The Latent Talents of The Industrial Work-Horse of the Microbial World: Bacillus
Authors: Vipin Chandra Kaliaa,b*, Subhasree Raya,b, Shikha Koula,b, Jyotsana Prakasha,b, Ravi Kumara
aMicrobial Biotechnology and Genomics, CSIR - Institute of Genomics and Integrative Biology (IGIB), Delhi University Campus, Mall Road, Delhi-110007.
bAcademy of Scientific & Innovative Research (AcSIR), 2, Rafi Marg, Anusandhan Bhawan, New Delhi- 110001

The Industrial Work-Horse

Bacillus is a truly omnivorous organism, being present in the air, soil and water. Most Bacillus spp. especially B. subtilis, B. licheniformis and B. amyloliquefaciens are categorized as GRAS (Generally Regarded As Safe) organisms. These organisms have enormous abilities to grow to high cell densities and produce virtually all kinds of enzymes at commercially economic scales. The different species can easily metabolize a large number of pollutants which are present in the environment. The protease enzymes produced by Bacillus spp. are an important component of the washing detergents, which find use even in leather industry. Starch metabolizing enzyme – amylase, is extensively used in Paper and Textile industries. Other applications of Bacillus include: (i) Bioremediation of fat-rich waste waters through lipases, (ii) Biocontrol agent against fungi, (iii) Food processing industries – natto, a soyabean based product is very popular in Japan, (iv) bioactive molecules – carotenoid pigments, vitamins, flavouring agents (v) Plant growth promoters, etc. In contrast to the most favourite, Escherichia coli – as host for genetic engineering among Gram-negative organisms, Bacillus has been gaining importance as host for recombinant technological research among Gram-positive organisms. Recent research works have elucidated the novel talents of this Industrial Work-Horse of the Microbial World: (i) Bio fuel generator, (ii) Biodegradable plastics producer, and (iii) Antipathogens.

Biofuels

Hydrogen (H2) has been recognized as a non-polluting fuel for the future. The potential of Bacillus as biological hydrogen producer started getting attention in the 1980-1990s. The metabolic pathway elucidated among Clostridia, proceeds through the combination of enzymes: (i) pyruvate-Fd oxidoreductase (POR), and (ii) H2-POR. On the other hand, among fermentative bacteria such as Escherichia coli, the following set of enzymes is necessary: (i) pyruvate-formate lyase, (ii) formate dehydrogenase and (iii) hydrogenase. The exact metabolic pathway responsible for H2 production by Bacillus is still to be elucidated. Preliminary research works indicate the involvement of nitrogenase and hydrogenase enzymes – operative in nitrogen fixation. Theoretically, 12 moles of H2 can be generated from 1 mole of glucose (Hexose sugars). However, in practice up to 4.0 mol/mol hexose sugar can be achieved. Bacillus spp. have been reported to generate up to 2.6 mol H2/ mol glucose (pure substrate). Bacillus coagulans can metabolize cellobiose to yield 5.6 mol H2/mol feed.Bacillus sp. can utilize agricultural and food waste to yield up to 1.5 molH2 /mol (hexose). Bacillus along withBrevundimonas or with Clostridium strains as co-cultures were 2-4 times more effective than their individual H2 yielding capacities. Bacillus strains can ferment apple waste, pea-shells, damaged wheat grains, crude glycerol to H2 to the tune of up to 150 L/kg total solids.

Biodegradable plastic

Bacillus species have been the most studied polyhyxoyalkanoate (PHA) producers among Gram-positive group of organisms. A wide range of Bacillus spp. such as B. licheniformis, B. subtilis, B. thurigiensis, B. megaterium, etc., metabolize sugars, volatile fatty acids, alcohols and biowastes (pea-shells, date syrup, molasses, potato and jiggery wastes) to PHA, which may constitute 64-83% of the dry cell mass. Compared to homo-polymers, the commercial value of co-polymers of PHA is much higher because of their high stability and higher molecular weight. Bacillus spp. can switch their metabolic activity depending up on the substrates used as feed. On nutrient rich medium, homopolymers of PHB are produced. Addition of propionate or valerate enable Bacillus strains such as B. amyloliquefaciens strain DSM7 and B. circulans DSM1529 to synthesize PHA co-polymers - P(3HB-3HV) having 45-50% as 3-hydorxyvalerate. B. cereus UW85 could metabolize the combined feed to tercopolymer composed of 3HB, 3HV along with 6HHx. Recent works have used mixed wastes, where onion peel hydrolysate proved instrumental in enhancing the co-polymer yield and composition of pea-shell slurry. The best part of using Bacillus spp., is their ability to produce PHA even at high N content i.e. the process is independent of N limitation, which is a pre-requisite for all Gram-negative bacteria tested so far. Another feature, which make Bacillus as a strong contender for commercial production of PHA is that most of them lack the PHA-depolymerase enzyme. Thus, the risk of its getting metabolized immediately on completion of the PHA biosynthetic process is minimized. And finally, the arrangement of genes of the PHA synthetic pathway, i.e. phaABC of the PHA operon, which is necessary for high Mw PHB, is inherently present in B. megaterium, B. thuringiensis, and B. cereus strains.

Antipathogens

Antibiotics have been the life-line for human beings for almost 7-8 decades. The situation is changing at an alarming rate, as bacteria are undergoing genetic changes in response to stress caused by usage of heavy doses of antibiotics. Now, it has reached a stage where commercial production of antibiotics is proving to be uneconomical. The latest approach is to prevent bacteria from expressing their pathogenicity / virulence factors rather than killing them out rightly. About 65% of the infectious diseases are caused by biofilm forming bacteria. Biofilm formation takes place through the biosynthesis and release of signal molecules by bacteria. Once the bacteria sense that the signal molecules are present in high concentration, they equate this to the presence of their population densities to be high. At high cell densities, they express their pathogenicity / virulence factors, which prove lethal to the host cell. This high cell density dependent phenomenon has been termed as quorum sensing (QS). The QS mediated biofilm provides additional protection to bacteria against antibiotics. Bacterial cells can be made susceptible to lower doses of antibiotics by disrupting or preventing the formation of the biofilm.

Bacillus spp. such as B. anthracis, B. thruingiensis, B. cereus, B. mycoides, B. subtilis, B. sonorensis, and B. marcoestinctum have been found to possess gene (aiiA) for producing enzyme such as acyl-homoserine lactone (AHL) – lactonase. This enzyme opens up the ring structure of the AHL signal molecules and inactivates them. Inactivated AHL signal molecules do not allow QS mechanism to proceed. Another unique feature of usingBacillus as source of AHL-lactonases is the fact that aiiA gene from Bacillus species have been expressed in diverse hosts: Pseudomonas, Burkholderia, Escherichia, Erwinia, Serratia and a few eukaryotes. Genetically engineered tobacco and potato plants become resistant to plant pathogen – Erwinia cartovora. Fish mortality caused by Aeromonas hydrophila was significantly reduced Bacillus lactonases. Co-inoculation of Bacillus with Vibrio harveyi and A. hydrophila protected fresh water prawn from getting attacked by these pathogens. Thus, combination of the QS inhibitors (as antipathogens) and low doses of antibiotics can prevent the bacteria from expressing their pathogenic behavior. AHLs and AHL-lactonases thus have become the novel drug targets and therapeutic agents, respectively, to prevent infectious diseases.

Conclusion

The latent talents of Bacillus like bio-H2, PHA and AHL-lactonase production can be exploited in future for novel biotechnological applications.

References:

1. Agarwala M, Choudhury B, Yadav RNS (2014) Comparative study of antibiofilm activity of copper oxide and iron oxide nanoparticles against multidrug resistant biofilm forming uropathogens. Indian J Microbiol 54:365â€"368. doi: 10.1007/s12088-014-0462-z

2. Alipiah NM, Shamsudin MN, Yusoff FM, Arshad A (2015) Membrane biosynthesis gene disruption in methicillin-resistant Staphylococcus aureus (MRSA) as potential mechanism for reducing antibiotic resistance. Indian J Microbiol 54:41-49. doi: 10.1007/s12088-014-0488-2

3. Arasu MV, Al-Dhabi NA, Rejiniemon TS, Lee KD, Huxley VAJ, Kim DH, Duraipandiyan V, Karuppiah P, Choi KC (2015) Identification and characterization of Lactobacillus brevis P68 with antifungal, antioxidant and probiotic functional properties. Indian J Microbiol 55:19-28. doi: 10.1007/s12088-014-0495-3

4. Gui Z, Wang H, Ding T, Zhu W, Zhuang X, Chu W (2014) Azithromycin reduces the production of α-hemolysin and biofilm formation in Staphylococcus aureus. Indian J Microbiol 54:114-117. doi: 10.1007/s12088-013-0438-4

5. Hema M, Balasubramanian S, Princy SA (2015) Meddling Vibrio cholerae murmurs: A neoteric advancement in cholera research. Indian J Microbiol 55:121â€"130. doi:10.1007/s12088-015-0520-1

6. Huma N, Shankar P, Kushwah J, Bhushan A, Joshi J, Mukherjee T, Raju SC, Purohit HJ, Kalia VC (2011) Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus. J Microbiol Biotechnol 21:1001-1011. doi:10.4014/jmb.1105.05056

7. Kalia VC (2013) Quorum sensing inhibitors: an overview. Biotechnol Adv 31:224â€"245. doi:10.1016/j.biotechadv.2012.10.004

8. Kalia VC (2014) In search of versatile organisms for quorum‐sensing inhibitors: acyl homoserine lactones (AHL)‐acylase and AHL‐lactonase. FEMS Microbiology Letters 359:143. doi: 10.1111/1574-6968.12585

9. Kalia VC (2014) Microbes, antimicrobials and resistance: The battle goes on. Indian J Microbiol 54:1-2. doi: 10.1007/s12088-013-0443-7

10. Kalia VC (2015) Microbes: The most friendly beings? In: Quorum Sensing vs Quorum Quenching: A Battle with No End in Sight, 1-5. Editor: VC Kalia. Springer India. doi:10.1007/978-81-322-1982-8_1

11. Kalia VC, Prakash J, Koul S (2016) Biorefinery for glycerol rich biodiesel industry waste. Indian J Microbiol. 56:113-125. doi: 10.1007/s12088-016-0583-7

12. Kalia VC, Purohit HJ (2011) Quenching the quorum sensing system: potential antibacterial drug targets. Crit Rev Microbiol 37:121-140. doi: 10.3109/1040841X.2010.532479

13. Kalia VC, Raju SC, Purohit HJ (2011) Genomic analysis reveals versatile organisms for quorum quenching enzymes: Acyl-homoserine lactone-acylase and â€"lactonase. Open Microbiol J 5:1-13. doi:10.2174/1874285801105010001

14. Kaur G, Rajesh S, Princy SA (2015) Plausible drug targets in the Streptococcus mutans quorum sensing pathways to combat dental biofilms and associated risks. Indian J Microbiol 55:349-357. doi:10.1007/s12088-015-0534-8

15. Kumar P, Patel SKS, Lee JK, Kalia VC (2013) Extending the limits of Bacillus for novel biotechnological applications. Biotechnol Adv 2013;31:1543â€"1561. doi:10.1016/j.biotechadv.2013.08.007

16. Kumar P, Ray S, Patel SKS, Lee JK, Kalia VC (2015) Bioconversion of crude glycerol to polyhydroxyalkanoate by Bacillus thuringiensis under non-limiting nitrogen conditions. Int J Biol Macromols 78:9-16. doi: 10.1016/j.ijbiomac.2015.03.046

17. Kumar P, Sharma R, Ray S, Mehariya S, Patel SKS, Lee JK, Kalia VC (2015) Dark fermentative bioconversion of glycerol to hydrogen by Bacillus thuringiensis. Bioresour Technol 182:383-388. doi: 10.1016/j.biortech.2015.01.138.

18. Kumar P, Singh M, Mehariya S, Patel SKS, Lee JK, Kalia VC (2014) Ecobiotechnological approach for exploiting the abilities of Bacillus to produce co-polymer of polyhydroxyalkanoate. Indian J Microbiol 54:151â€"157. doi: 10.1007/s12088-014-0457-9

19. Kumar P, Mehariya S, Ray S, Mishra A, Kalia VC (2015) Biodiesel industry waste: A potential source of bioenergy and biopolymers. Indian J Microbiol. 55:1-7. doi: 10.1007/s12088-014-0509-1

20. Moroeanu VI, Vamanu E, Paun G, Neagu E, Ungureanu OR, Eremia SAV, Radu GL, Ionescu R, Pelinescu DR (2015) Probiotic strains influence on infant microbiota in the in vitro colonic fermentation model GIS1. Indian J Microbiol 55:423-429. doi: 10.1007/s12088-015-0542-8

21. Patel SKS, Kalia VC (2015) Integrative biological hydrogen production: an overview. Indian J Microbiol 53:3-10. doi: 10.1007/s12088-012-0287-6

22. Prakasham RS, Kumar BS, Kumar YS, Kumar KP (2014) Production and characterization of protein encapsulated silver nanoparticles by marine isolate Streptomyces parvulus SSNP11. Indian J Microbiol 54:329-336. doi: 10.1007/s12088-014-0452-1

23. Saxena A, Mukherjee M, Kumari R, Singh P, Lal R (2014) Synthetic biology in action: Developing a drug against MDR-TB. Indian J Microbiol 54:369-375. doi:10.1007/s12088-014-0498-0

24. Shang Z, Wang H, Zhou S, Chu W (2014) Characterization of N-acyl-homoserine lactones (AHLs)-deficient clinical isolates of Pseudomonas aeruginosa. Indian J Microbiol 54:158-162. doi: 10.1007/s12088-014-0449-9

25. Wang R, Fang S, Xiang S, Ling S, Yuan J, Wang S (2014) Generation and characterization of a scFv antibody against T3SS needle of Vibrio parahaemolyticus. Indian J Microbiol 54:143-150. doi: 10.1007/s12088-013-0428-6


About Author / Additional Info:
Researcher in Microbial Biotechnology and Genomics at CSIR-IGIB, Delhi