Microbial Production Of Hyaluronic Acid Research Articles

A Review of the Production of Hyaluronic Acid in the Context of Its Integration into GBAER-Type Biorefineries

Biorefineries (BRFs) that process the organic fraction of municipal solid waste and generate bioproducts and bioenergies have attracted attention because they can simultaneously address energy and environmental problems/needs. The objective of this article was to critically review the microbial production of hyaluronic acid (MPHA) and its production profile for its integration into a GBAER-type BRF (a type of BRF based on organic wastes) and to identify the environmental and economic sustainability aspects of the modified BRF that would confirm it as a sustainable option. It was found that the MPHA by selected strains of pathogenic Streptococci was moderate to high, although the trend to work with genetically transformed (GT) (innocuous) bacteria is gaining momentum. For instance, A GT strain of Corynebacterium glutamicum reached a maximum HA production of 71.4 g L−1. MPHA reports that use organic wastes as sources of carbon (C) and nitrogen (N) are scarce. When alternative sources of C and N were used simultaneously, HA production by S. zooepidemicus was lower than that with conventional sources. We identified several knowledge gaps that must be addressed regarding aspects of process scale-up, HA industrial production, economic feasibility and sustainability, and environmental sustainability of the MPHA.

Microbial hyaluronic acid production in the 21 century: a roadmap toward high production, tailored molecular weight

Hyaluronic acid is a biopolymer that has gained attention from both researchers and the public since its discovery in 1934. In 2022, its global annual demand was 667 MT, with a market value of 9.4 billion dollars and an expected compound growth rate of 7.7% from 2024 and 2030. Such popularity lies in its many applications, which ranges from pharmaceutical to Tissue Engineering. Its molecular weight (MW) determines which application hyaluronic acid is better suited for: high MW (over 1 MDa) is used in medical applications such as osteoarthritis and ophthalmology, and low MW (less than 0.05 MDa) are suited for moisturizing cosmeceuticals. This makes hyaluronic acid molecular weight an important parameter for its production. The first industrial process was based on extraction from rooster combs. However, it shifted toward microbial production with Streptococcus zooepidemicus. Studies focus on improving its production and tailoring its molecular weight by accessing optimum temperature, pH, aeration, agitation, carbon, and nitrogen sources concentration and origin. his review aims to provide an overview of the current knowledge on the effects of metabolic and environmental factors in microbial hyaluronic acid production. The review shows that there has been extensive research in optimizing cultivation medium composition, temperature, pH, and aeration between 2018 and 2023, but further studies are required to optimize the metabolism of S. zooepidemicus towards maximum biopolymer production and molecular weight.

Microbial Hyaluronic Acid Production: A Review

Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid.

Evaluation of Agave tequilana by-products for microbial production of hyaluronic acid

The tequila industry generates enormous amounts of solid byproducts originated from the agave plant such as leaves (AL) and bagasse (AB). Agave leaves are often left on the fields for natural degradation and agave bagasse is usually burned. An ideal option for the use of these residues is obtaining high commercial value products such as hyaluronic acid (HA). In this contribution the production of HA with Streptococcus zooepidemicus was evaluated using the juice from AL, and with enzymatic hydrolyzates. The hydrolyzates were produced from untreated fibers from both AB and AL and acid-pretreated fiber from AB and AL. The juice obtained from AL had the highest amounts of monosaccharides (42.05 g/L). However, a higher production of HA (4.23 g/L) was obtained from hydrolyzates of pretreated fibers from AL. This research shows that residues from tequila industry are a promising source of monosaccharides for production of hyaluronic acid with Streptococcus zooepidemicus.

Optimization and kinetic modeling of media composition for hyaluronic acid production from carob extract with Streptococcus zooepidemicus.

Hyaluronic acid (HA), a mucopolysaccharide belonging to the glycosaminoglycan family, consists of repeating disaccharide units and has been used directly or indirectly in numerous human health practices. This study focused on evaluating carob pods for microbial HA production and kinetic modeling of HA fermentation. Therefore, the optimal medium composition was determined using Plackett-Burman Design (PBD) for HA production from carob extract with Streptococcus zooepidemicus. Maximum HA production of shake flask fermentation was 2.6g/L (1.25 × 106) in the optimum medium, comprising 10°Bx of carob pods extract, 0.5g/L of MgSO4.7H2O, 10.0g/L of casein, 2.5g/L of KH2PO4, 2.0g/L of NaCl, 1.5g/L of K2HPO4, 0.002g/L of FeSO4 and 10.0g/L of beef extract. In the continuation of the study, the fermentation performed with the optimal medium composition was modeled using three different models including the logistic model for biomass production, the Luedeking-Piret model for HA production, and the modified Luedeking-Piret model for substrate consumption. Based on the results, the experimental HA production data agreed with the Luedeking-Piret model with an R2 of 0.989. Since the α value was 63-fold higher than the value of β, the HA production is growth-associated. Consequently, carob extract can be evaluated as a promising carbon source for producing HA.

Incremental Model Identification of Bio-processes from Data: Application to Microbial Production of Hyaluronic Acid

The development of reliable kinetic models of bioprocesses from data is a challenging task. In this work, a systematic approach to develop a kinetic model of bioprocess involving single biomass from concentration data is proposed without imposing any kinetic model a priori. The proposed incremental model identification approach decomposes the model-building task into a set of sub-tasks such as determining the yield coefficients and maintenance coefficient, specific growth rate structure identification, and parameter estimation. It is shown that the proposed approach allows identifying the mechanism of product formation. The proposed approach is applied to the microbial production of Hyaluronic acid (HA), an important biopolymer, using a recombinant Lactococcus lactis MKG6. An unstructured kinetic model is developed for the HA production from data. It is shown that HA production is a growth-associated process. Further, the specific growth rate of HA production is identified from a set of rate candidates. It is revealed that the specific growth rate in the HA production follows the non-competitive HA inhibition model. The parameters obtained by the incremental identification are further refined to obtain statistically optimal estimates using the simultaneous model identification. Validation of the identified kinetic model of HA production on new experimental data shows that the proposed approach leads to a reliable kinetic model with the optimal parameter estimates.

Microbial production of hyaluronic acid: the case of an emergent technology in the bioeconomy

AbstractIn 1988 a novel fermentation route to high molecular weight hyaluronan mediated by Streptococcus zooepidemicus was first reported. In a few years, following rapidly expanding demand for hyaluronan as a key ingredient of facial dermal fillers, streptococcal production was commercialized, first in Western Europe and North America, and subsequently in China. Outlining the case for the microbial production of hyaluronan, an emergent technology in the bioeconomy, the purpose of this study is to further advance the field of sustainable, high‐added‐value, biobased production. The study recounts how this bioproduction actually developed, its advantages, and its current limitations. The research and educational outcomes of the study offer useful lessons to bioeconomy scholars and practitioners. © 2021 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.

Sucrose-modified iron nanoparticles for highly efficient microbial production of hyaluronic acid by Streptococcus zooepidemicus

Nanoparticles (NPs) were hypothesized to enhance fermentation processes and assist microorganisms in producing valuable biopolymers. Donors of trace iron, i.e., FeSO4·7H2O, zero-valence iron nanoparticles (Fe NPs), and ferric oxide nanoparticles (α-Fe2O3 NPs), were tested to study the impact on hyaluronic acid (HA) production. The bioprocess with the addition of 30 mg/L Fe NPs produced higher HA than the other groups. However, Fe NPs were limited by the synergistic effect of geomagnetism and high surface energy, resulting in obvious agglomeration behavior. To address this, we developed novel sucrose-modified iron nanoparticles (SM-Fe NPs), which showed effective improvement of dispersion and agglomeration. Concerning the SM-Fe NP additives, an adequate supply of nutrients and trace elements provided sufficient substrates and energy for the reproduction of Streptococcus zooepidemicus. Furthermore, the highest HA production with the addition of 30 mg/L SM-Fe NPs was 0.226 g/L, and the dry weight of the produced HA increased 3.28 times compared with the control group (0.069 g/L). This work significantly improved HA production and presented promising opportunities for industrial production.

Metabolic Engineering of Escherichia coli for the Production of Hyaluronic Acid From Glucose and Galactose.

Hyaluronic acid is a glycosaminoglycan biopolymer widely present throughout connective and epithelial tissue, and has been of great interest for medical and cosmetic applications. In the microbial production of hyaluronic acid, it has not been established to utilize galactose enabling to be converted to UDP-glucuronic acid, which is a precursor for hyaluronic acid biosynthesis. In this study, we engineered Escherichia coli to produce hyaluronic acid from glucose and galactose. The galactose-utilizing Leloir pathway was activated by knocking out the galR and galS genes encoding the transcriptional repressors. Also, the hasA gene from Streptococcus zooepidemicus was introduced for the expression of hyaluronic acid synthase. The consumption rates of glucose and galactose were modulated by knockout of the pfkA and zwf genes, which encode 6-phosphofructokinase I and glucose-6-phosphate dehydrogenase, respectively. Furthermore, the precursor biosynthesis pathway for hyaluronic acid production was manipulated by separately overexpressing the gene clusters galU-ugd and glmS-glmM-glmU, which enable the production of UDP-glucuronic acid and UDP-N-acetyl-glucosamine, respectively. Batch culture of the final engineered strain produced 29.98 mg/L of hyaluronic acid from glucose and galactose. As a proof of concept, this study demonstrated the production of hyaluronic acid from glucose and galactose in the engineered E. coli.

Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance heterologous production of hyaluronic acid in engineered Bacillus subtilis.

In microbial cultivations for hyaluronic acid (HA) production, oxygen can be a limiting substrate due to its poor solubility in aqueous medium and the substantial increase in culture viscosity at relatively low HA titers. Shear stress due to the high agitation and aeration rates required to overcome oxygen limitation may reduce the quality (i.e., molecular weight) of HA, and production costs associated with power consumption and supplemental oxygen may be excessive. Here, we report the application of oxygen vectors to the heterologous production of HA in engineered Bacillus subtilis, leading to significantly improved culture performance. We first derived an improved HA-producing strain of B. subtilis through engineering of the promoter driving coexpression of seHas and tuaD, leading to high-level HA production. Out of seven potential oxygen vectors evaluated in a preliminary screening, significant improvements to the HA titer and/or cell density were observed in cultures containing n-heptane, n-hexadecane, perfluoromethyldecalin, and perfluoro-1,3-dimethylcyclohexane. Adjustments to the vector concentration, timing of vector addition, and the agitation rate resulted in further enhancements, with the HA titer reaching up to 4.5 g/L after only 10 hr cultivation. Moreover, our results indicate that certain vectors may alter the functional expression of Class I hyaluronan synthase (HAS) in B. subtilis, and that higher shear rates may drive more carbon flux through the HA biosynthetic pathway without negatively affecting the MW. Our study demonstrates the efficacy of oxygen vectors to enhance heterologous HA production in B. subtilis, and provides valuable insight for future bioprocess development in microbial HA production.

Real-time monitoring of hyaluronic acid fermentation by in situ transflectance spectroscopy.

Hyaluronic acid (HA) is a high-value polysaccharide with many biomedical applications. Microbial production of HA is now replacing the traditional extraction method from rooster combs. Production of medical-grade HA with defined characteristics requires controlled process conditions because there are many fermentation process parameters that affect the microbial synthesis of HA. This necessitates the development of online tools for monitoring multiple analytes during microbial fermentation. Here, we describe the application of in situ transflectance spectroscopy for online quantification of seven major fermentation analytes, viz. biomass, glucose, lactate, formate, ethanol, acetate and HA in metabolically engineered Lactococcus lactis fermentations. The near-infrared spectral information acquired from synthetic mixtures and untransformed L. lactis fermentations were used to develop chemometric models. Based on principal component analysis and partial least squares regression methods, analyte-specific models were developed for quantification. These models were then independently validated for fermentation analytes from four different recombinant L. lactis strains. The chemometric model developed for HAbased on recombinant L. lactis fermentation data and pure HA standardscould accurately predict HA concentrations under homolactic conditions. The online estimation of HA was found to be poor under heterolactic conditions due to the overlapping absorbance of acetate produced in these cultures. Alternatively, an independent model based on yield correlation was successfully developed for indirect real-time quantification of HA.

Microbial production of hyaluronic acid from agro-industrial by-products: Molasses and corn steep liquor

Agro-industrial by-products are being explored as alternative low-cost nutrients to produce hyaluronic acid (HA) by Streptococcus zooepidemicus. In this study, we formulated three culture media containing corn steep liquor (CSL) and sugarcane molasses (M), to produce microbial HA using batch bioreactor conditions (pH 6.7, 500rpm and 1vvm aeration). Final HA concentrations of 3.48gL−1 were produced in culture medium containing corn steep liquor (10% v/v) and glucose, being comparable (3.60gL−1) to the control medium containing tryptone and glucose. The use of molasses (10% v/v) as carbon source produced a marked inhibition of S. zooepidemicus growth and HA production due to a low sugar consumption. The HA produced in CSL culture media had a high molecular weight of 3.8×103kDa, greater than HA produced in tryptone-containing medium (3.0×103kDa).This is the first report achieving HA productions comparable to synthetic a medium in a batch bioreactor using CSL as the main nitrogen source. However, further optimization of culture conditions must be carried out towards using this agricultural by-product for the sustainable industrial production of HA.

Heterologous Production of Hyaluronic Acid in an ε-Poly-L-Lysine Producer, Streptomyces albulus.

Hyaluronic acid (HA) is used in a wide range of medical applications, where its performance and therapeutic efficacy are highly dependent on its molecular weight. In the microbial production of HA, it has been suggested that a high level of intracellular ATP enhances the productivity and molecular weight of HA. Here, we report on heterologous HA production in an ε-poly-l-lysine producer, Streptomyces albulus, which has the potential to generate ATP at high level. The hasA gene from Streptococcus zooepidemicus, which encodes HA synthase, was refactored and expressed under the control of a late-log growth phase-operating promoter. The expression of the refactored hasA gene, along with genes coding for UDP-glucose dehydrogenase, UDP-N-acetylglucosamine pyrophosphorylase, and UDP-glucose pyrophosphorylase, which are involved in HA precursor sugar biosynthesis, resulted in efficient production of HA in the 2.0 MDa range, which is greater than typical bacterial HA, demonstrating that a sufficient amount of ATP was provided to support the biosynthesis of the precursor sugars, which in turn promoted HA production. In addition, unlike in the case of streptococcal HA, S. albulus-derived HA was not cell associated. Based on these findings, our heterologous production system appears to have several advantages for practical HA production. We propose that the present system could be applicable to the heterologous production of a wide variety of molecules other than HA in the case their biosynthesis pathways require ATP in vivo.

Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis.

Microbial production of hyaluronic acid (HA) is an attractive substitute for extraction of this biopolymer from animal tissues. Natural producers such as Streptococcus zooepidemicus are potential pathogens; therefore, production of HA by recombinant bacteria that are generally recognized as safe (GRAS) organisms is a viable alternative that is being extensively explored. However, plasmid-based expression systems for HA production by recombinant bacteria have the inherent disadvantage of reduced productivity because of plasmid instability. To overcome this problem, the HA synthesis genes (hasA-hasB and hasA-hasB-hasC) from has-operon of S. zooepidemicus were integrated into the chromosome of Lactococcus lactis by site-directed, double-homologous recombination developing strains VRJ2AB and VRJ3ABC. The chromosomal integration stabilized the genes and obviated the instability observed in plasmid-expressed recombinant strains. The genome-integrated strains produced higher molecular weight (3.5-4 million Dalton [MDa]) HA compared to the plasmid-expressed strains (2 MDa). High molecular weight HA was produced when the intracellular concentration of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) and uridine diphosphate-glucuronic acid (UDP-GlcUA) was almost equal and hasA to hasB ratio was low. This work suggests an optimal approach to obtain high molecular weight HA in recombinant strains.

Microbial production of hyaluronic acid: current state, challenges, and perspectives

Hyaluronic acid (HA) is a natural and linear polymer composed of repeating disaccharide units of β-1, 3-N-acetyl glucosamine and β-1, 4-glucuronic acid with a molecular weight up to 6 million Daltons. With excellent viscoelasticity, high moisture retention capacity, and high biocompatibility, HA finds a wide-range of applications in medicine, cosmetics, and nutraceuticals.Traditionally HA was extracted from rooster combs, and now it is mainly produced via streptococcal fermentation. Recently the production of HA via recombinant systems has received increasing interest due to the avoidance of potential toxins. This work summarizes the research history and current commercial market of HA, and then deeply analyzes the current state of microbial production of HA by Streptococcus zooepidemicus and recombinant systems, and finally discusses the challenges facing microbial HA production and proposes several research outlines to meet the challenges.

Microbial production of hyaluronic acid from agricultural resource derivatives

Agricultural resource derivatives (ARDs) such as hydrolysate soy protein concentrate (HSPC), whey protein concentrate (WPC), and cashew apple juice (CAJ) were studied with focus on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Supplementation of the media with corn steep liquor (CSL) was also evaluated. Synthetic medium containing glucose and yeast extract was used as control. CAJ was a promising medium for the production of HA. It produced the highest amount of HA (0.89 g L −1), similar to that of the control (0.86 g L −1). WPC and HSPC media were the most effective for the production of biomass. CSL did not influence the production of HA when HSPC and WPC were used. However, in the synthetic medium it doubled the yield of HA from glucose. The average molecular weight of HA ranged from 10 3 to 10 4 Da for the ARDs and 10 7 Da for the synthetic medium.

Hyaluronic acid production by Streptococcus zooepidemicus in marine by-products media from mussel processing wastewaters and tuna peptone viscera.

BackgroundHyaluronic acid is one of the biopolymers most commonly used by the pharmaceutical industry. Thus, there is an increasing number of recent works that deal with the production of microbial hyaluronic acid. Different properties and characteristics of the fermentation process have been extensively optimised; however, new carbon and protein sources obtained from by-products or cheap substrates have not yet been studied.ResultsMussel processing wastewater (MPW) was used as a sugar source and tuna peptone (TP) from viscera residue as a protein substrate for the production of hyaluronic acid (HA), biomass and lactic acid (LA) by Streptococcus zooepidemicus in batch fermentation. Commercial medium formulated with glucose and tryptone was used as the control. The parametric estimations obtained from logistic equations and maintenance energy model utilized for modelling experimental data were compared in commercial and low-cost media. Complete residual media achieved high production (3.67, 2.46 and 30.83 g l-1 of biomass, HA and LA respectively) and a high molecular weight of HA (approximately 2500 kDa). A simple economic analysis highlighted the potential viability of this marine media for reducing the production costs by more than 50%.ConclusionsThe experimental data and mathematical descriptions reported in this article demonstrate the potential of media formulated with MPW and TP to be used as substrates for HA production by S. zooepidemicus. Furthermore, the proposed equations accurately simulated the experimental profiles and generated a set of interesting parameters that can be used to compare the different bacterial cultures. To the best of our knowledge, this is the first work in which a culture media formed by marine by-products has been successfully used for microbial HA production.

Statistical modeling and optimization for enhanced hyaluronic acid production by batch culture of Sreptococcus zooepidemicus via the supplement of uracil

This work is aimed to achieve the optimal hyaluronic acid (HA) production by batch culture of Streptococcus zooepidemicus via the supplement of nucleotide bases using response surface methodology (RSM). First, the influence of nucleotide bases (adenine, guanine, cytosine, thymine, and uracil) on microbial HA production was investigated using fractional factorial design (FFD). By a 25-2 FFD, uracil was found to be the most significant factor for cell growth and HA production, while the other nucleotide bases were shown to have no significant effects on cell growth and HA production. Also, the impact of uracil on cell growth and HA production was further investigated by RSM, where two variables were considered: uracil concentration and supplement time. The optimal uracil concentration and supplement time were found to be 0.051 g/L and 7 h, respectively, and the predicted maximal HA production reached 6.42 g/L. The maximal HA production increased from 5.0 g/L of the control without uracil supplement to 6.31 g/L at the optimal conditions in validation experiments.

Application of a novel cavern volume controlled culture model to microbial hyaluronic acid production by batch culture of Streptococcus zooepidemicus

With the unique physio-chemical and biological properties, hyaluronic acid (HA) finds a wide range of applications in biomedical, cosmetic and healthcare fields. The microbial HA production is limited by poor mixing performance and low oxygen mass transfer rate. In this work, a novel cavern volume controlled culture model was proposed and applied to microbial HA production by batch culture of Streptococcus zooepidemicus. In the cavern model-controlled culture process, the complete mixing was achieved via the dynamic control of agitation speed in the range from 50 rpm to 470 rpm, and HA yield increased from 5.5 ± 0.1 g/L of the control to 6.2 ± 0.1 g/L. There was not significant influence of the model-controlled culture on the molecular weight of HA compared with the control, in which the agitation speed was kept at 294 rpm. With better mixing performance and improved oxygen mass transfer efficiency, the cavern volume controlled culture model may be helpful for the production of the other microbial biopolymers like xanthan gum, curdlan and poly(glutamic acid).

Enhancement of Hyaluronic Acid Production by Batch Culture of Streptococcus zooepidemicus with N-Dodecane as an Oxygen Vector

This study aimed to examine the influence of adding an oxygen vector, n-dodecane, on hyaluronic acid (HA) production by batch culture of Streptococcus zooepidemicus. Owing to the high viscosity of culture broth, microbial HA production during 8-16 h was limited by the oxygen transfer coefficient K(L)a, which could be enhanced by adding n-dodecane. With the addition of n-dodecane to the culture medium to a final concentration of 5% (v/v), the average value of K(L)a during 8-16 h was increased to 36+/-2 h(-1), which was 3.6 times that of the control without n-dodecane addition. With the increased K(L)a and dissolved oxygen (DO) by adding 5% (v/v) of n-dodecane, a 30% increase of HA production was observed compared with the control. Furthermore, the comparison of the oxygen mass transfer in the absence and presence of n-dodecane was conducted with two proposed mathematical models. The use of n-dodecane as an oxygen vector, as described in this paper, provides an efficient alternative for the optimization of other aerobic biopolymer productions,where K(L)a is usually a limiting factor.