Biosurfactants refer to the microbial origin or any kind of surfactants. Maximum bio surfactants have been produced by the microbes that are synthesized in the form of extracellularly. It can be produced in that situation when the nitrogen is limited during the phase of stationary of the biomass growth (Parthipan et al. 2017, p.193).
Biosurfactants have been applied to increase the surface area of the hydrophobic. Biosurfactants can be used to enhance the bioavailability of the substrates in the context of hydrophobic via solubilization. They have controlled the attachment and have removed the microorganisms from the upper surfaces (Rane et al. 2017, p.492).
There are several microbes in the environment that have been used for the production of biosurfactants. Yeast, Fungi and bacteria belong from the various species and from strains are used for the production of biosurfactants of a large variety of the molecular structure. A number of microorganisms can produce the extracellular compounds (Rane et al. 2017, p.492). Bacillus Subtilis and other species of Bacillus can be used for the production of biosurfactants in the food industries.
According to Bouassida et al. (2018, p.71), Bacillus subtilis strains can be used for production of lipo heptapeptide surfactin. This Bacillus subtilis strain is the most useful producer of biosurfactants. It can be used to reduce the surface tension of the water from 70 to less than 32 mN.
In this study, it has been used in the two experiments of fermentation. First experiment containing the fermentation has been conducted by applying the shake flasks. Second experiment has occurred in the bioreactor method is a scaled up method. These two fermentation experiments have been performed applying the batch method and three types of measurements have been performed such as pH, surface tension and the density of optical.
1st experiment has used the batch method and applied the shake flasks. It is a method has been used by microbiologists (Parthipan et al. 2017, p.193). Biotechnologists are applied in the academy field and bio-industry. This method uses three measurements such as pH, density of optical and surface tension. Nutrient broth of 100 ml in the flask of 500 ml has been used for the experiment. It has been incubated at the temperature of twenty five or thirty seven degree celsius.
500 ml flask has been prepared and the nutrient broth has been used as medium. 500 ml flask contains 100 ml of nutrient broth (Rane et al. 2017, p.492). The nutrient broth needs the nutrients or elements such as sodium chloride; extract of yeast quantity is 6gm per litre, peptone in 1 g per litre and glucose.
The nutrient broth has been incubated for the three days and the fermentation has occurred at the room temperature of 25 degree Celsius thirty seven degree Celsius and thirty degree Celsius. In this paper, the flask has been incubated for thirty degree Celsius. Bacillus subtilis has been incubated overnight. After the time limit of 24 hours, 48 hours and after 72 hours the flasks can be removed from the shaker or incubated and stored in the refrigerator (Rane et al. 2017, p.492)
Execution of the process of the measurement through different methods required 1ml of the broth to be taken at three different times of the experiment. For individual flasks, the measurements had been performed thrice. The average of the readings obtained had been plotted in a graphical manner.
A spectrophotometer had been used to measure the optical density of the sample. Water has been used to calibrate the spectrometer. During the practical analysis method, three 1 ml culture of the broth is to be taken to measure the optical density at 600 nm. Optical density has been measured using the spectrophotometer.
The growth of the biosurfactant, Bacillus subtilis had been measured at different times of the experiment with varying temperatures. 10 ml of the culture sample had been centrifuged. The remaining broth after the extraction of the inoculums containing the culture is had been centrifuged with the speed of 3000 revolutions per minute for a time period of 5 minutes. The supernatant liquid had been decanted into a new tube.
A nutrient broth containing 3 liters of the culture along with the fermenter conditions was taken. The optimal temperature for the experiment has been decided by the shaking flask culture data. The time taken for the incubation for the nutrient broth is three days. For the conduction of the experiment, 30 ml of overnight culture containing the Bacillus subtilis is going to constitute the inoculum which is going to be used in the fermentation process. At the time of the analytical method, 10 ml of sample 1 at time 0 had been taken from the fermenter to be processed for analysis. Consecutively, 10 ml of Sample 2 had been extracted at 24 hours. 10 ml of sample 3 had been taken at 48 hours. Lastly, 10 ml of sample 4 had been taken at 72 hours. All the extraction process was followed by extraction of the sample from the fermentor and placing them in the refrigerator to go forth with the analysis procedure.
During the practical analysis, 1ml of the culture from each of the extracted fluids was taken in a culture broth.
3 cuvettes containing 1 ml of the culture broth had been taken for the measurement of the optical density of the sample. Before the initiation of the experiment, calibration was performed making use of the water. The culture broth had been measured using the spectroscope at 600nm. The optical density had been measured by using the spectrophotometer which had been calibrated using water.
Tensiometer had been used to measure the surface tension of the sample culture. 2ml of the supernatant fluid had been transferred to the glass vessels. Three similar glass vessels had been prepared out of the supernatant fluid. Making use of the tensiometer the interfacial tension between the two liquids has been determined.
Optical density is measured as the refractive index of the optical component or the refractive medium. The measurement of the optical density reflects the ability of the component to slow or delay the rates of the transmission of the light through the medium (Yang et al. 2018, p.10). High levels of optical density are generally reflected through the slower speed through which the light travels. The results of the optical density of both the experiments are presented in Figure 1. In the first experiment, the optical density of three similar samples was measured at four different time intervals.
At 25 degrees centigrade, the optical density of the sample taken for analysis displayed the minimum optical density. The range of the value was from 0.0 to 0.10. With the increase in temperature, when the temperature was raised to 30degree centigrade, the optical density showed a slight increase to 0.016 being the maximum value. In contrast, when the temperature was 37 degrees centigrade, the optical density of all the samples analyzed across the three days resulted in 0.
With the progression of time, the sample collected in 24 hours at varying temperatures stated to show a rigging optical density. The highest optical density is observed when the temperature is 30dgree centigrade, followed by 37 degrees centigrade. Observation of the optical density of the analyzed sample at 48-degree centigrade showed a consistent rise in the optical density in all three days was assayed. Assessment of the optical density after 72 hours, the optical density was still on the rise but the nature of growth was considerably lower than the increase that was observed in 48 hours. From the results that have been observed in the shake flask test, the optimal temperatures can be determined to be 30 degrees centigrade.
In the second experiment the biosurfactant fermentation making use of the automated fermenter, the optical density was measured using the optimal temperature which had been determined to be 30 degree centigrade. In the first 24 hours, the growth had been minimum which is reflected in the optical density values obtained from the spectrophotometer. The rise in the optical density values gets initiated when the sample is collected after a time period of 48 hours. The rise in the optical density values keeps on continuing as the sample is collected after a time period of 72 hours. In both the experiments that had been performed exponential growth had been observed in the samples that had been assayed after a time period of 72 hours.
In both the experiments, temperature constitutes a crucial factor for the determination of the optimal growth of bacteria along with the detection of the growth pattern using the two experiments using the shake flasks and automated fermenter. The first experiment included the use of the shake flasks, made use of varying levels of temperatures to determine the optimal temperature for the growth of the Bacillus subtilis bacteria. Increasing the temperature distinctly increased the values obtained in the spectrometry. In contrast, when the temperatures were increased, the surface tension values decreased considerably.
A comparative analysis of the two experiments based on the difference of the surface tension has been performed has been reflected in figure 2. As can be perceived from the graphical figure, the surface tension reduces with the progression of time. In both the experiments there is an observable accentuated decrease from 50.9 to 39.6 in the experiment using the shake flasks. Simultaneously in the second experiment, the surface tension showed a decrease from 57.5 to 22.6 in the automated fermenter device.
In the first experiment using the shake flasks, the surface tension which is measured at 25 degrees centigrade displayed a consistent decrease in all three days when the sample had been assayed. The decrease is noted down to be 39.6 being the lowest from 52.2. As the temperatures were increased, the decrease in the surface tension over the four time periods when the samples were collected, displayed a decrease in value. The decrease in the value is noted down to be 27.4 from 54.1. Similarly, when the temperatures were raised to 37 degrees a similar trend of decrease was observed. In the second experiment, which included fermentation of the biosurfactant using the automated fermenter there was a consistent decrease in the surface tension as the samples were collected during different times. The maximum decrease was observed in the sample which was collected after a time period of 24 hours. After 24 hours the surface tension values decreased gradually without showing and steep decline. From figure 2 it can be said that the decrease is considerably more in the first experiment which was conducted using the shake flask method.