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Increasing Antibiotic Yield for the Method of Dispersed Growth of Streptomyces in Liquid Culture

Introduction
Streptomyces is a genus of filamentous bacteria. It is in the family Streptomycetaceae, they include over 500 species, which occur in both soil and water (Hodgson, 2000). A lot of the species are important in the process of decomposition of organic matter in soil, these species contribute to the earthy odour of soil and also of decaying leaves, it also contributes to the fertility of soil (Hodgson, 2000). Some of the species are notable for their production of broad-spectrum antibiotics (Blaskovich et al., 2017), chemicals that the bacteria are able to naturally produce to kill or are able to inhibit the growth certain microorganisms (Hodgson, 2000). The antibiotic producers include: S. aureofaciens (yielding chlortetracycline), S. rimosis (oxytetracycline; see tetracycline), S. griseus (streptomycin), S. erythraeus (erythromycin), and S. venezuelae (chloramphenicol) (Hodgson, 2000). Streptomyces is categorised as gram-positive aerobic bacteria and they are of a complex form (Hodgson, 2000).
Streptomyces is one of the most vital group of industrial microorganisms as a producer of bioactive compounds and are the main bacteria used for the production of antibiotics (Blaskovich et al., 2017).
Within liquid culture, Streptomyces grows in the form of a pellet (Vecht-Lifshitz et al., 1990). The pellets will have limitations due to mass transfer, this produces a solute gradient through the sphere (Feeney, 2015). In the centre of the pellets the cells will be nutrient limited (Hobbs et al., 1989). This growth method means that problems arise for the physiology of the microbial (Feeney, 2015). Due to cells containing Streptomyces to have a variety of physiological states. It isn’t clear whether dispersed growth happens at a phenotypic level or a genotypic level (Hobbs et al., 1989). Whichever one it is the instability genetically means that there’s a very high chance that changes will happen if there is prolonged continuous growth (Hobbs et al., 1989). Pellet formation is not exclusive to Streptomyces, its seen in liquid cultures of fungi (Vecht-Lifshitz, Magdassi and Braun, 1990). In a number of fungal species, the incorporation of high weight molecular weight polyanions into the growth media has solved the problem of pellet formation in liquid cultures (Hobbs et al., 1989. The polymeric compounds are thought to act by inducing electrostatic repulsion between the spores or the cells, this prevents initial aggregation of spores in the inocula as well as clumping of mycelia that is in the growth culture (Guthrie et al., 1998).
The production of oxytetracycline increased after immobilization of Streptomyces rimosus cells in calcium alginate gels in comparison with free cells in a study by Enshasy et al. (1996). Studies have shown that the use of immobilized cells that adhered on glass wool for both rifamycins and oxytetracycline production for 5 repeated batches had been looked in to (Feeney, 2015). The aim of the paper by Enshasy et al. (1996) was to describe the optimal conditions for repeated batch production of oxytetracycline by immobilized cells, and the productivity of immobilized cells for 10 repeated batches in comparison with free cells (Enshasy et al., 1996).
The use of culture systems where plant cells are immobilized on a stationary support is acknowledged as a way by which the cell environment can be manipulated, and therefore the yields of specific secondary metabolites increased the cells suspended in liquid (Dervakos and Webb, 1991). So that microbial contamination is kept to a minimum, the number of steps in the immobilised procedure have to be kept to a minimum (Doleyres and Lacroix, 2005).
Using immobilised cells rather than free cells has many advantages such as enhanced biological stability, high biomass concentration, improved mass transfer, advantageous partition effects Increased product yields, increased product stability, integration with downstream processing Advantages due to cell proximity Increased reaction selectivity and versatility in the selection of the reactor (Lindsey et al., 1983). There are a number of different methods that has been used for immobilising bacteria, these include physical entrapment in polymeric networks, attachment / adsorption to a preformed carrier, membrane entrapment and microencapsulation (Doleyres and Lacroix, 2005).
Immobilisation offer a number of advantages for biomass and metabolite production (Leenen et al., 1996) this is compared to free-cell systems which include high cell density, reuse of biocatalysts, improved resistance to contamination and bacteriophage attack, enhancement of plasmid stability, prevention from washing-out during continuous cultures, and physical and chemical protection of cells (Lindsey et al., 1983). Immobilised cells have the advantage of being alive but not necessarily replicating. This is the perfect state for antibiotic producing cells (Leenen et al., 1996). living, non-reproducing cells, retained by a membrane or fixed to a carrier, are used (Wandrey, 1996).
Biotechnological processes based on immobilized whole cells have developed quickly over the last few decades, mainly using viable, metabolically active microorganisms (Chaumeil, 2015). Natural immobilized cell (IC) structures, ie, biofilms, are being increasingly investigated at the cellular level owing to their importance for human health and various areas of industrial and environmental relevance (Junter and Jouenne, 2017).
A number of immobilisation techniques have been described over the last 40 years, in mainly in books and journals (Kandimalla, 2008). Immobilising cell systems can be separated into either artificial or naturally occurring (Jauron and Granstrom, 1989). In the artificial category, microbial/eukaryotic cells will be artificially entrapped inside of or attached to numerous matrices or supports where they are kept in or not kept in a viable state, this depend on the amount of harmfulness of the immobilisation procedure itself (Jauron and Granstrom, 1989).
Polysaccharide gel matrices, such as Ca-alginate hydrogels, are one of the most regularly used materials for harmless cell entrapment (Kandimalla, 2008). Cell attachment to an organic or inorganic substratum may be attained by forming covalent bonds between cells and the support using cross-linking agents (Junter and Jouenne, 2017). This immobilisation method is commonly incompatible with cell viability. The unstructured adsorption of the microbial cells to the different forms of carrier gives natural immobilised cell systems in which cells are attached to their support by non-covalent bonds, generally not specific interactions for example electrostatic interactions (Junter and Jouenne, 2017). In appropriate environmental conditions, this initial adsorption step may be followed by colonisation of the support, leading to the formation of a biofilm in which microorganisms are entrapped within a matrix of extracellular polymers secreted by themselves (Junter and Jouenne, 2017). Biofilms are securely attached to their substratum rather than purely adsorbed cells. therefore, they offer more practical as an immobilised cell system. Surface colonisation to procedure biofilms is a universal bacterial strategy for survival, and undesirable biofilms may occur on inert or living supports that may be in natural or in biological surroundings and also in industrial systems (Junter and Jouenne, 2017).
Aim of the Project
This research will build on the methods discussed in the paper written by Hobbs et al. (1989). The aim will be to use dispersed growth of Streptomyces in a liquid culture but will also aim to increase the antibiotic yield from this research by using immobilised cells. Coke (a form of coal) will be used to immobilise the cells, and will be used and looked at to determine whether the once the cells were immobilised will they grow to gain a higher yield of antibiotics. The cells will be immobilised but they will not be dead, they will not necessarily be able to replicate in this state which is the perfect state and condition for the antibiotic producing cells.
Objectives of the Project
Determine if antibiotic yield can be increased.
Optimise previous method.
Apply method of increasing antibiotic yield.
Determine whether immobilisation will increase the yield of antibiotic.

Method
Methods taken from paper written by Hobbs et al. (1989) this method will be built upon by using the principle of immobilising the cells with the use of coke to determine whether or not antibiotic yields will be improved and increase (Lebeau, 1996). Whilst using the method from the study by Hobbs et al. (1989) before growth conditions are set the carrier (coke) will be prepared and then the Streptomyces will be fixed to the carrier, then the method will continue to be carried out
(El-Enshasy et al., 1996).***
.
Work plan
Below is a table of a proposed schedule and work plan for the research. It is imperative that all the work is carried out carefully and that there is plenty of time for all the work to be carried out to the best standard.
Resources
Equipment and/or Resources
Costing (approximate number)
Laboratory equipment
£10,000
Consumables
£4,000
Computer hardware and software
£2,000
Travelling expenses
£1,000
Laboratory Technician x2
£15,000
Biologist x2
£30,000
Total
£62,000
Impact statement
The use of polyanions and immobilised cells will be used to reduce the problems associated with the growth in pellet form will be studied and the research will increase the biomass yield and also it will give an increased yield of actinorhodin which is an antibiotic that is blue pigmented and is a secondary metabolite. This research will look to increase the yield of the antibiotic (actinorhodin/OD unit).
Increasing the antibiotic yield using dispersed growth of Streptomyces in a liquid culture will mean that as well as the benefits of dispersed growth of Streptomyces in a liquid culture there will also be more antibiotics produces whereas in the initial method there was no increase in antibiotic yield. This will also open up the capability of immobilised cells and may be beneficial in the scientific field as a method that can be applied to similar microorganisms.
An increase in the understanding of Streptomyces, a microorganism group, will be a benefit of this study by using a method of growing it that allows to understand the physiology of the cell and to look at how immobilised cells optimise this and allow further understanding of how the antibiotic grows and why the yield would increase when cells were immobilised.
References
AMR (2016). THE REVIEW ON ANTIMICROBIAL RESISTANCE. [online] HM Government. Available at: https://amr-review.org/sites/default/files/160518_Final paper_with cover.pdf.
(AMR, 2016)
Blaskovich, M., Butler, M. and Cooper, M. (2017). Polishing the tarnished silver bullet: the quest for new antibiotics. Essays In Biochemistry, 61(1), pp.103-114.(Blaskovich et al., 2017)
Chaumeil, M., Najac, C. and Ronen, S. (2015). Studies of Metabolism Using 13C MRS of Hyperpolarized Probes. Methods in Enzymology, pp.1-71.
(Chaumeil, 2015)
Dervakos, G. and Webb, C. (1991). On the merits of viable-cell immobilisation. Biotechnology Advances, 9(4), pp.559-612.
(Dervakos and Webb, 1991)
Doleyres, Y. and Lacroix, C. (2005). Technologies with free and immobilised cells for probiotic bifidobacteria production and protection. International Dairy Journal, 15(10), pp.973-988.
(Doleyres and Lacroix, 2005)
El-Enshasy, H., Farid, M. and El-Diwany, A. (1996). Oxytetracycline production by free and immobilized cells of Streptomyces rimosus in batch and repeated batch cultures. Immobilized Cells – Basics and Applications, Proceedings of an International Symposium organized under auspices of The Working Party on Applied Biocatalysis of the European Federation of Biotechnology Noordwijkerhout, pp.437-443.(El-Enshasy et al., 1996)
Feeney, M. (2015). How does one culture streptomyces?. [online] Quora. Available at: https://www.quora.com/How-does-one-culture-streptomyces [Accessed 3 Dec. 2018].
(Feeney, 2015)
Guthrie, E., Flaxman, C., White, J., Hodgson, D., Bibb, M. and Chater, K. (1998). A response-regulator-like activator of antibiotic synthesis from Streptomyces coelicolor A3(2) with an amino-terminal domain that lacks a phosphorylation pocket. Microbiology, 144(3), pp.727-738.(Guthrie et al., 1998)
Hobbs, G., Frazer, C., Gardner, D., Cullum, J. and Oliver, S. (1989). Dispersed growth of Streptomyces in liquid culture. Applied Microbiology and Biotechnology, 31(3).
(Hobbs et al., 1989)
Hodgson, D. (2000). Primary metabolism and its control in streptomycetes: A most unusual group of bacteria. Advances in Microbial Physiology, pp.47-238.(Hodgson, 2000)
Jaurin, B. and Granstrom, M. (1989). ?-Glucosidase genes of naturally occurring and cellulolytic Streptomyces species: characterization of two such genes in Streptomyces lividans. Applied Microbiology and Biotechnology, 30(5).(Jauron and Granstrom, 1989)
Junter, G. and Jouenne, T. (2017). Immobilized Viable Cell Biocatalysts: A Paradoxical Development ?. Reference Module in Life Sciences.
(Junter and Jouenne, 2017)
Kandimalla, V., Tripathi, V. and Ju, H. (2008). Biosensors based on immobilization of biomolecules in sol-gel matrices. Electrochemical Sensors, Biosensors and their Biomedical Applications, pp.503-529.
(Kandimalla, 2008)
Lebeau, T., Jouenne, T., Mignot, L. and Junter, G. (1996). Double-chambered bioreactors based on plane immobilized-cell membrane structures. Immobilized Cells – Basics and Applications, Proceedings of an International Symposium organized under auspices of The Working Party on Applied Biocatalysis of the European Federation of Biotechnology Noordwijkerhout, pp.532-537.(Lebeau, 1996)
Leenen, E., Dos Santos, V., Tramper, J. and Wijffels, R. (1996). Characteristics and selection criteria of support materials for immobilization of nitrifying bacteria. Immobilized Cells – Basics and Applications, Proceedings of an International Symposium organized under auspices of The Working Party on Applied Biocatalysis of the European Federation of Biotechnology Noordwijkerhout, pp.205-212.(Leenen et al., 1996)
Lindsey, K., Yeoman, M., Black, G. and Mavituna, F. (1983). A novel method for the immobilisation and culture of plant cells. FEBS Letters, 155(1), pp.143-149.
(Lindsey et al., 1983)
Vecht-Lifshitz, S., Magdassi, S. and Braun, S. (1990). Pellet formation and cellular aggregation inStreptomyces tendae. Biotechnology and Bioengineering, 35(9), pp.890-896.(Vecht-Lifshitz et al., 1990)
Wandrey, C. (1996). Why immobilize?. Immobilized Cells – Basics and Applications, Proceedings of an International Symposium organized under auspices of The Working Party on Applied Biocatalysis of the European Federation of Biotechnology Noordwijkerhout, pp.3-16.
(Wandrey, 1996)

Strategies of Vitrification in Animals

Background of Vitrification
An important aspect in preserving female gametes for future use, permitting the female genetic material to be stored unfertilized until an appropriate mate is selected is known as cryopreservation of oocytes.5 In gametes and embryos, cryopreservation has become an essential part of assisted reproductive technologies (ART)3 as the extensive use of animal reproductive technologies is often dependent on the success of gamete and embryo cryopreservation.1 Sperm cryopreservation has had a high success rate when used in humans as well as a variety of mammalian species.3 Embryo cryopreservation allows the full genetic complement of sire and dam conservation, thus having potential for protecting and managing species population cohesion and heterozygosity.5 Embryo cryopreservation has also been used in clinical ART with a result in healthy baby deliveries, however, there are known setbacks as it requires the male to produce embryos. It has been prohibited in some countries due to ethical, legal, and religious beliefs. 3
Slow freezing (slow cooling) and vitrification are the two leading methods for cryopreservation. The standard technique which is favored in early procedures and used in various species for oocyte and embryo cryopreservation is known as slow freezing (slow cooling). The new method of vitrification is being widely used as it damages the oocytes and embryos less than slow freezing. Vitrification is known as the ultra-rapid cooling method.3 Vajta states that the “physical definition of vitrification is the solidification of a solution (glass formation) at low temperatures without ice crystal formation. The phenomenon can be regarded as an extreme increase of viscosity and requires either rapid cooling rates or the use of cryoprotectant solutions, which depress ice crystal formation and increase viscosity at low temperatures.”6
Vitrification in domestic animals6 (2000)
Vitrification is an important study as it leads to embryo cloning. There was no decrease for further developmental ability in bovine embryos in the pre-compaction stage along with blastomeres being successfully used as donors for nuclear transfer, allowing for a rapid increase in research for the cloning industry.6 The blastocyst rates were maintained as recipient cytoplasts for successful cryopreserved and full-term development were obtained. 6 There was a breakthrough in cryopreservation of bovine oocytes as the blastocyst rates after fertilization and in vitro culture stated to approach non-cryopreserved control values and live offspring. 6 It has been understood that the success of cryopreservation is dependent on the culture conditions as recent studies in culture methods have improved embryo quality and the traditional equilibrium freezing method may also be successfully applied. 6 A major encounter researchers found was to improve and stabilize the excellent survival rates in vitro and birth of some piglets while permitting large scale porcine embryo transfer as it has not been successful for long.6 There has also been challenge in establishing a universal standardized vitrification method, that may be successful for cryopreservation for different species in different developmental stages of oocytes and embryos.6
Caffeine and Oocyte: Sheep3 (2018)
There has been extensive research has been done on cryopreservation of metaphase II (MII) oocytes, where oocytes at this stage can disrupt the meiotic spindle, however, could be avoided by freezing the oocytes at the germinal vesicle (GV) stages. 3 There have been advantages in both human and animal reproduction of freezing oocytes. There have also been studies to cryopreserve GV-oocytes in mammalian species with live human and animal births and the blastocyst development rate is low. 3 With advantages, there are also many obstacles. One is the that there is no standard protocol established in both humans and some animals regarding frozen GV-oocytes prior to fertilization while being matured in vitro (IVM).3 Moawad et al state that “oocyte meiotic maturation is controlled by the level of two cytoplasmic protein kinases such as maturation promoting factor (MPF) and mitogen-activated protein kinase (MAPK).” 3 Previous studies show that immature ovine oocytes in vitrification decreased the level of both MPF and MAPK after IVM, where high activities of MPF and MAPK are responsible for the onset of GV breakdown and are essential for oocyte maintenance at MII-stage. 3
In the study they evaluated the effects of vitrification of ovine GV-oocytes on spindle assembly, MPF/MAP kinases activities, and preimplantation development following IVM and IVF along with elucidating the impact of caffeine supplementation during IVM on the quality and development of vitrified/warmed ovine GV-oocytes. 3 They use caffeine (1, 3, 7 – trimethylxanthine) as a phosphodiesterase inhibitor. 3 The study shows in sheep there was an increase in both MPF and MAPK kinases when treated with in vitro matured oocytes (IVM) along with improving both the frequencies of nuclear envelope breakdown and chromosome condensation of transferred nuclei, It also increased the total cell numbers while reducing the frequency of apoptotic nuclei in blastocyst embryos produced by somatic cell nuclear transfer. 3 It was also found that aged denuded ovine oocytes had an increase in blastocyst development and decrease in the frequency of polyspermy following IVF when using the caffeine treatment. 3 There was a negative impact of oocyte quality and development after IVM/IVF and embryo culture, when trying to restore the spindle integrities, MPF/MAPK activities and subsequent development of vitrified/warmed oocytes at the GV-stage by supplementing them in vitro maturation.3 There is still little known about the role of development of vitrified/warmed oocytes.
Non-Vitrified vs Vitrified in vitro-derived embryos are transferred in cattle1 (2018)
Do et al1 states that in the cattle industry, the slow freezing method is used more when producing embryos of superovulation in cryopreservation. When comparing slow freezing and vitrification of in vitro0derived embryos transferred to cattle, there was lower survival rates in slow freezing procedures.1 This was based on their capacity to re-expand and to hatch after being thawed or warmed and cultured in vitro making it an effective method used in cryopreservation in human oocytes along with showing promising results in bovine oocytes and embryos.1 There is no vigorous procedure for cattle embryos which optimize pregnancy through invitro-derived embryos being transferred into females. 1
Re-vitrification or storage in liquid nitrogen with pig embryos4 (2018)
An improved vitrification procedure, The open pulled straw (OPS) method is an improved vitrification procedure which provides high rate in vitro using post warming embryo hopefully leading to higher rates in pregnancy and farrowing in porcine embryos after surgical ET without any prior treatments. 4 There were similar survival rates after 24 h in culture between vitrified and fresh embryos. 4 Embryo transfer (ET) technology is on high demand in the swine industry.4 There was a loss of interest in this technology for decades due the need of surgical procedures for embryo collection along with the difficulties arising when trying to use embryo cryopreservation in swine. 4 But thanks to new developments and safer non-surgical ET, vitrification is becoming the only effective method for porcine embryos when trying to preserve them for long periods as it avoids the formation of intracellular ice crystals. 4
As re-vitrification and air embryo shipment in the vapor phase of liquid nitrogen is a new method in cryopreservation, there are many unsolved factors. 4 As Nohalez et al states, “the number of warmed embryos exceed the number of embryos necessary to be transferred to the recipients or when some recipients cannot receive embryos due to health problems or difficulties during the insertion of the non-surgical ET catheter, a number of unexpected supernumerary embryos could have been warmed.” 4 They believe they could use re-vitrification as a way to restore extra warm embryos for future ET as it is has more flexibility for transfer allowing for an increase in number of transferred embryo per recipient.4 However the survival rates for post-warming embryos in pigs have not yet been discover for the effects of re-vitrification.4 But through this study, further information was found to help explain the re-vitrification process. 4 The results were detrimental to a significant proportion of embryos as more than 60% of porcine blastocyst derived from vitrified and warmed CCMs (cavitating morulae) and UBLs (unhatched blastocyst) survive re-vitrification and re-warming. 4 The embryos were either accidentally warmed or warmed embryos were not transferred into recipients due to special circumstances and may be re-vitrified and used in future ETs. 4
Vitrification of buffalo oocyte and embryos5 (2016)
In bovines, most methods of reproductive biotechnology have been applied, however, are not as efficient. 5 Buffaloes also have been reported to have poor response to superovulation treatments, exhibiting relatively low yield of in vivo-derived embryos. 5 For buffalo embryo cryopreservation both slow freezing and vitrification techniques are used. 5 In the future, vitrification will become the most suitable method of cryopreservation of any cells and tissues. Especially for buffalo oocytes as the extremely large single cells contain an excess amount of intracellular lipid making them sensitive to cryopreservation. 5
Conclusion
Compared to slow-freezing (slow-cooling) method, vitrification technologies applied on oocytes and embryos has become more successful along with MII-stage oocytes rather than GV stage for their higher membrane stability during freezing.5 Although there has been success, a major obstacle in vitrification is associated with chilling injury, osmotic stress, CPA toxicity, and ice crystallization.5More studies need to be done to further understand and identify key aspects in a protocol which will increase the GV-stage oocytes maturation rate, fertilization and blastocyst rate, as well as the survival and hatching rate of vitrified-thawed embryos.5

References
Do, V. H.; Catt, S.; Amaya, G.; Batsiokis, M.; Walton, S.; Taylor-Robinson, A. W. Comparison of pregnancy in cattle when non-vitrified and vitrified in vitro-derived embryos are transferred into recipients. Theriogenology 2018, 120, 105-110.
Kokotsaki, M.; Mairhofer, M.; Schneeberger, C.; Marschalek, J.; Pietrowski, D. Impact of vitrification on granulosa cell survival and gene expression. Cryobiology 2018.
Moawad, A. R.; Choi, I.; Zhu, J.; EL-Wishy, A. B. A.; Amarnath, D.; Chen, W.; Campbell, K. H. S. Caffeine and oocyte vitrification: Sheep as an animal model. International Journal of Veterinary Science and Medicine 2018, 6, S41-S48.
Nohalez, A.; Martinez, C. A.; Parrilla, I.; Maside, C.; Roca, J.; Gil, M. A.; Rodriguez-Martinez, H.; Martinez, E. A.; Cuello, C. Eventual re-vitrification or storage in liquid nitrogen vapor does not jeopardize the practical handling and transport of vitrified pig embryos. Theriogenology 2018, 113, 229-236.
Parnpai, R.; Liang, Y.; Ketudat-Cairns, M.; Somfai, T.; Nagai, T. Vitrification of buffalo oocytes and embryos. Theriogenology 2016, 86, 214-220.
Vajta, G. Vitrification of the oocytes and embryos of domestic animals. Animal Reproduction Science 2000, 60-61, 357-364.

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