Sustainable menaquinone-7 production through continuous fermentation in biofilm bioreactors.

Menaquinone-7 (MK-7), a vital vitamin with numerous health benefits, is synthesized and secreted extracellularly by the formation of biofilm, dominantly in Bacillus strains. Our team developed an innovative biofilm reactor utilizing Bacillus subtilis natto cells to foster biofilm growth on plastic composite supports to produce MK-7. Continuous fermentation in biofilm reactors offers a promising strategy for achieving sustainable and efficient production of Menaquinone-7 (MK-7). Unlike conventional batch fermentation, continuous biofilm reactors maintain a steady state of operation, which reduces resource consumption and waste generation, contributing to sustainability. By optimizing fermentation conditions, MK-7 production was significantly enhanced in this study, demonstrating the potential for sustainable industrial-scale production. To determine the optimal operational parameters, various dilution rates were tested. These rates were selected based on their potential to enhance nutrient supply and biofilm stability, thereby improving MK-7 production. By carefully considering the fermentation conditions and systematically varying the dilution rates, MK-7 production was significantly enhanced during continuous fermentation. The MK-7 productivity was found to increase from 0.12 mg/L/h to 0.33 mg/L/h with a dilution rate increment from 0.007 to 0.042 h-1). This range was chosen to explore the impact of various nutrient supply rates on MK-7 production and to identify the optimal conditions for maximizing productivity. However, a further increase in the dilution rate to 0.084 h-1 led to reduced productivity at approximately 0.16 mg/L/h, likely due to insufficient retention time for effective biofilm formation. Consequently, a dilution rate of 0.042 h-1 exhibited the highest productivity of 0.33 mg/L/h, outperforming all investigated dilution rates and demonstrating the critical balance between nutrient supply and retention time in continuous fermentation. These findings validate the feasibility of operating continuous fermentation at a 0.084 h-1 dilution rate, corresponding to a 48 h retention time, to achieve the highest MK-7 productivity compared to conventional batch fermentation. The significant advancements achieved in enhancing Menaquinone-7 (MK-7) productivity through continuous fermentation at optimal dilution rates in the present work indicate promising prospects for even greater efficiency and sustainability in MK-7 production through future developments.

Effects of pullulan additive and co-culture of Aureobasidium pullulans on bacterial cellulose produced by Komagataeibacter hansenii.

Bacterial cellulose (BC) exhibits a unique combination of porosity, tensile strength, reticulated crystal structure and biocompatibility useful for many applications in the food, biomedical and other industries. Polysaccharide addition has been shown to improve the production and the mechanical properties of BC nanocomposites. This study examined the effect of pullulan on BC fermentation as well as the co-culturing of the BC producer with Aureobasidium pullulans, a fungal strain that produces pullulan as an exopolysaccharide. Results showed that a 1% pullulan addition improved Young's modulus of BC pellicles for sixfold. Addition of pullulan at 1.5% and 2% levels could increase the BC production from 0.447 to 0.814 and 1.997 g/L, respectively. The co-culture fermentation demonstrated a mixed effect on the aggregation and bundling of BC while resulting in a significant improvement in mechanical properties. The study provided a polysaccharide additive and a novel fermentation method to produce BC with improved properties.

Salt and nitrogen amendment and optimization for cellulase and xylanase production using dilute acid hydrolysate of distillers' dried grains with solubles (DDGS) as the feedstock.

Distillers' dried grains with solubles (DDGS) is a by-product of dry-mill corn ethanol production comprising a high nutritional value due to residual fiber, protein, and lipid contents. The fiber content of DDGS is high enough to be considered a valuable source for the production of hydrolytic enzymes, such as cellulase and xylanases, which can be used for hydrolysis of lignocellulosic feedstock during ethanol production. The DDGS-based medium prepared after acid hydrolysis provides adequate sugars for enzyme production, while additional macronutrients, such as salts and nitrogen sources, can enhance the enzyme production. Therefore, this study was undertaken to evaluate the effect of salts (KH2PO4, CaCl2·2H2O, MgSO4·7H2O, FeSO4·7H2O, CoCl2·6H2O, and MnSO4·H2O), peptone, and yeast extract on enzyme secretion by four different Aspergillus niger strains and to optimize the nitrogen source for maximum enzyme production. Yeast extract improved the cellulase production (0.38 IU/ml) for A. niger (NRRL 1956) as compared to peptone (0.29 IU/ml). However, maximum cellulase productions of 0.42 IU/ml and 0.45 IU/ml were obtained by A. niger (NRRL 330) and A. niger (NRRL 567), respectively, in presence of ammonium sulfate. The optimized nitrogen amounts resulted in a significant increase in the cellulase production from 0.174 to 0.63 IU/ml on day 9 of the fermentation with A. niger (NRRL 330). The composite model improved both cellulase and xylanase production. In conclusion, the optimization of all three nitrogen sources improved both cellulase and xylanase production in the DDGS-based media.

Development of bioactive solid support for immobilized Lactobacillus casei biofilms and the production of lactic acid.

Polypropylene was modified to contain chitosan and evaluate its ability to generate Lactobacillus casei biofilms and their lactic acid production. Biofilm formation was carried out in either rich or minimal media. The chitosan-modified polypropylene harbored ~ 37% more cells than the control polypropylene. The biofilms from the chitosan-modified polypropylene grown in rich medium produced ~ 2 times more lactic acid after 72 h of incubation than the control suspended cells. There was no significant difference in the production of lactic acid after 72 h by L. casei biofilms on the chitosan-modified polypropylene grown in minimal media as compared with cells in suspension after 48 h and 72 h of incubation. Infrared spectroscopy confirmed higher deposition of nutrients and biomass on the chitosan-modified polypropylene as compared to the chitosan-free polypropylene. Electron and atomic force microscopy confirmed thicker biofilms when rich media were used to grow them as compared to minimal medium.

A Review on the Utilization of Lignin as a Fermentation Substrate to Produce Lignin-Modifying Enzymes and Other Value-Added Products.

The lignocellulosic biomass is comprised of three major components: cellulose, hemicellulose, and lignin. Among these three, cellulose and hemicellulose were already used for the generation of simple sugars and subsequent value-added products. However, lignin is the least applied material in this regard because of its complex and highly variable nature. Regardless, lignin is the most abundant material, and it can be used to produce value-added products such as lignin-modifying enzymes (LMEs), polyhydroxyalkanoates (PHAs), microbial lipids, vanillin, muconic acid, and many others. This review explores the potential of lignin as the microbial substrate to produce such products. A special focus was given to the different types of lignin and how each one can be used in different microbial and biochemical pathways to produce intermediate products, which can then be used as the value-added products or base to make other products. This review paper will summarize the effectiveness of lignin as a microbial substrate to produce value-added products through microbial fermentations. First, basic structures of lignin along with its types and chemistry are discussed. The subsequent sections highlight LMEs and how such enzymes can enhance the value of lignin by microbial degradation. A major focus was also given to the value-added products that can be produced from lignin.

Distillers' dried grains with solubles (DDGS) and its potential as fermentation feedstock.

Distillers' dried grain with solubles (DDGS) is a byproduct of bioethanol fermentation, which uses the dry milling technology for starch-rich grains such as corn, wheat, and barley. The current interest in bioethanol is increasing due to the need for renewable liquid fuels specifically in the transportation sector. Since DDGS is rich in crude protein, fat, fiber, vitamins, and minerals, it is currently used as aquaculture, livestock, and poultry feeds. In recent years, DDGS has been used as feedstock in the production of value-added products via microbial fermentation. Numerous studies reported the production organic acids, methane, biohydrogen, and hydrolytic enzymes using DDGS. While DDGS contains remarkable amounts of macronutrients, pre-treatment of DDGS is required for release of the fermentable sugars. The pre-treatment methods such as chemical, physical, and biological origin are either solely used or combined to obtain maximal yields for different applications. Therefore, this review summarizes some of the most prominent pre-treatment processes generating high fermentable sugar yields for the productions of value-added products in the last 5 years. A special focus has been given to the effect of the variability of DDGS on the final product. Integration of hydrolytic enzyme production with the traditional bioethanol production facilities has been discussed for further improvement of bioethanol, methane, and biohydrogen using DDGS as fermentation feedstock.Key points• Distillers' dried grain with solubles (DDGS) has high nutritional value, but the nutritional profile is variable.• DDGS can be used for microbial fermentation feedstock to produce value-added products.• A review of the microbial products using DDGS is given for the last 5 years.• DDGS has the potential to replace expensive feedstocks of value-added products.

Biofilm reactors as a promising method for vitamin K (menaquinone-7) production.

Menaquinone-7 (MK-7) is the most potent subtype of vitamin K with extraordinarily high half-life in the circulatory system. Therefore, MK-7 plays a critical role in promoting human wellbeing today. Studies on MK-7 every year show more and more magnificent benefits of it in preventing cardiovascular diseases and osteoporosis to battling cancer cells, Alzheimer's and Parkinson's diseases. Thus, it needs to be supplemented to daily diet for accumulative and long-term benefits. Chemical synthesis of MK-7 produces a significant cis-isomer form of it, which has no biological activity. Fortunately, due to its key role in electron transfer in bacteria, trans-MK-7 is biosynthesized by especially Gram-positive strains mainly Bacillus genus. Concordantly, MK-7 could be produced via solid or liquid state fermentation strategies. In either regime, when static fermentation is applied in the absence of agitation and aeration, operational issues arise such as heat and mass transfer inefficiencies. Thus, scaling up the process becomes a challenge. On the other hand, studies have indicated that biofilm and pellicle formation that occur in static fermentations are key characteristics for extracellular MK-7 secretion. Therefore, this review covers the most recent discoveries of the therapeutic properties of MK-7 and optimization attempts at increasing its biosynthesis in different media compositions and effective growth parameters as well as the cutting-edge use of biofilm reactors where B. subtilis cells have the infrastructures to form mature biofilm formations on plastic composite supports. Biofilm reactors therefore can provide robust extracellular MK-7 secretion while simultaneously enduring high agitation and aeration rates, which then address the scale-up and operational issues associated with static fermentation strategies.

Effects of medium components in a glycerol-based medium on vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Menaquinone-7 (MK-7) as the most important form of Vitamin K has been reported to have miraculous benefits such as preventing cardiovascular diseases and osteoporosis along with antitumor effects. Therefore, there have been numerous studies in the past decades to improve MK-7 production via microbial fermentation. Unfortunately, both solid and liquid state fermentation strategies that are utilized for MK-7 production, face fundamental operational and scale-up issues as well as intense heat and mass transfer problems during fermentation. In this regard, biofilm reactors seem to be a practical solution to overcome these issues and enhance the production in agitated liquid fermentation. Therefore, this study was undertaken to utilize biofilm reactors in investigating and optimizing different media components in a glycerol-based medium. Using response surface methodology, the effects of glycerol, yeast extract, and soytone were studied in the fermentation medium on MK-7 production in biofilm reactor. With a composition of 48.2 g/L of glycerol, 8.1 g/L of yeast extracts, 13.6 g/L of soytone and 0.06 g/L of K2HPO4, MK-7 concentrations could reach 14.7 ± 1.4 mg/L in biofilm reactors, which was 57% higher compared to the MK-7 concentration achieved in suspended-cell reactors under similar conditions, while glycerol was depleted by the end of the fifth day in biofilm reactors, but glycerol was never depleted in suspended-cell reactors. Evidently, biofilm reactors present a reliable strategy to address the operational issues that occur during MK-7 biosynthesis on an industrial scale production.

Implementation of fed-batch strategies for vitamin K (menaquinone-7) production by Bacillus subtilis natto in biofilm reactors.

Recent studies show the essential health benefits associated with vitamin K, especially menaquinone-7 (MK-7). These benefits include reducing risks of cardiovascular diseases, osteoporosis, and even cancer. However, MK-7 production on an industrial level is only possible through bacterial fermentation and also current static fermentation strategies are not potent enough with difficulties to scale up. Biofilm reactors, however, may be a practical alternative. Biofilm reactors provide a controlled environment for the microorganisms to form mature and robust biofilms that enable them to produce value-added products with enhanced efficiencies. In this study, fed-batch addition of glucose and glycerol were investigated to the base media in biofilm reactors, as carbon source addition seemed crucial in batch fermentations. Results indicated that fed-batch strategies can be significantly effective in glucose-based medium, increasing the end-product concentrations to 28.7 ± 0.3 mg/L of MK-7 which was 2.3 fold higher than the level produced in suspended-cell bioreactors and renders the biofilm reactors as a potential replacement for static fermentation strategies. Moreover, morphological changes of B. subtilis were tracked during the 12-day long fermentation runs and finally, SEM investigations confirmed significant biofilm and extracellular matrices formed on the plastic composite support (PCS) in the biofilm reactors. In conclusion, biofilm reactors especially with fed-batch fermentation regimes seem to be an effective tool for MK-7 production at industrial scales.

Optimization of Bacillus subtilis natto growth parameters in glycerol-based medium for vitamin K (Menaquinone-7) production in biofilm reactors.

Menaquinone-7 (MK-7) is the key form of vitamin K used as a dietary supplement and its production revolves around Bacillus subtilis natto. Current fermentation strategies, which suggest static fermentations without aeration and agitation, can be problematic for large scale MK-7 production due to biofilm formation. The use of biofilm reactors, therefore, is proposed in the present study, which could utilize both agitation and aeration without interrupting MK-7 secretion. In this study, biofilm reactors were constructed using the selected plastic composite support (PCS) and B. subtilis natto strain for MK-7 production. Using response surface methodology (RSM), optimum growth parameters including temperature, pH, and agitation were determined in a glycerol-based medium. Results were presented in a statistical model (R 2 = 0.90), leading to optimum growth conditions of temperature (35 °C), agitation (200 rpm) and pH (6.58). Model-predicted MK-7 concentration was validated and MK-7 concentration of 12.09 mg/L was produced in the biofilm reactor. The obtained concentration was 58% higher as compared to the suspended-cell culture (7.67 mg/L). The results of this study will provide a critical step towards improved industrial scale production of MK-7.

Strain and plastic composite support (PCS) selection for vitamin K (Menaquinone-7) production in biofilm reactors.

Menaquinone-7 (MK-7), a subtype of vitamin K, has received a significant attention due to its effect on improving bone and cardiovascular health. Current fermentation strategies, which involve static fermentation without aeration or agitation, are associated with low productivity and scale-up issues and hardly justify the commercial production needs of this vitamin. Previous studies indicate that static fermentation is associated with pellicle and biofilm formations, which are critical for MK-7 secretion while posing significant operational issues. Therefore, the present study is undertaken to evaluate the possibility of using a biofilm reactor as a new strategy for MK-7 fermentation. Bacillus species, namely, Bacillus subtilis natto, Bacillus licheniformis, and Bacillus amyloliquifaciens as well as plastic composite, supports (PCS) were investigated in terms of MK-7 production and biofilm formation. Results show the possibility of using a biofilm reactor for MK-7 biosynthesis. Bacillus subtilis natto and soybean flour yeast extract PCS in glucose medium were found as the most potent combination for production of MK-7 as high as 35.5 mg/L, which includes both intracellular and extracellular MK-7.

Production and application of menaquinone-7 (vitamin K2): a new perspective.

Menaquinone-7, a highly valuable member of the vitamin K series, has significant effects on preventing osteoporosis and cardiovascular disease besides its positive effects on blood coagulation. In this review, chemical and biological aspects of menaquinone-7 are briefly summarized followed by a critical review on upstream and downstream processing developments for its production and recovery, including solid versus liquid fermentations, static versus agitated fermentations and online versus post-production recovery. Latest research outcomes for improving industrial scale production of menaquinone-7 are summarized and recommendations are given for areas of future research.

Recent advances for the production and recovery methods of lysozyme.

Lysozyme is an antimicrobial peptide with a high enzymatic activity and positive charges. Therefore, it has applications in food and pharmaceutical industries as an antimicrobial agent. Lysozyme is ubiquitous in both animal and plant kingdoms. Currently, egg-white lysozyme is the most commercially available form of lysozyme. The main concerns of egg-white lysozyme are high recovery cost, low activity and most importantly the immunological problems to some people. Therefore, human lysozyme production has gained importance in recent years. Scientists have developed transgenic plants, animals and microorganisms that can produce human lysozyme. Out of these, microbial production has advantages for commercial productions, because high production levels are achievable in a relatively short time. It has been reported that fermentation parameters, such as pH, temperature, aeration, are key factors to increase the effectiveness of the human lysozyme production. Moreover, purification of the lysozyme from the fermentation broth needs to be optimized for the economical production. In conclusion, this review paper covers the mechanism of lysozyme, its sources, production methods and recovery of lysozyme.

Enhancement and modeling of microparticle-added Rhizopus oryzae lactic acid production.

Lactic acid has a wide industrial application area and can be produced by fungal strains. However, excessive bulk growth form of fungi during the fermentations is a major problem, which limits the fermentation performance. Microparticles are excellent tools to prevent bulk fungal growth and provide homogenized fermentation broth to increase uniformity and the prediction performance of the models. Therefore, in this study, addition of aluminum oxide and talcum microparticles into fermentations was evaluated to enhance the production of lactic acid by Rhizopus oryzae. The results showed that the bulk fungal growth was prevented and the lactic acid concentration increased from 6.02 to 13.88 and 24.01 g/L, when 15 g/L of aluminum oxide or 10 g/L of talcum was used, respectively, in the shake-flask fermentations. Additionally, substrate concentration, pH, and agitation were optimized in the bioreactors using response surface methodology, and optimum values were determined as 126 g/L of glucose, 6.22 pH, and 387 rpm, respectively. Under these conditions, lactic acid production further increased to 75.1 ± 1.5 g/L with 10 g/L of talcum addition. Also, lactic acid production and glucose consumption in the batch fermentation were successfully modeled with modified Gompertz model and modified logistic model. RMSE and MAE values for lactic acid production were calculated as 2.279 and 1.498 for the modified Gompertz model; 3.6 and 4.056 for the modified logistic model. Additionally, modified logistic model predicted glucose consumption with -2.088 MAE and 2.868 RMSE, whereas these values were calculated as 2.035 and 3.946 for the modified Gompertz model.

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation.

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.

Strain selection and medium optimization for glucoamylase production from industrial potato waste by Aspergillus niger.

BACKGROUND: Glucoamylase is one of the most common enzymes used in the food industry to break down starch into its monomers. Glucoamylase production and its activity are highly dependent on medium composition. Starch is well known as a glucoamylase inducer, and utilization of industrial starchy potato waste is an inexpensive way of improving glucoamylase production. Since glucoamylase production is highly dependent on medium composition, in this study medium optimization for glucoamylase production was considered to enhance glucoamylase activity. RESULTS: Among the evaluated microbial species, Aspergillus niger van Tieghem was found to be the best glucoamylase-producing fungus. The Plackett-Burman design was used to screen various medium ingredients, and malt extract, FeSO4 .7H2 O and CaCl2 ·2H2 O were found to have significant effects on glucoamylase production. Finally, malt extract, FeSO4 .7H2 O and CaCl2 .2H2 O were optimized by using a central composite design of response surface methodology. The results showed that the optimal medium composition for A. niger van Tieghem was 50 g L(-1) industrial waste potato mash supplemented with 51.82 g L(-1) malt extract, 9.27 g L(-1) CaCl2 ·2H2 O and 0.50 g L(-1) FeSO4 .7H2 O. CONCLUSION: At the end of optimization, glucoamylase activity and glucose production were improved 126% and 98% compared to only industrial waste potato mash basal medium; 274.4 U mL(-1) glucoamylase activity and 41.7 g L(-1) glucose levels were achieved, respectively. © 2015 Society of Chemical Industry.

Current and future trends for biofilm reactors for fermentation processes.

Biofilms in the environment can both cause detrimental and beneficial effects. However, their use in bioreactors provides many advantages including lesser tendencies to develop membrane fouling and lower required capital costs, their higher biomass density and operation stability, contribution to resistance of microorganisms, etc. Biofilm formation occurs naturally by the attachment of microbial cells to the support without use of any chemicals agent in biofilm reactors. Biofilm reactors have been studied and commercially used for waste water treatment and bench and pilot-scale production of value-added products in the past decades. It is important to understand the fundamentals of biofilm formation, physical and chemical properties of a biofilm matrix to run the biofilm reactor at optimum conditions. This review includes the principles of biofilm formation; properties of a biofilm matrix and their roles in the biofilm formation; factors that improve the biofilm formation, such as support materials; advantages and disadvantages of biofilm reactors; and industrial applications of biofilm reactors.

Effects of fed-batch and continuous fermentations on human lysozyme production by Kluyveromyces lactis K7 in biofilm reactors.

Lysozyme is a lytic enzyme, which has antimicrobial activity. It has been used for food and pharmaceutical applications. This study was undertaken to evaluate fed-batch and continuous fermentations for the human lysozyme production in biofilm reactor. Results showed that addition of lactose the mid-log phase to make the concentration back to the initial level generates higher lysozyme production (177 U/ml) compared with lactose addition in late-log phase (174 U/ml) (p < 0.05). Moreover, fed-batch fermentation with glucose as initial carbon source and continuous addition of lactose with 0.6 ml/min for 10 h demonstrated significantly higher lysozyme production (187 U/ml) compared to the batch fermentation (173 U/ml) (p < 0.05). In continuous fermentation, biofilm reactor provided significantly higher productivity (7.5 U/ml/h) compared to the maximum productivity in suspended cell bioreactor (4 U/ml/h), because the biofilm reactor provided higher cell density at higher dilution rate compared to suspended cell reactor (p < 0.05).

Microparticle-enhanced Aspergillus ficuum phytase production and evaluation of fungal morphology in submerged fermentation.

Phytase can be used in animal's diets to increase the absorption of several divalent ions, amino acids and proteins and to decrease the excessive phosphorus release in manure to prevent negative effects on the environment. This study aimed to enhance the current submerged fungal phytase productions with a novel fermentation technique by evaluating the effect of the various microparticles on Aspergillus ficuum phytase production. It was observed that microparticles prevented bulk fungal pellet growth, decreased average fungal pellet size and significantly increased phytase activity in the submerged fermentation. Microbial structure imaging results showed that the average fungal pellet radius decreased from 800 to 500 and 200 µm by addition of 15 g/L aluminum oxide and talcum, respectively, in shake-flask fermentation. Also, addition of 15 g/L of talcum and aluminum oxide increased phytase activity to 2.01 and 2.93 U/ml, respectively, compared to control (1.02 U/ml) in shake-flask fermentation. Additionally, phytase activity reached 6.49 U/ml within 96 h of fermentation with the addition of 15 g/L of talcum, whereas the maximum phytase activity was only 3.45 U/ml at 120 h of fermentation for the control in the 1-L working volume bioreactors. In conclusion, microparticles significantly increased fungal phytase activity and production yield compared to control fermentation.

Enhanced Aspergillus ficuum phytase production in fed-batch and continuous fermentations in the presence of talcum microparticles.

This study aimed to enhance Aspergillus ficuum phytase production in fed-batch and continuous fermentations with addition of talcum microparticles. Phytase activity almost doubled in fed-batch and continuous fermentations by addition of 15 g/l of talcum compared to the control. Effect of talcum on fungal morphology was also shown that addition of talcum provided smaller fungal pellets and more homogenized fermentation broth compared to the control. Average fungal pellet radius decreased from 500 to 100 µm by addition of 15 g/l of talcum in the bioreactors. Also, 15 g/l talcum addition increased phytase productivity and optimum dilution rate in the continuous fermentations from 0.293 to 0.621 U/ml/h and from 0.09 to 0.1/h, respectively, compared to control.