Enhanced oxygen transfer rate and bioprocess yield by using magnetite nanoparticles in fermentation media of erythromycin
© Labbeiki et al.; licensee BioMed Central Ltd. 2014
Received: 10 August 2014
Accepted: 7 September 2014
Published: 16 September 2014
Magnetite nanoparticles have widespread biomedical applications. In the aerobic bioprocesses, oxygen is a limiting factor for the microbial metabolic rate; hence a high availability of oxygen in the medium is crucial for high fermentation productivity. This study aimed to examine the effect of using magnetite nanoparticles on oxygen transfer rate in erythromycin fermentation culture.
Magnetite nanoparticles were synthetized through co-precipitation method. After observing the enhanced oxygen transfer rate in deionized water enriched with magnetite nanoparticles, these nanoparticles were used in the media of by Saccharopolyspora erythraea growth to explore their impact on erythromycin fermentation titer. Treatments comprised different concentrations of magnetite nanoparticles, (0, 0.005, 0.02 v/v).
In the medium containing 0.02 v/v magnetite nanoparticles, KLa was determined to be 1.89 time higher than that in magnetite nanoparticle-free broth. An improved 2.25 time higher erythromycin titer was obtained in presence of 0.02 v/v nanoparticles.
Our results, demonstrate the potential of magnetite nanoparticles for enhancing the productivity of aerobic pharmaceutical bioprocesses.
Oxygen is one of the most important substrate influencing productivity of aerobic bioprocesses . While oxygen has a low solubility in most fermentation media, the uptake of the major amount of oxygen by microorganisms during the fermentation decreases the dissolved oxygen level in liquid to less than the critical concentration, rendering oxygen the limiting factor for productivity. A significantly low rate of oxygen transfer from gas into liquid, will lead to a decreased microbial metabolic rate, thereby low fermentation performance. Therefore an adequate supply of O2 is required for achieving a high fermentation productivity, particularly in high-cell density bioprocesses  such as erythromycin fermentation by Saccharopolyspora erythraea.
The Oxygen Transfer Rate (OTR) is usually determined by volumetric mass transfer coefficient (KLa). This variable is affected by several factors, including composition of medium, and geometrical and operating characteristic of the bioreactor . Several methods have been proposed for improving KLa, including use of more effective agitation and aeration systems, enriching air with pure oxygen, reducing gas bubbles' size and enhancing gas hold up , and modifying physical properties of the medium by adding dispersed phases containing particles in size of μm  capable of solubilizing O2 more than water.
Recent studies have shown that nanomaterials have potential to positively influence the variables affecting biochemical processes. For instance, Olle et al.  showed that O2 mass transfer improves in the presence of colloidal nanoparticle dispersion. In addition, Nagi et al.  reported an enhanced oxygen mass transfer rate in the presence of nano-size particles.
Following this growing line of research, in the present study, first aqueous solution of magnetite nanoparticles (MNPs) was prepared. The solution was then added to the fermentation media of S. erythraea to examine the possible effects of magnetite nanoparticles on O2 transfer rate and final titer of fermentation product.
The erythromycin-producing strain Saccharopolyspora erythraea PTCC 1685 was obtained from Persian Type Culture Collection I-124, Iran. Soybean flour was supplied from Maxsoy Co., Iran. Chemical reagents and media were purchased from Merck or Sigma.
Synthesis of nanoparticles
Several procedures have been developed for synthesis of iron oxide nanoparticles ,. In this study, co-precipitation technique was used for synthesis of magnetite nanofluid, which is based on the simultaneous precipitation of Fe3+, Fe2+ ions in basic aqueous media . Some advantages of this method include being straightforward, cheap, and environment-friendly, and producing a uniform size distribution of nanoparticles.
Due to their strong magnetic properties, the synthetic MNPs were aggregated near the magnet. MNPs were then washed with deionized water three time sat the end of process. Next, NPs were dried in oven at 80°C overnight to be characterized by XRD and TEM analysis .
Experimental determination of the volumetric mass transfer coefficient (KLa) by dynamic method
Media and cultural method
Purification of erythromycin was carried out by centrifugation of the samples (4000 rpm, 20 min) followed by using magnet to separate the possibly remaining magnetite nanoparticles from the supernatant. The supernatant was then diluted with 0.2 M carbonate-bicarbonate buffer of pH9.6 and the total erythromycin was extracted with chloroform. The extracted erythromycin was then mixed with the bromophenol blue reagent. The absorbance of organic phase was measured at 415 nm by spectrophotometry -.
T-test was used to examine the significance of the mean differences. KLa was calculated from regression analysis. P < 0.05 was considered as the statistical significance.
Results and discussion
NPs' crystal structure
Effect of nanoparticles on mass transfer coefficient
Effect of MNPs on erythromycin titer
The chief objective of the present study was to explore the impact of MNPs on OTR and thereby titer of bioprocess product. MNPs are strong oxygen absorbent due to their increased surface area at nano scale. These nanoparticles have proven useful when used as recyclable oxygen carriers in aerobic fermentation . A number of mechanisms proposed for the positive impact of MNPs on OTR include enhancing oxygen solubility in the fermentation medium, inducing microconvection in the surrounding fluid by Brownian motion , and enlarging the gas-liquid interfacial surface through being adsorbed on the air bubbles, preventing them from coalescence .
Results obtained in this study, which are in close agreement with the findings from previous researches ,-, add weight to the notion of MNPs being candidate members of O2-vectors that promote the oxygen transfer in stirred aerobic bioprocesses.
Composition of fermentation medium plays an important role in the titer of secondary metabolites and the cost of fermentation product . Because the erythromycin producing bacterium, S. erythraea is an aerobic actinomycete, proper oxygenation is crucial to achieve a high yield of this substance. Our results clearly indicate that presence of MNPs remarkably enhance the erythromycin production by S. erythraea.
Possible toxicity of nanoparticles is a major concern, particularly when used in pharmaceutical bioprocesses ,. The fact that MNPs can be efficiently separated from fermentation medium, virtually eliminate the risk of possible toxic effects. On the other hand, the wide use of MNPs in environmental applications, including pollutant removal, toxicity mitigation, and water and waste treatment  indicates the safety of its use at limited doses. These advantages together with their positive impact on fermentation productivity as supported in this study, introduce use of them as a viable strategy for an improved pharmaceutical bioprocessing.
The metabolic rate and growth of microbial biocatalysts are controlled by oxygen; hence, for an improved yield of aerobic bioprocesses, there is a need for strategies enabling a high rate oxygen transfer, while maintain affordable energy consumption. Our results indicate that MNPs can improve the efficiency of oxygen transfer in the fermentation medium. Use of MNPs in the erythromycin fermentation culture enhanced erythromycin titer, presumably via promotion of microbial growth and viability. The straightforward and inexpensive synthetize of these biocompatible, non-toxic and non-volatile nanoparticles, together with their positive impact on oxygen transfer rate, introduce them as promising agents for achieving an enhanced productivity of the industrial bioprocesses.
The authors wish to thank Dr. Meysam Mobasheri for participating in statistical analysis and revision of the manuscript.
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