Continuous Feeding Strategy Research Articles

Enhancement of ε-poly-L-lysine production by Streptomyces albulus FQF-24 with feeding strategies using cassava starch as carbon source.

ε-Poly-L-lysine (ε-PL) is a natural and wide-spectrum antimicrobial additive. In this study, the production of ε-PL by Streptomyces albulus FQF-24 using cassava starch (CS) as carbon source and the effects of different feeding methods were investigated in a fermenter. The initial shake flask experiments demonstrated the efficient production of ε-PL with CS, achieving the ε-PL production of 1.18g/L. Subsequent investigations in the fermenter identified that the ideal pH was 3.8 during the ε-PL synthesis phase. Under this condition, the production of ε-PL reached 1.35g/L. When the pH was maintained at 3.8, the investigation of improvement of feeding composition was carried out in a 5 L fermenter. The intermittent feeding containing CS, inorganic and organic nitrogen sources resulted in the maximum ε-PL production and dry cell weight (DCW) reaching 17.17g/L and 42.73g/L. Additionally, continuous feeding with the composition of CS, organic and inorganic nitrogen sources, and inorganic salts further increased ε-PL production and DCW to 27.56g/L and 38.5g/L. Summarily, the above results indicate that the fermentation using low-cost CS and continuous feeding strategy with whole medium composition can provide a beneficial reference for the efficient production of ε-PL.

Continuous feeding strategy for polyhydroxyalkanoate production from solid waste animal fat at laboratory- and pilot-scale.

Bioconversion of waste animal fat (WAF) to polyhydroxyalkanoates (PHAs) is an approach to lower the production costs of these plastic alternatives. However, the solid nature of WAF requires a tailor-made process development. In this study, a double-jacket feeding system was built to thermally liquefy the WAF to employ a continuous feeding strategy. During laboratory-scale cultivations with Ralstonia eutropha Re2058/pCB113, 70% more PHA (45 gPHA L-1 ) and a 75% higher space-time yield (0.63 gPHA L-1 h-1 ) were achieved compared to previously reported fermentations with solid WAF. During the development process, growth and PHA formation were monitored in real-time by in-line photon density wave spectroscopy. The process robustness was further evaluated during scale-down fermentations employing an oscillating aeration, which did not alter the PHA yield although cells encountered periods of oxygen limitation. Flow cytometry with propidium iodide staining showed that more than two-thirds of the cells were viable at the end of the cultivation and viability was even little higher in the scale-down cultivations. Application of this feeding system at 150-L pilot-scale cultivation yielded in 31.5 gPHA L-1 , which is a promising result for the further scale-up to industrial scale.

Assessing Intermediate Formation and Electron Competition during Thiosulfate-Driven Denitrification: An Experimental and Modeling Study.

There is increasing interest in thiosulfate-driven denitrification for low C/N wastewater treatment, but the denitrification performance varies with the thiosulfate oxidation pathways. Models have been developed to predict the products of denitrification, but few consider thiosulfate reduction to elemental sulfur (S0), an undesirable reaction that can intensify electron competition with denitrifying enzymes. In this study, the model using indirect coupling of electrons (ICE) was developed to predict S0 formation and electron competition during thiosulfate-driven denitrification. Kinetic data were obtained from sulfur-oxidizing bacteria (SOB) dominated by the branched pathway and were used to calibrate and validate the model. Electron competition was investigated under different operating conditions. Modeling results reveal that electrons produced in the first step of thiosulfate oxidation typically prioritize thiosulfate reduction, then nitrate reduction, and finally nitrite reduction. However, the electron consumption rate for S0 formation decreases sharply with the decline of thiosulfate concentration. Thus, a continuous feeding strategy was effective in alleviating the competition between thiosulfate reduction and denitrifying enzymes. Electron competition leads to nitrite accumulation, which could be a reliable substrate for anammox. The model was further evaluated with anammox integration. Results suggested that the branched pathway and continuous supply of thiosulfate are favorable to create a symbiotic relationship between SOB and anammox.

Continuous Feeding Reduces the Generation of Metabolic Byproducts and Increases Antibodies Expression in Chinese Hamster Ovary-K1 Cells.

Chinese hamster ovary (CHO) cells are the most important host system used for monoclonal antibody (mAb) expression. Moreover, the fed-batch culture mode is the most widely used method to increase mAb expression in CHO cells by increasing the amount of feed. However, a high amount of culture feed results in the production of metabolic byproducts. In this work, we used a continuous feeding strategy to reduce metabolic byproducts and improve mouse–human chimeric anti-epidermal growth factor receptor vIII (EGFRvIII) antibody C12 expression in Chinese hamster ovary-K1 cells. Moreover, the effects of the feeding strategy on the cell culture and monoclonal antibody production were evaluated in chemically defined suspension cultures of recombinant CHO-K1 cells. Compared with bolus feeding methods, the continuous feeding method did not have any advantages when the feeding amount was low, but with a high feeding amount, the continuous feeding method significantly reduced the concentrations of lactate and NH4+ in the later culture stage. At the end of the culture stage, compared with bolus feeding methods, the lactate and NH4+ concentrations under the continuous feeding mode were reduced by approximately 45% and 80%, respectively. In addition, the antibody C12 expression level was also increased by almost 10%. Compared to the bolus feeding method, the antibody C12 produced by the continuous feeding method had a lower content of high-mannose glycoforms. Further analysis found that the osmolality of the continuous feeding method was lower than that of the typical fed-batch bolus feeding method. Conclusively, these results indicate that the continuous feeding method is very useful for reducing metabolic byproducts and achieving higher levels of mAb production.

Feeding strategies for small-scale rearing black soldier fly larvae (Hermetia illucens) as organic waste recycler

Processing organic waste using black soldier fly (BSF)-based technology offers a promising alternative for sustainable organic waste management and urban sanitation. This study was conducted to assess the influence of feeding strategies on the efficacy of BSF larvae to recycle organic wastes into value products. Fruit waste and chicken manure were used as organic waste samples while commercial chicken feed was used as a control, and were processed for 15 days in circular plastic containers (Ø 30 × 12 cm) with 50; 100; 150; and 200 mg/larva/day continuous and batch feeding diets, using 500 four-day-old larvae per diet, repeated four times. Larval survival rates were not significantly affected by the feeding strategies. However, average larval biomass of 83.69 ± 13.04 g and 82.46 ± 08.52 g was achieved for the continuous and batch feeding strategies, respectively, under favorable conditions. Larval feed reduction rates ranged from 24.65 ± 03.48% to 72.78 ± 01.48% and 24.52 ± 0.27% to 72.25 ± 12.13% with continuous and batch feeding strategies, respectively, and were significantly affected by the different daily diets. On the other hand, the bioconversion rates ranged from 13.34 ± 0.26% to 50.82 ± 02.27%, and the highest values were observed with the continuous feeding diets. This study confirms the efficacy of BSF larvae to thrive in different organic substrates and shows that the continuous feeding strategy can be better and enhance a sustainable small-scale organic waste management.

Development of a Strategy to Improve the Stability of Culture Environment for Docosahexaenoic Acid Fermentation by Schizochytrium sp.

A stable culture environment is the key for optimal growth and metabolic activity of microorganisms, especially in marine species, and intermittent feeding during DHA production using Schizochytrium sp. generates an unstable culture environment. To investigate the effect of unstable culture environment on the cells' physiological status and DHA synthesis, fermentations with different feeding strategies were performed on the lab scale. The intermittent feeding strategy caused fluctuations of substrate concentration and osmotic pressure, which had a negative effect on cell division and product synthesis. The physiological status and metabolic level of Schizochytrium sp. were relatively stable under a continuous feeding strategy with a relatively stable substrate concentration of 20-25g/L, which was beneficial for the efficient transformation of substrate, leading to an improvement of DHA productivity. This strategy was further applied to pilot scale, whereby the DHA content, DHA productivity, convert ratio of glucose to lipid and DHA reached 55.02%, 320.17mg/(L·h), 24.35%, and 13.40%, respectively. This study therefore provides an efficient strategy for ensuring a stable culture environment for the production of DHA and similar metabolites. Graphical Abstract.

High-level production of 3-hydroxypropionic acid from glycerol as a sole carbon source using metabolically engineered Escherichia coli.

As climate change is an important environmental issue, the conventional petrochemical-based processes to produce valuable chemicals are being shifted toward eco-friendly biological-based processes. In this study, 3-hydroxypropionic acid (3-HP), an industrially important three carbon (C3) chemical, was overproduced by metabolically engineered Escherichia coli using glycerol as a sole carbon source. As the first step to construct a glycerol-dependent 3-HP biosynthetic pathway, the dhaB1234 and gdrAB genes from Klebsiella pneumoniae encoding glycerol dehydratase and glycerol reactivase, respectively, were introduced into E. coli to convert glycerol into 3-hydroxypropionaldehyde (3-HPA). In addition, the ydcW gene from K. pneumoniae encoding γ-aminobutyraldehyde dehydrogenase, among five aldehyde dehydrogenases examined, was selected to further convert 3-HPA to 3-HP. Increasing the expression level of the ydcW gene enhanced 3-HP production titer and reduced 1,3-propanediol production. To enhance 3-HP production, fed-batch fermentation conditions were optimized by controlling dissolved oxygen (DO) level and employing different feeding strategies including intermittent feeding, pH-stat feeding, and continuous feeding strategies. Fed-batch culture of the final engineered E. coli strain with DO control and continuous feeding strategy produced 76.2 g/L of 3-HP with the yield and productivity of 0.457 g/g glycerol and 1.89 g·L-1 ·h-1 , respectively. To the best of our knowledge, this is the highest 3-HP productivity achieved by any microorganism reported to date.

Continuous cultivation strategy for yeast industrial wastewater-based polyhydroxyalkanoate production

Polyhydroxyalkanoates (PHAs) present an eco-friendly alternative for conventional plastics. Industrial wastewater from the food industry is a copious source of organic carbon that can be recovered in the form of PHA. However, the wastewater composition varies considerably among the different industries demanding for an industry-specific investigation of the PHA production process. Wastewater from the yeast industry, besides its high concentration in organic carbon, also contains a high ammonium concentration which might decrease the PHA production. Thus, this study aims to investigate PHA production using yeast industry wastewater via an enriched mixed microbial culture (MMC). A less explored cultivation strategy, i.e., continuous cultivation for PHA production was evaluated and compared to the widely used batch cultivation. PHA accumulating MMC was enriched using a sequencing batch reactor (SBR) operated under aerobic dynamic feeding. An MMC dominated by the Thauera species was successfully enriched in the SBR. Experimental results showed that ammonium is indeed required for PHA accumulation and the complete absence of ammonium negatively affects the accumulation process. Using wastewater, batch and continuous feeding strategies, respectively, yielded the PHA accumulation of 72 % and 65 % per dry cell weight. Despite the slightly lower PHA accumulation with continuous cultivation, four times more biomass growth was produced. Consequently, higher theoretical PHA production (270 t/year) can be expected using continuous cultivation in half of the reactor volume (45m3). Therefore, this study asserts the viability of continuous cultivation as a feasible investigatory tool and PHA production strategy.

Improved fed-batch production of high-purity PHB (poly-3 hydroxy butyrate) by Cupriavidus necator (MTCC 1472) from sucrose-based cheap substrates under response surface-optimized conditions.

Experimental investigations were carried out for Cupriavidus necator (MTCC 1472)-based improved production of poly-3 hydroxy butyrate (PHB) through induced nitrogen limiting fed-batch cultivation strategies. Initially Plackett-Burman design and response surface methodology were implemented to optimize most influencing process parameters. With optimized process parameter values, continuous feeding strategies ware applied in a 5-l fermenter with table sugar concentration of 100g/l, nitrogen concentration of 0.12g/l for fed-batch fermentation with varying dilution rates of 0.02 and 0.0461/h. To get enriched production of PHB, concentration of the sugar was further increased to 150 and 200g/l in feeding. Maximum concentrations of PHB achieved were 22.35 and 23.07g/l at those dilution rates when sugar concentration maintains at 200g/l in feeding. At maximum concentration of PHB (23.07g/l), productivity of 0.58g/lh was achieved with maximum PHB accumulation efficiency up to 64% of the dry weight of biomass. High purity of PHB, close to medical grade was achieved after surfactant hypochlorite extraction method, and it was further confirmed by SEM, EDX, and XRD studies.

DNA-SIP based genome-centric metagenomics identifies key long-chain fatty acid-degrading populations in anaerobic digesters with different feeding frequencies

Fats, oils and greases (FOG) are energy-dense wastes that can be added to anaerobic digesters to substantially increase biomethane recovery via their conversion through long-chain fatty acids (LCFAs). However, a better understanding of the ecophysiology of syntrophic LCFA-degrading microbial communities in anaerobic digesters is needed to develop operating strategies that mitigate inhibitory LCFA accumulation from FOG. In this research, DNA stable isotope probing (SIP) was coupled with metagenomic sequencing for a genome-centric comparison of oleate (C18:1)-degrading populations in two anaerobic codigesters operated with either a pulse feeding or continuous-feeding strategy. The pulse-fed codigester microcosms converted oleate into methane at over 20% higher rates than the continuous-fed codigester microcosms. Differential coverage binning was demonstrated for the first time to recover population genome bins (GBs) from DNA-SIP metagenomes. About 70% of the 13C-enriched GBs were taxonomically assigned to the Syntrophomonas genus, thus substantiating the importance of Syntrophomonas species to LCFA degradation in anaerobic digesters. Phylogenetic comparisons of 13C-enriched GBs showed that phylogenetically distinct Syntrophomonas GBs were unique to each codigester. Overall, these results suggest that syntrophic populations in anaerobic digesters can have different adaptive capacities, and that selection for divergent populations may be achieved by adjusting reactor operating conditions to maximize biomethane recovery.

Loss-in-Weight Feeding Trials Case Study: Pharmaceutical Formulation

This article presents a case study of a continuous feeding strategy for five pharmaceutical components (active pharmaceutical ingredient (API), Prosolv HD90, crospovidone, magnesium stearate, and colloidal silicon dioxide), for the purpose of developing a direct compression continuous manufacturing system. Feeding options for each of these five powders were examined. Prosolv HD90 and crospovidone easily resulted in optimal feeding conditions, whereas the remaining powders in the formulation presented some challenges. The API displayed flow issues, leading to clogging of the screen with small openings. Magnesium stearate exhibited shear sensitivity; increasing shear of the powder due to hopper agitation resulted in drifting feeding performance. Colloidal silicon dioxide exhibited electrostatic issues that render most tooling options unsuitable for steady operation. All of these difficulties were resolved using the methods described in this article.

Statistical Modeling of Batch Versus Continuous Feeding Strategies for Pollutant Removal by Tropical Subsurface Flow Constructed Wetlands

In this study, six mesocosms, including three subsurface flow constructed wetlands (SSF CWs) and three sand filters (without plants) were set up at the campus of Nanyang Technological University, Singapore. The objective of this study was to compare the removal efficiencies of chemical oxygen demand (COD), nitrogen (N) and total phosphorus (TP) under batch and continuous operational modes. Three factors, namely, with/without the presence of vegetation, operational modes (batch and continuous) and hydraulic residence time were investigated by two statistical models including factorial design and its rationale analysis and a quadratic polynomial regression model with ANOVA, to find the relationships between the contaminant removal efficiencies and the affecting factors, as well as to determine the dominant variables and how each of them interact for each parameter.

Batch versus continuous feeding strategies for pharmaceutical removal by subsurface flow constructed wetland

This study evaluated the effect of continuous and batch feeding on the removal of 8 pharmaceuticals (carbamazepine, naproxen, diclofenac, ibuprofen, caffeine, salicylic acid, ketoprofen and clofibric acid) from synthetic wastewater in mesocosm-scale constructed wetlands (CWs). Both loading modes were operated at hydraulic application rates of 5.6 cm day−1 and 2.8 cm day−1. Except for carbamazepine, clofibric acid and naproxen, removal in CWs was significantly (p < 0.05) enhanced under the batch versus continuous mode. For all compounds tested except naproxen, values for first-order decay constants (k) for drain and fill operation were higher than that for the continuous mode of operation. Correlation between the distribution coefficient (log Dow) and removal efficiencies of pharmaceutical compounds in the CWs, showed that pharmaceutical removal efficiency was significantly (p < 0.1) and inversely correlated with log Dow value, but not with log Kow value.

Refeeding in anorexia nervosa

This paper reviews recent publications about the physiology associated with adaptation to malnutrition and refeeding (including the refeeding syndrome) and clinical outcomes of refeeding paradigms. A number of recent reviews and original publications have highlighted important differences from the assumptions underpinning the current refeeding guidelines for patients with anorexia nervosa. The notion of 'starting low and going slow' with the prescription of daily calories seems unlikely to be important in preventing refeeding syndrome. Recent publications suggest this approach does not necessarily add to safety in the refeeding process but rather the contrary. It typically results in weight loss and protracts hospitalization and nutritional recovery. Rather, the composition of macronutrients, in particular avoiding a high proportion of calories from carbohydrates, appears to be more important than the absolute number of calories. The means of initial refeeding appears increasingly important in this process, particularly following descriptions of postprandial hypoglycemia. The study supports a review of the current guidelines. Evidence for the use of continuous feeding strategies with less than 40% of calories from carbohydrates is presented. This approach has important implications for the prevention of the refeeding syndrome as well as the safety and efficiency with which refeeding may occur for children and adolescents with anorexia nervosa in hospital.

Nitrous oxide emissions from surface flow and subsurface flow constructed wetland microcosms: Effect of feeding strategies

The effects of continuous and intermittent feeding strategies on nitrogen removal and N 2O emission from surface flow and subsurface flow constructed wetlands were evaluated in this study. Microcosm wetlands planted with Phragmites australis were constructed and operated with different feeding strategies for the 4-month experiment. Results showed the intermittent feeding strategy could enhance the removal of ammonium effectively in the subsurface flow constructed wetlands, although it had no significant effect for the surface flow wetlands. And the intermittent feeding mode could promote the emission of N 2O. The amount of N 2O–N emission from the subsurface flow constructed wetlands with intermittent feeding mode was about 5 times higher than that with continuous feeding strategy and the emission rate ranged from 0.09 ± 0.03 to 7.33 ± 1.49 mg/m 2/h. Compared with the surface flow constructed wetlands, the N 2O emission in the subsurface flow constructed wetlands was affected significantly by the intermittent feeding mode.

Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: Effect of substrate composition and feeding regime on PHA productivity, composition and properties

In this study, the possibility of manipulating biopolymer composition in mixed culture polyhydroxyalkanoate (PHA) production from fermented molasses was assessed by studying the effects of substrate volatile fatty acid (VFA) composition and feeding regime (pulse wise versus continuous). It was found that the use of a continuous feeding strategy rather than a pulse feeding strategy can not only help mitigate the process constraints of the pulse-feeding strategy (resulting in higher specific and volumetric productivities) but also be used as means to broaden the range of polymer structures. Continuous feeding increased the hydroxyvalerate content by 8% relatively to that obtained from the same feedstock using pulse wise feeding. Therefore, the feeding strategy can be used to manipulate polymer composition. Furthermore, the range of PHA compositions, copolymers of P(HB-co-HV) with HV fraction ranging from 15 to 39%, obtained subsequently resulted in different polymer properties. Increasing HV content resulted in a decrease of the average molecular weight, the glass transition and melting temperatures and also in a reduction in the crystallinity degree from a semi-crystalline material to an amorphous matrix.

Optimization of l-methionine feeding strategy for improving S-adenosyl-l-methionine production by methionine adenosyltransferase overexpressed Pichia pastoris

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-L: -methionine (SAM). Two L: -methionine (L: -Met) addition strategies were used to supply the precursor: the batch addition strategy (L: -Met was added separately at three time points) and the continuous feeding strategies (L: -Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l(-1) h(-1), respectively). SAM accumulation, L: -Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 +/- 0.31 g l(-1), 41.7 +/- 1.4%, and 0.18 +/- 0.01 g l(-1) h(-1) with the best continuous feeding strategy (0.2 g l(-1) h(-1)), respectively. The bottleneck for SAM production with the low L: -Met feeding rate (0.1 g L(-1) h(-1)) was the insufficient L: -Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing L: -Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the L: -Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the L: -Met feeding rate reached 0.5 g l(-1) h(-1).

Functional biosynthesis of an allophycocyan beta subunit in Escherichia coli

Allophycocyanin is a phycobiliprotein with various biological and pharmacological properties. An expression vector was constructed using CpeS as the bilin lyase for the allophycocyanin beta subunit, resulting in overexpression of a fluorescent allophycocyanin beta-subunit in Escherichia coli. A high-density cell culture was developed using a continuous feeding strategy. After 16 h of culture, the dry cell density reached 21.4 g l(-1), the expression of the allophycocyanin beta-subunit was 0.86 g l(-1) broth, and the relative chromoprotein yield was 81.4%. The recombinant protein showed spectral features similar to native allophycocyanin, which provide an efficient methodology for large-scale production of this valuable fluorescent protein.

Enhancing xanthan fermentations by different modes of glucose feeding

This paper is the fourth in a series aimed at improving the understanding and operation of conventional agitated fermenters for the production of the commercially important gum, Xanthan. In the first, reproducible fermentations were established and this protocol was used in studies of different agitator types and of bulk mixing and dissolved oxygen concentration in the next two. Here, building on the previous work, the influence of different glucose feeding strategies on Xanthan production in a 20-L agitated fermenter under equivalent conditions of agitation and dissolved oxygen is reported. The biological performances in three types of fed-batch cultures (a two-step glucose addition, multiple glucose-pulse feeding and continuous feeding of glucose) are compared to two batch fermentations with different initial glucose concentrations. The work confirmed that improved performance cannot be achieved by increasing the initial glucose concentration above 50 g/L nor by a single 10 g/L pulse addition (initial glucose concentration of 40 g/L) while significant nitrogen is still present. On the other hand, the simple pulse and continuous feeding strategies, after nitrogen has been essentially exhausted and under conditions of nonlimiting dissolved oxygen and similar bulk mixing, can result in a greatly enhanced performance compared to batch fermentations. Using the final Xanthan gum concentration, the yield on glucose and the overall productivity as performance indices, values of 62 g/L, 0.82 g of Xanthan/g of glucose, and 0.72 g/(L.h), respectively, were obtained compared to literature values for conventional stirred bioreactors of 15-30 g/L, 0.27-0.86 g of Xanthan/g of glucose, and 0. 12-0.43 g/(L.h).

Foraging strategy and predator avoidance behaviour: an intraspecific approach

Relationships between predator avoidance behaviour (scanning and flocking) and foraging were studied in Calidris alpina, to test predictions regarding the effect of foraging techniques on such behaviours. The scanning hypothesis predicts that individuals with a tactile hunting technique and individuals with a visual hunting technique (both continuous searchers) do not differ in any variable related to scanning behaviour. The flocking hypothesis predicts that visually hunting individuals witl tend to form smaller flocks than tactile-foraging individuals. The two continuous feeding strategies did not differ among individuals in vigilance rate, nor in vigilance time or mean scan duration. However, with respect to flocking behaviour, visual foragers differed from tactile foragers in foraging flock size. The relationships between flocking behaviour and foraging strategy are discussed. The pattern found at the intraspecific level are the same as those found at interspecific level.