brands of RMBDs that were previously investigated were re-tested, as well as a newly introduced high-pressure processing (HPP) product. Raw beef sausage for humans was included for comparison. In total, 40 animal RMBDs (five batches per product) were tested for the presence of colony-forming units (CFUs), Salmonella spp., and Escherichia coli directly after defrosting and 4 h later, as well as thyroid hormone. Results: Exceeded EU standards for CFUs and Salmonella bacteria were present in several samples. In the HPP product, bacteria were still present; however, the counts were lower. There were no differences in CFUs directly after defrosting and 4 h later. The human raw meat product was negative for bacteria. Thyroid hormone could be detected in 20 out of 37 samples. In seven of these samples, the levels were >0.75 µg/g, which have been associated with hyperthyroidism. Conclusions: The hygiene (including the use of HPP production) and accurate removal of thyroid tissue during the production of RMBDs still need attention to prevent the presence of zoonotic bacteria, high CFUs, and diet-induced hyperthyroidism. Full article

Myoglobin (MG) is a heme-binding protein and can be used as a color and flavor additive for artificial meat. After the selection of stable constitutive expression, although the synthesis of porcine myoglobin (pMG) was achieved through the application of a modified GAP promoter (G1 promoter) in Komagataella phaffii, the lower titer of pMG cannot meet the requirements of commercial production. Herein, another powerful constitutive promoter (GCW14 promoter) was chosen and modified through randomizing its core region for the first time, leading to an increase of 1.18 to 6.01 times in strength. In addition, under the control of a mutated P_GCW14 promoter (P_GCWm-121), the titer of pMG was further enhanced by optimizing the integrated copy numbers of the pMG gene and knocking out the Yps1-1 protease. Applying the best engineered strain and suitable fermentation conditions, the highest titer of pMG (547.59 mg/L) was achieved in fed-batch fermentation using a cheap and chemically synthesized medium. Furthermore, the obtained pMG had similar peroxide-specific activity (427.50 U/mg) with the extracted natural product after the food-grade purification. The applied strategy can be utilized to synthesize other high value-added hemoproteins, enriching the applications of functional components in the field of artificial meat. Full article

The significant microbiota variability represents a key feature that makes the full comprehension of the functional interaction between microbiota and the host an ongoing challenge. To overcome this limitation, in this study, fish intestinal microbiota was analyzed through a meta-analysis, identifying the core microbiota and constructing stochastic Bayesian network (BN) models with SAMBA. We combined three experiments performed with gilthead sea bream juveniles of the same hatchery batch, reared at the same season/location, and fed with diets enriched on processed animal proteins (PAP) and other alternative ingredients (NOPAP-PP, NOPAP-SCP). Microbiota data analysis disclosed a high individual taxonomic variability, a high functional homogeneity within trials and highlighted the importance of the core microbiota, clustering PAP and NOPAP fish microbiota composition. For both NOPAP and PAP BNs, >99% of the microbiota population were modelled, with a significant proportion of bacteria (55–69%) directly connected with the diet variable. Functional enrichment identified 11 relevant pathways expressed by different taxa across the different BNs, confirming the high metabolic plasticity and taxonomic heterogeneity. Altogether, these results reinforce the comprehension of the functional bacteria–host interactions and in the near future, allow the use of microbiota as a species-specific growth and welfare benchmark of livestock animals, and farmed fish in particular. Full article

The Sierra Peña Blanca (SPB) region in Chihuahua, Mexico contains a significant uranium deposit representing about 40% of the country’s reserves. Common uranium minerals in this area include uranophane, schoepite, and weeksite/boltwoodite, with several superficial occurrences. Mining activities in the 1980s left unprocessed uranium ore exposed to weathering, with potential transport towards Laguna del Cuervo. This study presents an experimental simulation of uranium transport in SPB sediments using three approaches: (i) a batch experiment to evaluate the ideal adsorption of (UO₂)²⁺ by fine sediment; (ii) a column system fed with 569 mgU L⁻¹ UO₂(NO₃)₂ to simulate adsorption by different sediment particle sizes; (iii) a column system with an upper horizon of uranophane from the area, fed with deionized water, to simulate uranium weathering and transport in particulate material, determined by liquid scintillation counting, revealed that the clay fraction had the highest adsorption capacity for U. X-ray Absorption Fine Structure (XAFS) analysis at the U L₃ edge confirmed the U(IV) oxidation state and the fittings of the extended XAFS spectra confirmed the presence of the uranophane group of minerals. X-ray tomography further corroborated the distribution of particulate minerals along the column. The results suggest that the primary transport mechanism in SPB involves the fragmentation of uranium minerals, accompanied by eventual dissolution and subsequent adsorption of U onto sediments. Full article

Steam explosion (SE) is an effective lignocellulose pretreatment technology for second-generation L-lactic acid (L-LA) production. In this study, targeted to produce high-concentration L-LA from corn stover (CS), the fed-batch simultaneous saccharification and fermentation (SSF) of acidic, SE-pretreated CS was developed and demonstrated in a 5 L scale bioreactor under non-strict conditions (without detoxification and sterilization). The results indicated that the fed-batch SSF, with a simple pH control, realized a higher tolerance of the strains to the toxic by-products of hydrolysate, in comparison to the conventional sequential hydrolysis and fermentation (SHF), allowing for 153.8 g L⁻¹ of L-LA production, along with a productivity of 1.83 g L⁻¹ h⁻¹ in a system with a total of 40% (w/v) solid loading. The mass balance indicated that up to 449 kg of L-LA can be obtained from 1 t of dried CS. It exhibited obvious superiorities and laid down a solid foundation for the industrialization of second-generation L-LA production. Full article