Bacillus thermoamylovorans

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Introduction

Do you know what could be in your milk? Food pasteurization and sterilization techniques are an important aspect of the food industry as it helps to keep us safe from potentially pathogenic microbes that would make us sick otherwise. The potential benefits have not yet been discovered (1). Typically found in a warmer environment such as hot- water springs with pH between 5.4-8.5, Bacillus thermoamylovorans, undergo facultative anaerobic fermentation. Facultative anaerobic fermentation allows Bacillus thermoamylovorans to grow either in the presence of oxygen or without, thus they can metabolize organic substrates such as carbohydrates (glucose and lactose) (2). With the potential to harm humans as a thermotolerant microbe, Bacillus thermoamylovorans can endure pasteurization and other food sterilization processes that are meant to kill off microbes due to its spores, creating issues for the food industry. In pasteurization, Bacillus thermoamylovorans can live between 40 ̊C and 58 ̊C, with the possibility of survival at 37 ̊C and no survival at 30 ̊C (1).

Classification

Higher Order Taxa Bacterial; Firmicutes; Bacilli; Bacillales; Bacillaceae Species Bacillus thermoamylovorans, type strain LMG 18084T

NCBI: Taxonomy

JGI:GOLD

Phylogenetic Relatedness

Bacillus thermoamylovorans is a member of the Genus Bacillus, meaning it is comprised of a component that allows for the bacteria’s survival in many extreme conditions (5). 16S rRNA almost complete gene sequences were used to comprise the phylogenetic tree, also using maximum likelihood methods. Cultures were grown on Bacillus fumarioli agar (BFA) and TSA over several days are other methods used for compiling this phylogenetic tree. The closest relative of Bacillus thermoamylovorans according to the phylogenetic tree below is Bacillus thermolactis. Based on the 16S rRNA gene sequence analysis, Bacillus thermoamylovorans are 93.9% genetically similar to the species Bacillus thermolactis (1).

Ecological Habitat

B. thermoamylovorans was originally isolated from a hot spring in South Sumatera, Indonesia (11), and since then have been found in dairy farm processing equipment, and animal and human guts (6). Although there is not much information on B. thermoamylovorans hot-spring habitat, it is known that most hot-springs have a temperature well above 50 ˚C. This microbe can be unfavorable to humans as they form heat resistant spores which cause the spoilage of certain dairy farm foods. The foods become inedible, due to the bacteria’s spore-forming which allows it to withstand extreme temperature environments and grow vegetatively after treatment (1). This microbe poses a huge threat to the food processing industry, effecting many products that individuals require to be in the stores, such as cheeses, milk, and any other dairy products. Not only can the bacterium be found in hot-springs where both humans and thermophilic mirobes coexist in, and dairy farms, it can also be found in sewage sludge, degrading organic material. Bacillus thermoamylovorans form creamy white colored colonies. Bacillus thermoamylovorans can live in the presence of oxygen or without oxygen, in about 37-58 ̊C environments (6). B. thermoamylovorans optimally grow at pH 7.0 but can grow within a pH range of 5.4-8.5 (2).

Significance to the Environment

As a microbe that was originally isolated in a hot spring (11), B. thermoamylovorans has now piqued interest in the milk department. B. thermoamylovorans contaminate specific food ingredients, such as cocoa powder, by being present in animal feed, processing equipment, and other farm environments that go through the sterilization process. B. thermoamylovorans live in many different environments, but it has recently been known to make an impact on humans by spoiling milk after pasteurization (1). As a spore-forming Bacillus sp. this microbe can survive heat sterilization processes; when the spores contaminate milk during pasteurization, the vegetative cells begin growing in the milk and start breaking down fats, proteins, and peptides using protease and lipase enzymes. These heat-stable enzymes then remain active after pasteurization causing spoilage, such as flavor and odor imperfections after extended periods of storage (12). Between the sugars that are produced, fermented, and cycled back into the dairy products, B. thermoamylovorans may be one of the newest threats to the dairy industry where not much research has been conducted on the microbe. Most sterilization process on dairy farms only increase the quantity of this microbe, as their heat-resistant spores aid in biofilm formation making it harder to get sterilize the microbe out (7). Human induced changes that influence this microbe include climate change, as the earths atmospheric temperature increase so does the chance of survival of this microbe, and the processing methods used influence the microbe’s ability to survive especially is the proper techniques are not being implemented such as raw milk (12).

Ecological Lifestyle and Interactions

Bacillus thermoamylovorans is a facultative anaerobic organism that can ferment in the absence of oxygen but can also yield ATP if oxygen is present. B. thermoamylovorans are genetically similar to the species Bacillus thermolactis which is also a thermotolerant facultative anaerobe that can be isolated from milk (1). The production of the acids and enzyme activity is one of the main ways that these microbes spoil milk and other dairy products (7). The genus Bacillus can be found in raw milk along with other Gram-positive genera such as Streptococcus and Staphylococcus just to name a few. Milk can also contain Gram-negative bacteria such as Pseudomonas and Klebsiella. The Gram-positive lactic acid producing bacteria such as B. thermoamylovorans are known contributors of food spoilage. However yogurt and cheese products made from lactic acid bacterial fermentation are not considered negative forms of food spoilage (12). There also is not sufficient research on the relationship between the host and this type of thermophilic bacteria (9), however other microorganisms that can be found in milk have pathogenic tendencies such as S. aureus and Staphylococcus argenteus, causing foodborne illnesses such as food poisoning (12).

Significance to Humans

Based off of current research, Bacillus thermoamylovorans is known to interact with humans in a detrimental way. Many individuals drink raw Cow’s which milk already contains a fair amount of nutrients, amino acids like lysine, proteins that support human growth, and contains up to 5.5g/100g of fat and up to 75g of saturated fatty acids (8). Due to its production of lipolytic enzymes B. thermoamylovorans cause spoilage of sterilized milk (7). Since this microbe has endospores which allow it to become resistant to any of the extreme heat processes used, posing a threat to humans who may come in contact with the bacteria from raw unpasteurized milk, that is sold in just about every store. If an individual consumes a large sum of raw unpasteurized milk, they can develop an antibiotic resistant gene if not properly stored. If the antimicrobial resistant genes that are found in raw milk are passed to a pathogen, they have the potential to become so strong that drugs will not be able to treat any serious infections or disease (10). Although there is an emphasis on the bacteria’s presence in the food industry (1), it also has the potential for some health benefits as it shares common fermentation products with lactobacilli suggesting that it is important for probiotic use. Probiotics are living microorganisms that can be ingested to provide specific health benefits, and B. thermoamylovorans can be used to monitor nutrient utilization by digestion of compost (9).

Cell Structure

Bacillus thermoamylovorans are Gram positive bacteria whose isolated colonies are white circular shapes that are 2-3 mm in diameter (7). The microbe’s cells are 0.45 µm in width and 4.0 µm in length and can be characterized as slightly motile due to having flagella. They also live in some pretty extreme temperatures with an optimum temperature of 50 ̊C which is close to temperatures that would burn human skin. B. thermoamylovorans, can produce acid using starches, glucose, and carbohydrates, and possibility of nitrate reduction. There is no data in relation to the salinity of this microbe but there is data on other characteristics such as it’s positive ability for motility, catalase, and aerobic and anaerobic growth. This bacterium is also positive for acid production from L-Arabinose, D-Glucose, Glycogen, D-Mannose, Salicin, Starch, and D-xylose. It is also positive for Nitrate reduction at pH 6, 7, and 8 (5). This bacteria’s unique heat resistant properties such as flagella and spores, allows it to be sneaky and get past sterilization processes, and allows it to protect itself by making a slime like environment. Life as a thermophilic bacterium living on dairy farm processing equipment can be quite easy and hot. To protect itself from the high temperatures, Bacillus thermoamylovorans has endospores, these endospores allows protection of the cell by resisting the extreme temperatures meant for sterilization of the microbe.

Genome Structure, Content, and/or Gene Expression

B. thermoamylovorans was sequenced using 16S rRNA because of its involvement in the hydrolytic phase of biomass degradation processes. It has been found in isolated samples of sewage sludge that were undergoing a process called biodrying. After this genome was sequenced, scientists were able to discover that the thermophilic, heat-resistant microbe could degrade organic materials from sewage sludge such as proteins and starches, in which the product can then be turned into possible biofuel (3). With a linear Genome total length of 3,708,331 bp and a % GC of 38.8 ± 0.02 mol, and its unique spores are the reason it can survive so well in extreme conditions that microbes without spores cannot (5). The gene length/ genome ratio is 81.1 % and the genome contains 4317 coding DNA sequences, 3564 of which have predicted functions. 9.53% of the cluster of orthologous groups of proteins (COG) signified amino acid transport and metabolism, while 8.14% of COGs characterize carbohydrate transport and metabolism of the species. B. thermoamylovorans contain 96 carbohydrate-active enzymes, 33.3% were glycoside hydrolysis, 27.1% were glycosyl transferases, 20.8% carbohydrate esterase’s, 4.2% carbohydrate binding, and 14.6% auxiliary activities. Examination of the strain showed 99 % identity demonstrates how recent the genome has been sequenced. B. thermoamylovorans unique genetic components tell us that that the microbe is capable of processing and metabolizing persistent yet readily degradable sewage sludge biodrying materials. Finally, after completing the biodrying process there was noticeable decrease of 66.3% to 50.18% moisture content of the biodrying material (3).

Interesting Feature

The species name thermoamylovorans means using starch at high temperatures, this meaning makes the bacterium easier to understand and remember, as it describes one of many cool characteristics of this microbe (5). As a facultative anaerobe, Bacillus thermoamylovorans undergo anerobic respiration if oxygen is present, or they also can ferment sugars and other nutrients in the absence of oxygen. One unique thing about Bacillus thermoamylovorans is that they optimally grow in more nutrient deprived environments (6). This bacterium also has the potential to degrade organic materials such as proteins and carbohydrates from sewage sludge, in which the product can then be used for biofuel (3). Although there is not much information on how this microbe cycles its nutrients in the environments, it has been discovered that B. thermoamylovorans have increased chances of germination on nonnutrient germinant. It is said that they can grow whether vitamins and nutrients are present or not because they only stimulate the growth as they are not essential to the process (5). Although there is not much research to the topic, this bacterium also has the potential to serve as a probiotic for health benefits, which seems super exciting if they can cumulate enough data to make a product from the bacteria (9).

References

1.Coorevits, A., Logan, N.A., Dinsdale, A.E., Halket, G., Scheldeman, P., Heyndrickx, M., Schumann, P., Van Landoschoot, A., and De Vos, P. “Bacillus thermolactis sp. nov., isolated from dairy farms, and emended description of Bacillus thermoamylovorans”. International Journal of Systemic and Evolutionary Microbiology. 2011. Volume 61. p. 1954-1961. https://doi.org/10.1099/ijs.0.024240-0

2.Combet Blanc, Y., Ollivier, B., Streicher, C., Patel, B.K.C., Dwivedi, P.P., Pot, B., Prensier G., and Garcia, J.L. “Bacillus thermoamylovorans sp. nov., a moderately thermophilic and amylolytic bacterium”. International Journal of Systematic Bacteriology. 1995. Volume 45. p. 9-16.

3.Lu, C., Sheng-Wei, Z., Yu-Jun, Shen., Guo-Di, Z., Hong-Tao, L., and Zhi-Ying, W. “ Complete genome sequence provides insight into the biodrying-related microbial function of Bacillus thermoamylovorans isolated from sewage sludge biodrying material”. Bioresource Technology. 2018. Volume 260. p. 141-149. https://doi.org/10.1016/j.biortech.2018.03.121

4.Cultivation-independent approaches by Jeremy Dodsworth. Hedlund, BP., et al. “Impact of single-cell genomics and metagenomics on the emerging view of extremophile “microbial dark matter”. Extremophiles. 2014. Volume 18. p. 865-875. https://jgi.doe.gov/elucidating-extremophilic-microbial-dark-matter/

5.Logan, N.A., and De Vos, P. “Bacillus”. Bergey’s Manual of Systematics of Archaea and Bacteria. 2015. p. 1-164.

6.Berendsen, E.M., Krawczyk, A.O., Klaus, K., Jong, A., Boekhorst, J., et al. “Bacillus thermoamylovorans spores with very-high-level heat resistance germinate poorly in rich medium despite the presence of ger clusters but efficiently upon exposure to calcium-dipicolinic acid”. Applied and Environmental Microbiology. 2015. Volume 81. p. 7791-7801. doi: https://dx.doi.org/10.1138%2FAEM.01993-15

7.Flint, S., Gonzaga, Z., Good, J., and Palmer, J. “Bacillus thermoamylovorans- a new threat to the dairy industry – a review”. International dairy Journal. 2017. Volume 65. p. 38-43. https://doi.org/10.1016/j.idairyj.2016.10.002

8.Muehlhoff E, Bennett A, McMahon D, editors. Milk and dairy products in human nutrition. Rome, Italy: FAO; 2013.

9.Miyamoto, H., Seta, M., Horiuchi, S., Iwasawa, I., Naito, T., et al. “Potential probiotic thermophiles isolated from mice after compost ingestion”. Journal of Applied Microbiology. 2012. Volume 114. p. 1147-1157. doi: 10.1111/jam.12131

10.Amy Quinton. Raw milk may do more harm than good [internet]. UC Davis; 2020. Available from: https://www.ucdavis.edu/news/raw-milk-may-do-more-harm-good/

11.Yohandini, H., J., H. “Isolation and Phylogenetic analysis of thermophile community within Tanjung Sakti hot spring, South Sumatera, Indonesia”. HAYATI Journal of Biosciences. 2015. Volume 22. p. 143-148. http://dx.doi.org/10.1016/j.hjb.2015.10.006

12.Fusco, V., Chieffi, D., Fanelli, F., Logrieco, A., Cho, G.S., et al. “Microbial quality and safety of milk and milk products in the 21st century”. Comprehensive Revies in Food Science and Food Safety. 2020. Volume 19. p. 2013-2049. doi:10.1111/1541-4337.12568

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Edited by <Teresa Watkins>, a @MicrobialTowson student of Dr. Anne M. Estes at Towson University. Template adapted from templates by Angela Kent, University of Illinois at Urbana-Champaign and James W. Brown, Microbiology, NC State University.