Development Of A Fermentation Process In Lab

CASE II (DEVELOPMENT OF A FERMENTATION PROCESS IN LAB)

A company has isolated a new bacteria strain which can produce a novel antibiotic. The company asked your help to develop a fermentation process for the bacteria in shaking flasks. You need:

• Find the starting medium recipe for better optimization.
• List the factors requiring optimizations, and explain why the factors have impact on fermentation?

• Design experiment to optimize the fermentation factors.

Solution:

Certain microorganisms are capable of producing some organic compounds which are directly not involved in their growth and development, known as secondary metabolites. These secondary metabolites (e.g., antibiotics) are produced by plants, bacteria, fungi, etc.  Among all these groups, actinomycetes have a dominant place to produce secondary metabolites such as antibiotics. Actinomycetes are basically prokaryotes and gram positive bacteria, but their morphology has resemblance to fungi due to presence of finger shaped mycelia. Actinomycetes are widely distributed throughout the nature. They are present in natural and as well as in manmade environment. Moreover, they exist in freshwaters, soils, lakes, composts, manure and also in plants and food residues.  These micro-organisms are very prominent due to producing high value commercial products, industrially and as well as medically important compounds such as immunosuppressant, antibiotics and chemotherapeutics (Berdy, 2005, Vijayakumar et al., 2010, Bizuye et al., 2013).

Discovery of Pencillin by AlexandarFlemming has introduced a quite different era of medicine and science. Antibiotics has contributed to increase the life expectancy and also to save a huge numbers of lives. Probably, there is no need to repeat how significantly antibiotics have saved the lives and how they contributed to treat the infections and diseases, to decrease the top causes of mortality and morbidity of human (Aminov, 2010).

Actinomycetes produce antibiotics that have application in treating different human infections. They are also known due to their genetic material, because their DNA is enriched with >55% G+C (Guanine + Cytosine) content (González-Franco and Robles-Hernandez, 2009, Gurung et al., 2009, Ogunmwonyi et al., 2010). It was observed that almost 70% of naturally occurring antibiotics are isolated by different genus of actinomycetes. Among these genus, most important genus is Streptomyces for the production of antibiotics. Certain antibiotics such as erythromycin, penicillin and methicillin, they exhibit only one time potent or effective results against infection. Because bacteria has gained resistance against such antibiotics, that’s why they has become less effective(Raja et al., 2010).Isolation and screening of antibiotic producing psychrophilic actinomycetes and its nature from hill soil against viridians Streptococcus sp. (Raja et al., 2010). Then resistant strains of antibiotics were developed such as vancomycin and methicillin. But they cause certain side effects. Antibiotic resistant strains are increasing, so to prevent such happenings, this is a time to immediately develop novel antibiotics to replace these existing antibiotics by finding new approaches to isolate promising bacteria from soil(Ilić et al., 2005). Therefore, development of new antibiotics or using the combined antibiotics has delayed the climax of microbial resistance. There is new possibility to produce synergetic antibiotics to treat microbial infection. Antibiotic synergism is a novel approach between bioactive extracts and along with known antibiotics. By combining antibiotics, potential of drug to treat microbial infection in upgraded (Adwan and Mhanna, 2008). As discussed earlier, some antibiotics have side effects or expensive, thus, finding of novel antibiotics is an important necessity (Bizuye et al., 2013, Retnowati, 2010).  From actinomycetes, we can produce novel and effective antibiotics. The aim of this study is to isolate actinomycetes from soil samples and then screen them for the production of antibiotics.

Query:1

Isolation and screening of antibiotic producing actinomycetes from soil:

How to isolate and screen actinomycetes?

• Location of study

We studied different promising areas of India for actinomycetes producing soil and then we just focused on two regions i.e. garbage disposer areas of house kitchen and rhizosphere soil samples of different areas. Microbial community of soil is highly dependent on the status and composition of their soil habitat. Then we took different soil samples with different depths, from these two potent regions for further study.

• Sampling and isolation of actinomycetes

Samples were taken from two sources, one from garbage disposer and other from rhizosphere soil areas. During this study, 15 sterile samples were taken from 5 sites that have different depth (5, 8 and 11 cm) of the superficial layer from each location. Then soil samples were numbered accordingly and placed them in a sterilized paper sack, thenthese are transferred to Mycology laboratory, after that, homogenization of these samples carried out. Samples were spread out on trays in order to clean them out from leaves, roots and small stems. These samples were sieved in 2mm mesh and air dried them at room temp (i.e. 25°C) for 7 days. Then 5 grams of each sample was taken and poured into the test tubes that already have 10 ml of aseptic physiologic saline (0.9% NaCl) and then mix them for almost 3 minutes. Then incubate them for 15 minutes and then transfer 3 ml of supernatant to another aseptic test tube and then add 1.5 ml of streptomycin and chloramphenicol (0.2mgl/ml) and then stirrer them and  incubate for 30 minutes. This solution was shaken again by using vortex mixer. In next step one drop of sample was added to a Sabouraud’s dextrose agar medium and another drop was added to brain-heart infusion agar. These both media were containing 0.5 g/l of cycloheximide. They were now incubated at 35°C for 2-3 weeks. These colonies were selected and streaked on new plates of prepared culture media. Actinomycetes’ colonies were identified through their color and they were dried, rough with regular and irregular pattern and mentioned as convex colony. These plates were incubated at 35°C and analyzed up to 2-3 weeks for maximum growth. Although several colonies showed their maximum growth in first some days of incubation. In order to purify actinomycetes from other suspicious colonies, streak plate method was used. After that pure colonies were isolated by observing their colonial morphology, color of hyphae and aerial mycelia, and then these colonies were plated on the agar media singly.

Then cellophane tap and cover slip-buried methods were used to analyze morphology of actinomycetes. Light microscopy under oil immersion (1,000 x) was used to analyze the structure of mycelium, color and arrangement of arthrospore on the mycelium. Since a single method could not enough to identify all actinomycete thoroughly; therefore, a combination of different methods were important to identify actinomycetes isolates to the species level. If subdivided and fragile filaments specific of actinomycetes were examined, then they were isolated and inspected for partial acid-fastness by staining with the carbolfuchsin modified acid-fast stain with a weak (0.5-1%) sulfuric acid decolorizing solution. Furthermore, a standard biochemical tests and physiological criteria i.e ability to degrade the different organic compounds like casein, tyrosine, xanthine, hypoxanthine and starch as substrates, the use of urea and different carbon sources as well as growth in 4% gelatin medium were examined in order to get a possible classification to the species level. Gelatin hydrolysis was carried  by stabbing a loopful of organism about 1 cm into a nutrient gelatin tube (BBL), that were incubated at 35°C in air for up to 7 days.

Detection of hydrolysis was done by keeping the tubes at 4°C for 15 min and then tilting each tube at a 45° angle. The test was only viewed positive if the gelatin was liquefied.

For hydrolysis of urea, a heavy loopful of bacteria was suspended in 0.5 ml of a medium containing 1 g urea and 1 ml of a cresol red solution (0.5% [wt/vol]) in 100 ml phosphate buffer (0.005M, pH 6). Tubes were incubated at 35°C and checked after 1 and 2 days for a pink color shift showing a positive reaction.

• Primary screening

Actinomycetes isolated and analyzed from different soil samples, further samples were screened for the examination of their antimicrobial spectrum. Different test bacteria were used for primary screening, these bacteria were S. aureusATCC2923, Pseudomonas aeruginosa ATCC27857 (P.aeruginosa),Escherichia coli ATCC25922 (E. coli), Klebsiella pneumonia ATCC7000603 (K. pneumonia) and Salmonella typhi ATCC9289 (S. typhi). To ensure the Anti-fungal activity of actinomycetes, Saccharomyces cerevisiae(S. cerevisiae) was used as test organism. Activities were examined by using nutrient agar for culturing of bacteria and potato dextrose agar for fungi. Each plate was streaked with each isolated colony at the midpoint of a plate and incubated at 37 °C for 7 days. Then, fresh sub-cultured test organisms were perpendicular streaked to the actinomycete isolate. Then the plates were incubated for 24 hours at 37 °C for bacteria and incubated 48 h at 28 °C for fungi.After incubation, the zone of inhibition was measured and recorded.

• Fermentation and extraction of crude extracts

So, it is proved that solid state fermentation was fitter for the production of pigment by the strain D10 when it compared to submerged fermentation. And in this study, solid state fermentation was used for the production of crude pigment. Solid stat fermentation uses the solid substrates i.e bran, bagasse, and paper pulp. The major benefit of using these solid substrates is that nutrient-rich waste materials can be easily reused and recycled as substrates. In this technique of fermentation, the substrates are used very slowly and immoveable manner, so the same substrate can be utilized for long fermentation periods. Hence, this technique provides controlled release of nutrients.Based on the zone of inhibition in primary screening, actinomycetes isolates ( represented as Ab18, Ab28, Ab43) that have likely antimicrobial activity were selected for solid state fermentation and extraction, and then the crude extracts were analyze through agar well diffusion methods. In these study cultures of actinomycete isolates were grown in starch casein broth (200 mL) at 37 °C for 7days. Finally  10% of cultured broth was inoculated into sterilize Erlenmeyer flask that containing natural media (40 g wheat grain and 20 mL milk) on thermostat water bath at 37 °C for 7days. In order to concentrate the antimicrobial metabolite produced from isolates, equal volume of ethyl acetate (200 mL) was added in each solid state fermented cultures for 1 hour in thermostat water bath shaker at 37 °C. Then the active metabolite containing ethyl acetate was isolated from the solid residue with Whatman No.1 filter paper and extracts were concentrated through the help of rotavapour. The crude extracts collected from each isolates were dissolved in ethyl acetate (76 mg/mL) and utilized as stock concentration and ethyl acetate is used as control for the determination of actinomycetes activity against test pathogens. The wells (6 mm diameter) were cut using a sterile cork

Borer on Muller Hinton agar and potato dextrose agar. Twenty four hours young culture of S. aureus ATCC2923, methicillin resistant S. aureus (clinical isolates), E. coli ATCC25922, S. typhiATCC9289, K. pneumonia ATCC7000603, S. boydi ATCC9289) and 48 h young culture of Candia albicans (C. albicans, clinical isolates) these cultures were swabbed with sterilized cotton swab on the surface of prepared Muller Hinton agar for bacteria and potato dextrose agar for fungi. Sixty micro liters of dissolved crude extract was loaded into each well and left for 30 min unless the metabolite was diffused. Then the plates were incubated for 24 h at 37 °C for bacteria and 48 h at 28 °C for fungi. After incubation, the zone of inhibitions were measured and recorded.

Query:2

Optimization factors for maximum product output:

As the organisms under consideration are bacterial strains, so following factors must be taken into account during the growth optimization to obtain maximum quantities of the desired antibiotic.

• Carbon source:

The primary nutrient to run bacterial metabolic machinery is the carbon source and more specifically glucose. Glucose kick starts the process of glycolysis which is the cardinal growth sustaining pathway in all life forms. Normally LB medium used for bacterial culturing and fermentation contains Tryptone and yeast extract, which directly are not source of carbohydrates and only provide scant quantities of glucose. So in order to compensate this, 1-4 g/L of glucose is added to the medium recipe wherever  high metabolic activity is required especially in fermentation processes.

• pH value:

pH value has been categorized as of major interest for bacterial growth by (Juwarkar et al., 2006). For optimal bacterial growth, a steady pH value of 6-7.4 is recommended by nature protocols. The importance of this factor is of major interest because optimal pH is directly proportional to the growth of bacteria. The enzymes involved in bacterial division for example DNA polymerases work best at the perscribedpH. Any shock to the pH hinders the bacterial cell division or blocks it altogether. The key point to be noted here is that pH value determines the enzyme activity in all life forms. A drift in pH value can cause a serious damage to multiple metabolic processes if not stop them altogether. Therefore it is always advised to check the pH of the medium recipe prior to inoculation so that it can be adjusted.

• Temperature:

Another key factor is the temperature of incubation of bacterial cultures. The standardized temperature for optimal bacterial growth is 37 degrees celscius. However depending upon the type of bacteria and the environment from where it is isolated can also dictate the optimum temperature standards. For example bacteria that are isolated from dry harsh and warm regions can show optimum growths at even 50 degrees Celsius. The temperature is directly linked to the enzyme activity in bacteria. The more the temperature moves away from the optimal points the lesser the activity of enzymes is observed. However, unlike pH bacteria show increase in growth patterns as temperature is gradually increased up to a certain limit depending upon the type of bacteria after which it gradually declines.

• Inducers:

Inducers are the natural or synthetic biochemical entities that upregulate the production of a certain metabolite or gene product. These inducers interact with the specific metabolic pathway regulatory elements in such a way that the particular process keeps on producing the desired product without any inhibition at a multiple fold rate. The most common inducer used for protein production in bacteria is T7 RNA polymerase/ RNA promoter system. Among many other advantages, it has a wide variety of hosts and can easily be incorporated in diverse range of bacterial strains. As it is based on the promoter activation principle which entails that over activation of promoter of a metabolic pathway gene sequence will up regulate the expressive gene production which inturn will increase the quantities of the desired protein product.

• Medium additives:

Media additives vary according to the purpose of bacterial growth and desired product. As mentioned earlier, tryptone, yeast extract andNaCl dissolved in distilled water according to standard protocols is enough for a regular bacterial growth in broth medium. However, when the purpose is the production of a secondary metabolite, the metabolism of bacterial cell is enhanced for that an additional carbon source glucose/ dextrose is added as a supplement. Some studies have also shown that when specifically producing antibiotics, addition of 0.05% K2HPO4 significantly enhances the quantities of antibiotics produced by bacterial isolates(AL-GHAZALI and OMRAN, 2017).

• Aeration:

Aeration is also an important factor especially when aerobic bacteria are under consideration. It has been observed through various studies that an increase in the oxygen mass transfer coefficient can significantly increase the bacterial growth and the production of desired antibiotic up to 30 percent. The logic behind it is that the major  metabolic processes of an aerobic bacteria are driven by aerobic respiration. An increase in the index of available oxygen means an increase in the rate of aerobic respiration which in turn enhances the metabolism leading to an increased production of secondary metabolites and antibiotics(Wang and Zhang, 2007).

• Growth time:

Normally bacterial cultures start to show maximum growth within 8 hours which is also termed as growth or log phase. At this point the bacteria attain a maximum number of divided cells and shift towards a growth dormancy state also known as lag phase in which the number of newly grown cells decreases gradually. This behavior is an indicator that now a greater mass of bacterial cells is shifting from primary metabolism to secondary metabolism in which bacteria shift from rapidly growing phase to a survival mode. Once the bacterial cultures have attained the stationary phase, the production of secondary metabolites experiences a boost. This happens because now the bacteria are spending less energy in synthesizing peptidoglycans, primary proteins and nucleic acids. This residual energy and the capacity of bacteria to be less susceptible to the antibiotics in stationary phase are used for the production of secondary metabolites and other antibiotics. Normally this cycle starts after 8 hours of inoculation and attains a maximum productivity in 16-24 hours of initialinoculation.

Query 3

Experimental design for optimization of fermentation factors:

Normally for such experiments, that involve optimization of a process, the output production index dictates that what set of combined conditions must be applied to obtain maximum desired product rather than the standard values of protocols. In our case, we have 7-8 influential factors. Our next step is to determine significant factor/s that may affect the output production by Plackett-Burman method. Optimization of each studied factor is described following:

1. The first and foremost step is to screen out the factors that have a negligible effect when range is concerned logically. For example using inducers by standard protocols is acceptable as an increased quantity of inducer does not necessarily mean a higher output as mode of action of an inducer follows a qualitative pattern either an inducer induces an effect or it does not.
2. The second step involves the listing of all primary influential factors that will determine the direction of the experiment. In this case temperature and pH etc are the factors that will determine the optimum response.
3. The third step involves the determination of ranges. This step requires literature review to determine that what experimental ranges have been used in similar experiments for identical outputs. For example in our case the optimum temperature range can be 32-42 degrees Celsius, pH can be 6.6-7.6, added glucose concentration can be 0.5-4 g/L and growth time can be 16-24 hrs and so on. Once the experimental factors and their range values are established, it becomes easier to narrow down the set of conditions that can give the maximum output.
4. This step involves the intervention of biostatistical tools based on significant factors determined by PlackettBurman method. The specifications of our experiments are fulfilled by the Response Surface Methodology model (RSM). RSM uses a set of very specific tools which when combined determine the combined ranges of the experimental factors that can possibly give the maximum output i-e maximum quantity of produced antibiotic. RSM also rules out the factors that have a secondary role and do not significantly determine the output. The models which qualify the test of significance that is(P < 0.05) are then put to wet lab experimentation. A rough example for our experiment using RSM can be given as:

Runs	Temperature (Celsius)	pH	Glucose (g/L)	Growth time (hrs)	O2 mass transfer coefficient (h−1 )	Predicted Output
1	32	6.6	0.5	16	115	x
2	33	6.7	1	17	116	y
3	34	6.8	1.5	18	117	z

• Finally the results obtained from the statistical analysis will be used as the experimental ranges. Once the experiment is conducted and the quantity of antibiotic produced by each run is determined, a standard can be established by comparing the statistically predicted output and the actual output of the wet lab experiment. Such a technique will standardize the protocols for similar experiments that are to be conducted in the future(Wang et al., 2008).

References:

ADWAN, G. & MHANNA, M. 2008. Synergistic effects of plant extracts and antibiotics on Staphylococcus aureus strains isolated from clinical specimens. Middle-East Journal of Scientific Research, 3, 134-139.

AL-GHAZALI, L. H. & OMRAN, R. 2017. OPTIMIZATION OF MEDIUM COMPOSITION FOR ANTIBACTERIAL METABOLITE PRODUCTION FROM STREPTOMYCES SP. OPTIMIZATION, 10.

AMINOV, R. I. 2010. A brief history of the antibiotic era: lessons learned and challenges for the future. Frontiers in microbiology, 1, 134.

BERDY, J. 2005. Bioactive microbial metabolites. The Journal of antibiotics, 58, 1-26.

BIZUYE, A., MOGES, F. & ANDUALEM, B. 2013. Isolation and screening of antibiotic producing actinomycetes from soils in Gondar town, North West Ethiopia. Asian Pacific journal of tropical disease, 3, 375-381.

GONZÁLEZ-FRANCO, A. C. & ROBLES-HERNANDEZ, R. 2009. Actinomycetes as biological control agents of phytopathogenic fungi. Tecnociencia Chihuahua, 3, 64-73.

GURUNG, T. D., SHERPA, C., AGRAWAL, V. P. & LEKHAK, B. 2009. Isolation and characterization of antibacterial actinomycetes from soil samples of Kalapatthar, Mount Everest Region. Nepal Journal of science and Technology, 10, 173-182.

ILIĆ, S. B., KONSTANTINOVIĆ, S. S. & TODOROVIĆ, Z. B. 2005. UV/VIS analysis and antimicrobial activity of Streptomyces isolates. Facta universitatis series: Med Biol, 12, 44-46.

OGUNMWONYI, I. H., MAZOMBA, N., MABINYA, L., NGWENYA, E., GREEN, E., AKINPELU, D. A., OLANIRAN, A. O., BERNARD, K. & OKOH, A. I. 2010. Studies on the culturable marine actinomycetes isolated from the Nahoon beach in the Eastern Cape Province of South Africa. Afr J Microbiol Res, 4, 2223-2230.

RAJA, A., PRABAKARAN, P. & GAJALAKSHMI, P. 2010. Isolation and screening of antibiotic producing psychrophilic actinomycetes and its nature from Rothang hill soil against viridans Streptococcus sp. Res J Microbiol, 5, 44-49.

RETNOWATI, W. 2010. Identification of Streptomyces sp-MWS1 producing antibacterial compounds. Indonesian Journal of Tropical and Infectious Disease, 1, 82-85.

VIJAYAKUMAR, R., MURUGESAN, S. & PANNEERSELVAM, A. 2010. Isolation, characterization and antimicrobial activity of actinobacteria from point calimere coastal region, east coast of India. Int Res J Pharam, 1, 358-365.

WANG, Y.-H. & ZHANG, X. 2007. Influence of agitation and aeration on growth and antibiotic production by Xenorhabdus nematophila. World Journal of Microbiology and Biotechnology, 23, 221-227.

WANG, Y. H., FENG, J. T., ZHANG, Q. & ZHANG, X. 2008. Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology. Journal of applied microbiology, 104, 735-744.

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