Overview
We aim to make Clostridium tyrobutyricum facultative anaerobic and add it to animal feed. The engineered C. tyrobutyricum can adapt to aerobic environment so it doesn’t require strictly anaerobic condition while fermenting. At the same time, when it is excluded from animals, C. tyrobutyricum continuously produce butyric acid and can neutralize the alkaline soil. For practical concerns, the product is packed as a nutrient powder containing facultative anaerobic C. tyrobutyricum. Our product mainly serves the breeding and production industry. The powder is added into feed to improve animal health and animals' excreta containing our product will be beneficial to the alkaline soil.
1.Background & End users
We aim to make Clostridium tyrobutyricum facultative anaerobic and add it to animal feed. The engineered C. tyrobutyricum can adapt to aerobic environment so it doesn’t require strictly anaerobic condition while fermenting. At the same time, when it is excluded from animals, the production of butyric acid by C. tyrobutyricum can neutralize the alkaline soil. (If C. tyrobutyricum is still strictly anaerobic, it cannot survive in soil)
The product of our project is a nutrient solution containing facultative anaerobic C. tyrobutyricum. We have modified this strain, which can greatly improve its survival and activity in aerobic environment. According to our research, this product can mainly serve the breeding and production industry, and most animal feed processing plants can use this product. This product aims to make animals' excreta beneficial to the environment and not pollute the environment after eating these feedstuffs. Most of the existing feedstuffs are added with many antibiotics. Antibiotics are harmful to the environment and human health, but probiotics can greatly reduce this phenomenon. Although most land improvement products can improve the quality of land to a certain extent, they will also damage the environment and may have a negative impact on the crops on the land. Feed with C. tyrobutyricum can alleviate or even eliminate such problems, which is suitable for environmental development.
2.Product operation
Our product should be convenient to use. This principle is strictly followed in the whole process of designing. Our engineered bacteria are designed to be packed in a practical form and delivered with ease to the user. The user can directly add our product in to the fodder without any extra process.
At first, we considered to store our product as a form of nutrient solution that contains our engineered bacteria.
However, storing in the form of liquid has several limitations:
1.C. tyrobutyricum is active between 25℃ to 37℃. If stored under this temperature, the bacteria will experience excessive growth and go bad. Consequently, the solution has to be kept under -20℃ to make sure the bacteria is inactive. This will lead to much higher cost when trafficking and storing. [1]
2.Liquid is difficult to be mixed evenly with fodder. Nutrient solution will be quickly absorbed when added into dry fodder, causing uneven distribution.
3.The main constituent of nutrient solution is peptone, which is smelly. Livestock may find fodder unpalatable.
Therefore, we proposed an alternative way of storing: to keep the C.
tyrobutyricum in solid state, as powder or granule. This form brings several benefits:
1.The product can be stored for a long time.
2.It is convenient for transportation. Compared to nutrient solution, solid product with the same mass contains more C. tyrobutyricum.
3.When dealing with large amount of fodder, a packet of powder is much easier to use than a bucket of nutrient solution.
4.Solid feed additives need to be stored at low temperature, about below 15℃. Compared with liquid feed additives, which usually need to be stored at a temperature of -20℃, it has obvious advantages.[2]
However, to achieve this, the nutrient solution needs to be dried. We should find a proper humidity to make sure C. tyrobutyricum stop growing and go into dormancy. Also, the powder should not agglomerate. Otherwise, it will be hard to pour the powder out of its package or sprinkle it evenly over the fodder.
During the drying progress, we have to carefully control the temperature to make sure that C. tyrobutyricum does not die from heat and the liquid can be quickly dried. An optimal way is making the progress finish in a short time under the lowest temperature possible to make sure that our bacteria is not harmed badly from lethal temperature. In this regard, through a field investigation, we learned that our products can be granulated and dried using a spray drying device. By searching for information, we learned that we can granulate and dry the fermentation liquid via spray drying technology. [1] CY-6000Y spray dryer from Hangzhou Chuanyi Experimental Instrument company can realize instant spray drying under specific low temperature, ranging from 50℃ to 80℃, and can be utilized for our purpose.
After the livestock has taken the feed additive mixed with C. tyrobutyricum, after digestion, the excrement will contain a large amount of butyric acid, so the excrement discharged from the animal can be treated to solve the problem of land salinization. The feces can be directly added to the alkaline soil, or can be processed before transporting to the alkaline land. The farm can set up a manure storage tank to prevent pollution of the surrounding environment. Waste hay and cleaned calf litter can be added to the manure tank for composting. After fermentation and metabolism, the manure can be pulled and transported to the sports field for bedding. After complete metabolism, the feces are dried and can be collected and transported to the alkaline area for land improvement.[3][4]
3.Real-world implementation
Our products are mainly used in two scenarios. First, we made the modified C. tyrobutyricum into a feed additive, and used it in animal husbandry. Farmers purchased the feed additive containing C. tyrobutyricum and fed it to the livestock according to the proportion. It is necessary to strictly follow the operating requirements for product use. Secondly, after the livestock has eaten the feed containing C. tyrobutyricum, the excrement will contain butyric acid and live C. tyrobutyricum, which can be used to improve the salinized soil in the pasture or field.
Our product will probably face a few challenges. Firstly, our feed additives will encounter some challenges to meet the requirements of storage conditions, which most feed additives encounter as well. Generally, microbial feed additives need to be stored under low temperature and dry conditions. When the storage temperature is about 15℃ to 26℃, the unstable nutritional feed additives gradually lose the activity; and when the humidity in the air is high, the feed is prone to mold. And due to the reproduction of microorganisms, general feed additives are easy to absorb water, which will accelerate the deformation of additives. Our product needs to be stored at low temperature, below 15 ℃. Therefore, in order to store our product for a long period of time, we have designed a device that can store products at suitable temperature and humidity, and we are still in the process of making improvements to the original spray drying device to make it more suitable for our product.[2]
Secondly, in terms of market promotion, our product is very suitable for promotion at the economic level, because it can not only be used as a feed additive instead of antibiotics, but also has the effect of improving alkaline soil, which kills two birds with one stone.
However, our product may encounter some problems related to biosafety, because our C. tyrobutyricum is genetically modified and needs to be used by animals. Due to the regulations of our relevant national policies, further verification is required for its security, so it may encounter some challenges in market promotion.
If we want to carry out market promotion, we will first pass multiple rounds of strict safety verification and obtain relevant national approval certificates to ensure that our product will not bring biological safety problems to livestock.
4.Safety
We considered biosafety problems carefully. C. tyrobutyricum is a kind of bacteria that exist widely in nature, so it is generally safe.
However, the transgenetic C. tyrobutyricum may cause biological hazard if we do not preserve it properly according to the regulations on the safety administration of agricultural genetically modified organisms in China, as the transgenetic C. tyrobutyricum is in the secondary security level.
To solve this problem, we made a field research of a feed additive company in Nanjing and had an interview with Expert Li, a probiotic feed and animal nutrition expert. According to the result of the research and the interview, the transgenetic C. tyrobutyricum may cause some other harmful bacteria to form resistance genes. So, a device of a hermetic system in order to avoid bacteria divulging to the environment might be needed. However, since we are going to use it in feed and improving soil conditions, a better solution would be adding a plasmid that can make the transgenetic C. tyrobutyricum kill itself after it gets into the environment. For this aspect, further research is needed. [5][6]
5.Challenges and prospect
Although we may encounter challenges posed by biosafety issues, our product, if promoted, can be very effective in solving the problem of antibiotic abuse in feed additives and the problem of soil salinization to a large extent.
Reference
[1]张善亭,史燕,张淑丽,王海宽.丁酸梭菌的研究应用进展[J].生物技术通报.2013,(09)
[2]赵文翰.饲料添加剂的保管与贮存[J]. 农民科技培训.2004,(S1)
[3] https://zhidao.baidu.com/question/759673137140722044.html
[4] https://www.zhihu.com/question/446292497
[5]http://www.moa.gov.cn/ztzl/zjyqwgz/zcfg/202206/t20220607_6401864.htm
[6] http://www.gov.cn/zhengce/2020-12/26/content_5574537.htm
