Abstract:
This investigation is attempting to provide useful information in regard to the commercial use of bacterial starter cultures, and determine their optimal growth conditions. This is in order to establish the most economically viable production method, and controlling factors such as pH and atmospheric oxygen concentration in a Lactobacillus fermentation — for producing yogurt — can greatly improve yields by minimising stress and favouring a particular metabolic pathway. Experiments were conducted using two different growth mediums, and the atmosphere was controlled with the use of anaerobe jars and various atmosphere controlling sachets, to achieve aerobic (high oxygen levels), anaerobic (no or extremely low oxygen levels) and microaerophilic (low to moderate oxygen levels) conditions. Results showed that of the 6 bacterial species in the starter culture, 5 of them grew well in microaerophilic conditions on trypticase soy agar (TSA), and the last colony was able to be cultivated on universal agar. However, further research is required to grow them all under the same atmosphere and on the same media, as the interactions between the colonies are not yet fully understood, and this investigation was unsuccessful in growing all 6 on one medium.
Introduction:
Introduction to Starter Cultures:
Starter cultures are groups of several microorganisms (consortia) that are used in commercial and domestic setting to begin, accelerate or steer a specific fermentation process.[11] Common starter cultures include consortia of yeasts used in ethanol fermentation, as well as bread making and yoghurt making. Starter cultures are popular in settings where it is easier to simply purchase a starter culture, as opposed to researching and developing one – such as in developing nations [9]. Additionally, having a consortium of microbes in a fermentation system, as opposed to a single microbe, makes the system much more resistant to environmental changes, as it increases the likelihood of microbes having the traits needed to survive environmental change [3]. Furthermore, genetic diversity allows natural selection to occur, however, this will not necessarily favour the fermentation, as the desired products are bi-products of natural metabolic processes. Consortium fermentation can allow access to multiple metabolic pathways that may be more efficient [14]; this occurs when one microbe produces a waste product that is then converted to the desired product by another microbe.
Introduction to the inoculum:
An inoculum in the sample of desired microorganism that is added to a substrate in order to begin the fermentation. This study was investigating the growth media and atmosphere under which an unknown consortium of 6 bacterial species grew most efficiently, and the patterns of growth between species under aerobic, microaerophilic and anaerobic atmospheric conditions. This research was being done as the consortium in question is used in fermentation of Kefir – which is a fermented dairy product – and being able to determine the conditions at which the consortium has the highest yields of the fermentation product offers the opportunity to maximise the efficiency of the system, and make more profitable.
Introduction to atmosphere control:
In order to create and control a particular atmosphere, two techniques have been used. Atmosphere controlling sachets in anaerobe jars [5], as well as cultivating on universal agar. Both of these techniques are capable of growing strict anaerobes, with the sachets being able to create O2 concentration as low as 0.5%, and the universal agar being able to grow known strict anaerobes[6] — although the exact concentration created by universal agar are not known. The universal agar consists of schaedler broth agar, with additional antioxidants (Ascorbic acid and L-Glutathione) to absorb oxygen from the air and create an anaerobic environment [6]. Additional antioxidants such as uric acid can be added to create an even lower oxygen atmosphere, however that was not used for this investigation as Ascorbic acid and L-Glutathione produce a sufficiently anaerobic environment, and uric acid would be an expensive addition. This media also provides more nutrients than common media such as trypticase soy agar (TSA) [6].
Relevance to current science:
Similar studies have been conducted surrounding the survival of anaerobic bacterial species of clinical interest in oxygenated environments [13], and known strict anaerobes [12]. However, these investigations focused on the growth or survival of isolated species, and were focused around bacteria that were of interest to the scientific community. This investigation, however, is focusing on bacteria of which their status as anaerobes is not known. They were tested as a consortium to replicate the conditions of fermentation.
Methodology:
1) Initial Plating:
Before beginning the experiments with modified atmospheric conditions, an initial streak plate was conducted (Fig. 1) to determine the colony morphology present in the sample (Fig. 2). This is so that they could be identified via colony morphologyafter being incubated under different atmospheric conditions. Colony morphology is the analysis of elements of the colony (such as colour, shape, lustre and margin) with the naked eye [1]. Also, a three-part dilution series (10-1,10-2, 10-3) was created by diluting a suspension with sterile water, which was then \’spread plated’ to determine the ideal dilution to be used for the experiments. An ideal dilution grows between 30 and 300 colonies, minimising human error and statistical bias [15]. For the initial streak plate, TSA plates were inoculated with a sample from the initial starter culture, which was in an aqueous suspension with sterile, distilled water (SDW). This suspension was then streaked using sterile plastic inoculating loops.
For the spread plates, a serial dilution was conducted using 100µl of the aqueous suspension, which was serially diluted with 900µl of SDW until 10-1,10-2 and 10-3 Dilutions had been prepared. 100µl of these dilutions were plated onto TSA, and spread with sterile plastic spreaders. These initial plated were incubated aerobically at 28 °C for 48 hours before being assessed for colonies. They were incubated at 28 degrees and for 48 hours, as this is the known incubation conditions for the fermentation of this particular starter culture — as stated by the microbiologists familiar with the fermentation.
2) Experiment #1
The first experiment was conducted in order to investigate how the consortium grew under different atmospheric conditions when grown on the same media as in the initial streak plate and spread plates. For this experiment, 10-1, 10-2 and 10-3 dilutions were spread onto TSA plates, and incubated at 28 °C for 48 hours. The plates were incubated under aerobic, microaerophilic and anaerobic conditions, and the atmosphere was controlled with microaerophilic and anaerobic sachets which were placed in anaerobe jars with the plates to be grown in their respective conditions. The aerobic plates were incubated in a plastic tub within the incubator.
3) Microscopy
After experiment #2, the microbes that grew under aerobic conditions were gram stained and analysed under a microscope to assess their colony morphology. The gram stains were conducted as they provide information about a bacterium\’s cell membrane type (known as being gram negative or gram positive) by either turning pink or purple [10]. This was conducted over a steel sink with gloves to avoid staining equipment or skin. These slides were then examined under 400x magnification to determine the cell morphology of the microbes that grew in experiment #2 (fig.4), by comparing them to a known index of cell morphologies.
4) Experiment #2
The second experiment was investigating the impact of growth on universal agar, as opposed to TSA, in different atmospheric conditions. The universal agar was prepared by adjusting schaedler anaerobe broth to pH 7 with 10M KOH, and the adjusting an appropriate amount of ascorbic acid (1g/L) and l-glutathione (0.1g/L) to pH 7 with 10M KOH [6]. The antioxidants were then filter sterilised, and added to the schaedler broth and agar, which had been sterilised at 121 °C in an autoclave. 10-2 and 10-3 dilutions were spread plated onto separate universal agar plates. These plates were incubated under aerobic, microaerophilic and anaerobic conditions, and the atmosphere was controlled with microaerophilic and anaerobic sachets [5] which were placed in anaerobe jars with the plates to be grown in their respective conditions. The aerobic plates were incubated in a plastic tub within the incubator.
Results:
After the initial streak and spread plates 5 of the 6 colonies in the consortium were identified via colony morphology. A streak plate was used as it separated the colonies and allowed their colony morphology to be observed [15]. The final colony was identified later after it grew aerobically in experiment #2 (fig.1).
The cell morphology of the microbes in the consortium that grew aerobically in experiment #2 determined via microscopy and gram staining (Fig 2-5). 
| Microbe | Colony Morphology | Cell Morphology |
| m1 | Orange, opaque, glossy (1-2mm) | Single or double bacilli |
| m2 | Yellow, opaque, glossy (1-2mm) | Tetrad cocci |
| m3 | White, opaque, glossy (1-2mm) | Bacilli chains |
| m4 | Off-white, opaque, dull (2-5mm) | – |
| m5 | Yellow, translucent, glossy (1-2mm) | Single or bunched cocci |
| m6 | White, opaque, matte (10-20mm) | – |
Figure 6 – Colony and cell morphologies of microbes in the starter culture
The growth of each of the colonies under the variable conditions was determined by counting the number of colonies that grew on the plates spread with diluted samples [7]. These counts were then multiplied until the number of colony forming units (CFU) were able to be determined per ml of original sample (CFU/ml). CFU are the living cells that are present in a sample, and if plated on agar, would be able to replicate and produce a colony [2]. This can be seen for experiment #1 in fig 7 and for experiment #2 in fig 8.
Figure 8 – CFU of original sample experiment #2
Discussion:
Although the exact composition of the starter culture is unknown, it is known that it is used for the fermentation of dairy products to produce Kefir. The results from figures 4 and 5 show that 5 out of 6 species in the consortium grew well on TSA under microaerophilic conditions, this is likely as they are usually found in a microaerophilic environment (such as soil), and do not have high nutrient requirements.
Microbe 5 would only grow on the universal agar under aerobic conditions. This is possibly because it has higher nutrition requirements, or specific mineral requirements that TSA could not cater for, but universal agar could. Consequently, microbe number 5 would grow best aerobically on schaedler agar without the addition of ascorbic acid or l-glutathione, as these only serve to lower the oxygen concentration for the growth [6]. As such, depending on the necessity of microbe 5 in the overall fermentation, it would be worth investigating the growth of the entire consortium on schaedler broth, without the addition of oxygen scavengers.
However, considering microbe 5 grew on universal agar, which provides a microaerophilic atmosphere due to the antioxidants present [6], it would be more plausible that the entire consortium would grow on TSA under microaerophilic conditions, if some additional nutrients were supplied to sustain microbe 5, but not to the extent of schaedler broth where it is too rich for other organisms in the culture. It is possible that microbe 5 requires some trace nutrients provided in universal agar, such as Hemin and L-Cystine, which are both present at >0.5g/L to survive.[8]
Alternatively for microbe 5, it is possible TSA with additional nutrients in a microaerophilic environment would provide better growth conditions, as it is possible the combination of oxygen scavengers and microaerophilic atmosphere will create an environment that is too low in oxygen. However, if this were not possible due to m5 not surviving the low oxygen environment, the consortium may have to be grown aerobically on universal Agar, with alterations to the nutrients if necessary for microbes 4 and 6. Even on TSA microbes 4 and 6 did not grow in anaerobic or aerobic conditions, so it is plausible they require very specific conditions to grow; either due to the excess or specific nutrients present [8], or the low oxygen resulting from the antioxidants present [6].
When considering human error, the simplistic nature of counting colonies on agar plates means that there was no issue with equipment calibration, and as the plates counted had 30-300 colonies, counting them was not difficult. As determining a colony morphology can be difficult at times, due to competing colonies and phenotypic mutations [4], microscopy and gram stains were used to correctly identify the colonies from experiment two by comparing the cell morphology to known database, and with the assistance of experienced microbiologists; increasing the reliability of the investigation. The external validity of this investigation could be improved somewhat, by repeating the experiments multiple times, and being able to average consistent results. This would also enable for the identification of outliers or anomalies.
Potential continuations of this research could investigate other factors affecting the growth of the consortium (micronutrients, pH, incubation temperature, water activity etc.) as well as isolating the colonies in the consortium and determining their optimal growth conditions separately or in combinations of two or three to identify potentially unknown metabolic pathways for the fermentation. Additionally, if the colonies were identified (through 16S genetic sequencing, or colony and cell morphology) it would be possible to research the current literature on the species and further investigate options for optimising their growth as a consortium.
Conclusion:
This report was investigating the impact of atmospheric oxygen concentration on the growth of a Kefir fermentation consortium using different growth media, with the aim to determine the conditions under which the microbes in an unknown consortium will grow best. All of these microbes were identified by colony morphology as well as cell morphology for four of them, after being gram stained and examined under 1000x magnification to ensure reliability of judgement. After conducting multiple experiments at different atmospheric oxygen concentrations and with different growth media, it has been determined that 5 of the 6 microbes will grow well under a microaerophilic atmosphere on TSA. Due to the fact that microbe 5 did not grow under any atmosphere on TSA, the nutritional requirements of microbe 5 may be the reason it did not grow under these conditions, as opposed to the atmosphere. Further research is also required if microbes 4 and 6 were to be grown aerobically on universal agar. This would be optimal as these are the only conditions under which microbe 5 has grown, however all the microbes only grew under very specific conditions so it would be difficult.
Acknowledgements:
I would like to thank The Kings School and Mr Chris Piper for giving me the opportunity to conduct this research, as well as Dr Michelle Bull and Dr Belinda Chapman of Quantal Bioscience for facilitating the project, and for all their support and guidance.
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About the Author
Jack is an Australian student interested in a variety of applications of the life sciences. He is keen to pursue a career in research, and has been involved in various microbiology research projects. He has also worked in pharmaceutical compounding labs, and gained work experience in various other labs. Outside of science, Jack enjoys fishing, hiking and playing the guitar.