Dionn Carlo Silva
BIOL 6321 Applied Microbiology
Dr. Yemmireddy
Nov. 7, 2018
Exam – 2
Question #1
Vegetable fermentation is significant for the preservation, improved digestibility, increased nutritional value, and increased texture and flavor of a food. Vegetable fermentation can use lactic acid bacteria (LAB) which will help assist with complex microbial, biochemical, and physical reactions during the fermentation process. There are many factors that can affect vegetable fermentation, which include but are not limited to: technological factors, the types of ingredients used, the quality of the raw materials, and the type of native microbiota used (Matthews et al., 2005). Understanding these factors and applying them efficiently during the fermentation process can make the difference in a good vegetable fermentation. Vegetable fermentation can have many products, one of which is sauerkraut.
One product widely known from the process of vegetable fermentation is sauerkraut. Sauerkraut is a product produced because of lactic acid fermentation of shredded cabbage (Saxena, 2015). The concept behind sauerkraut is that the fermentation process is initiated by Leuconostoc mesenteroides and determines flavor development, Lactobacillus plantarum will cause propogation, and Lactobacillus brevis will be present at the completion of fermentatino (Sanexa, 2015). Many of the products from vegetable fermentation will undergo the same steps in a typical vegetable fermentation, and this process applies to sauerkraut as well. First, the vegetable undergoes blanching, peeling, or cooking. This blanching and cooking step is essential to inactivate enzymes in the vegetables and remove any gases. Next, the vegetable can be sliced, diced, shredded, or placed whole in a fermentation vessel where starter culture may also be added. Depending on the type of vegetable, starter culture is usually not added because then they will start competing with the natural microbiota of the vegetable. Next, the vegetables in the fermentation vessel are covered with brine, a mixture of salt and water, to inhibit the growth of Gram-negative bacteria (Waites et al., 2005). Following the addition of brine, the fermentation vessel is sealed to induce an anaerobic condition and prevent oxygen. The final fermentation time is dependent on a variety of factors: the type of vegetable used, the quality of ingredients used, the temperature, how much salt was added, sorbic acid, citric acid, and ascorbic acid (Matthews et al., 2005).

References for Question #1
Matthews, K.R., Montville, T.J. (2005). Food Microbiology: An Introduction. Washington D.C.: 2005. Print.

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Saxena, S. (2015). Applied Microbiology. Punjab, India: Springer., 2015. Print.

Waites, M.J., Morgan, N.L., Rockey, J.S., Higton, G. (2005). Industrial Microbiology: An Introduction. London: Blackwell Science Ltd., 2005. Print.

Question #2
Understanding the difference between biodegradation and bioremediation is important in understanding the various environmental applications of microorganisms. Biodegradation is a natural process in which material is biologically degraded. In contrast, bioremediation is an engineered process using the application of biological microbes to degrade material. Two environmental applications of these processes include fungal biodegradation and metal bioremediation.
Fungal biodegradation is the ability of a fungi to convert lignocellulosic material to essential metabolites for growth; these fungi secrete enzymes, including cellulases, hemicellulases, and ?-glycosidases (Soliman et al., 2013). This is significant, as large amounts of lignocellulosic “waste” are generated through agricultural practices which can generate an environmental problem (Soliman et al., 2013). Biological degradation of this waste using several fungal species would be significant in helping prevent and limit this environmental problem. In addition to biodegradation, metal bioremediation is also another environmental application of these processes.
Metal bioremediation is another environmental application and can occur by adsorption and by physio-bio-chemical mechanisms. Heavy metals can be biosorbed, a type of bioremediation mechanism, by fungal species at binding sites without using energy. Studies have showed that the metal binding behavior of extracellular polymeric substances (EPS) revealed the ability to complex heave metals through, various mechanisms, such as proton exchange and micro-precipitation of metals (Dixit et al, 2015; Comte et al., 2008; Fang et al., 2010). However, the limitations of heavy metal adsorption by various fungal species can be attributed to the lack of understanding of genetics and genome characteristics of these fungal species. For physio-bio-chemical mechanisms, several fungal species have been shown to absorb metals. Sacchromyces cerevisiae, for example, can act as a biosorbent for the removal of Zn (II) and Cd (II) through ion exchange mechanisms (Chen et al, 2007; Talos et al., 2009). Other fungal species like Aspergillus parasitica and Cephalosporium aphidicola can biodegraded Pb (II) contaminated soils using the biosorption process (Tunali et al., 2006; Akar et al., 2007).
References for Question #2
A, Shereen & A, Yahia & A, Abdou. (2013). Fungal Biodegradation of Agro-Industrial Waste. 10.5772/56464.

Akar, T.; Tunali, S.; Cabuk, A. Study on the characterization of lead (II) biosorption by fungus
Aspergillus parasiticus. Appl. Biochem. Biotech. 2007, 136, 389–406.

Chen, C.; Wang, J.L. Characteristics of Zn2+ biosorption by Saccharomyces cerevisiae. Biomed.

Dixit, R.; Wasiullah; Malaviya, D.; Pandiyan, K.; Singh, U.B.; Sahu, A.; Shukla, R.; Singh, B.P.; Rai, J.P.; Sharma, P.K.; Lade, H.; Paul, D.Bioremediation of Heavy Metals from Soil and Aquatic Environment: An Overview of Principles and Criteria of Fundamental Processes. Sustainability 2015, 7, 2189-2212.

Environ. Sci. 2007, 20, 478–482.

Fang, L.C.; Huang, Q.Y.; Wei, X.; Liang, W.; Rong, X.M.; Chen W.L.; Cai, P. Microcalorimetric and potentiometric titration studies on the adsorption of copper by extracellular polymeric substances (EPS), minerals and their composites. Bioresour. Technol. 2010, 101, 5774–5779.

of lead(II) from aqueous solutions by Cephalosporium aphidicola. Sep. Purif. Technol. 2006, 47, 105–112.

Talos, K.; Pager, C.; Tonk, S.; Majdik, C.; Kocsis, B.; Kilar F.; Pernyeszi, T. Cadmium biosorption on native Saccharomyces cerevisiae cells in aqueous suspension. Acta Univ. Sapientiae Agric.Environ. 2009, 1, 20–30.

Tunali, S.; Akar, T.; Oezcan, A.S.; Kiran, I.; Oezcan, A. Equilibrium and kinetics of biosorption.
Question #3
One microbe that has a significant agricultural application is Sinorhizobium meliloti. S. meliloti 1021 is a species of Rhizobium that infects plants such as rice and will promote growth by enhancing plant hormones and photosynthesis performance, giving the plant the ability to tolerate stress (Chi et al., 2010). Siniorhizobium species are also considered to be bacterial biofertilizers. These biofertilizers will cause increased root hairs, nodules, and nitrate reductase activity. Furthermore, these biofertilizers will undergo a mechanism called nitrogen cycling in order to provide nitrogen necessary for the microbial processes (Dhanasekar and Dhandapani, 2012). Siniorhizobium species will also produce plant hormones, such as indole acetic acid (IAA), gibberellins (GA), and cytokinins (CK) (Abd El-Fattah et al., 2013; Chi et al., 2010). This is important as plant hormones will significantly increase plant growth. Several studies have shown how biofertilizers will increase photosynthesis performance leading to plant tolerance to stress, increased resistance to pathogens, and thus increased crop improvement (Chi et al., 2010; Thamer et al., 2011; Sahoo et al., 2013).
Another microbe associated with agricultural applications are baculoviruses. Baculoviruses can be used as insecticides as they are highly specific to insect and closely related arthropods, they can continue to persist in the environment, and they can be easily mass produced (Beas-Catena et al., 2014). About 60 baculovirus-based pesticides have been used worldwide to control diverse insect pests (Beas-Catena et al., 2014). The mechanism for baculoviral infection is as follows: occlusion bodies ingested by an insect dissolve in the midgut, occlusion-derived viruses are released and infect the midgut epithelial cells, the virion will bud out of the cell and cause systematic infection throughout the insect in the early stages of infection but during the late stages of infection cells will die releasing more occlusion bodies (Rohrmann, 2013).
Lastly, the entomopathogenic fungus, Beauveria bassiana can potentially be used as a bio-control agent, specifically as a biopesticide. The importance of using Beauveria bassiana as a bio-control agent lies within the advantages. These advantages include: a high degree of specificity for pest control, lack of problems concerning pest resistance to the fungus, and high persistence in the environment even after infection will provide a long-term pest suppression (Pinnamaneni and Potineni, 2010). Unlike bacteria and viruses that pass through the gut wall, fungi have different mechanisms of infection. The mechanism for infection of this fungi is as follows: the fungal spore will adhere to the cuticle which will activate the spore and cause germination; an appressorium, a specialized cell used by fungi to infect a host, is formed and will penetrate the cuticle; the invasion of the epidermis and hypodermis occurs; lastly, tissue invasion and proliferation of cell bodies will occur in the host insect (Pinnamaneni and Potineni, 2010). Thus, the insect will die because of tissue invasion, depletion of nutrient resources, and toxicosis (Pinnamaneni and Potineni, 2010).

References for Question #3
Abd El-Fattah DA, Ewedab WE, Zayed MS, Hassaneina MK: Effect of carrier materials, sterilization method, and storage temperature on survival and biological activities of Azotobacter chroococcum inoculants. Ann Agric Sci 2013, 58:111–118.

Beas-Catena, A., Mirón, A., Camacho, F., Contreras-Gómez, A., & Molina-Grima, E. (2014). Baculovirus biopesticides: An overview. JAPS, Journal of Animal and Plant Sciences. 24. 362-373.

Chi F, Yang P, Han F, Jing Y, Shen S. Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021. Proteomics. 2010; 10:1861–1874. doi: 10.1002/pmic.200900694.

Dhanasekar R, Dhandapani R: Effect of biofertilizers on the growth of Helianthus annuus. Int J plant, Ani Environ Sci 2012, 2:143–147.

Pinnamaneni, R., Potineni, K. Mechanisms involved in the entomopathogenesis of Beauveria bassiana. Asian Journal of Environmental Science. 2010; 5:65-74.
Rohrmann GF. Baculovirus Molecular Biology Internet. 3rd edition. Bethesda (MD): National Center for Biotechnology Information (US); 2013. Chapter 3, The baculovirus replication cycle: Effects on cells and insects. 2013 Dec 12. Available from: https://www.ncbi.nlm.nih.gov/books/NBK138305/.
Sahoo RK, Bhardwaj D, Tuteja N: Biofertilizers: a sustainable eco-friendly agricultural approach to crop improvement. In Plant Acclimation to Environmental Stress. Edited by Tuteja N, Gill SS. LLC 233 Spring Street, New York, 10013, USA: Springer Science plus Business Media; 2013b:403–432.

Thamer S, Schädler M, Bonte D, Ballhorn DJ: Dual benefit from a belowground symbiosis: nitrogen fixing rhizobia promote growth and defense against a specialist herbivore in a cyanogenic plant. Plant Soil 2011, 34:1209–1219.

Question #4
A recent study (Hampton, 2018) shows that gut microbes can shape an individual’s response to cancer immunotherapy. Before this discovery, the effectiveness of certain cancer immunotherapy drugs was very individualistic and would be different for each patient. Routy et al. describes how patients receiving PD-1 inhibitors as cancer treatment and had taken antibiotics for differing conditions would have a lower overall survival compared with patients who had not taken antibiotics (Routy et al., 2017). In another study, Gopalakrishnan et al. describes that patients who had greater gut microbe diversity would respond better to their PD-1 inhibitor treatment for their melanoma (Gopalakrishnan et al, 2017). Both of these studies support the idea that a greater and more diverse gut microbe can make the difference in an individual’s response to cancer immunotherapy. The mechanisms by which these bacterial strains exert their immunomodulatory effects in the body is currently unknown and additional research is needed (Hampton, 2018). My perspective of this is this is very interesting and important research. If gut microbes can lead to more successful cancer treatments, then perhaps there are many other applications for gut microbes unknown to researchers currently. With this discovery that gut microbes can shape an individual’s response to cancer immunotherapy, it has laid a foundation for future research.

References for Question #4
Gopalakrishnan V , Spencer CN , Nezi L , et al . Gut microbiome modulates response to anti–PD-1 immunotherapy in melanoma patients. Science 2018;359:97 103.doi:10.1126/science.aan4236
Hampton T. Gut Microbes May Shape Response to Cancer Immunotherapy. JAMA. 2018;319(5):430–431. doi:10.1001/jama.2017.12857
Routy B , Le Chatelier E , Derosa L , et al . Gut microbiome influences efficacy of PD-1–based immunotherapy against epithelial tumors. Science 2018;359:91–7.doi:10.1126/science.aan3706
Extra Credit
One industrial product of microbial origin which uses submerged fermentation is antibiotics of the Penicillium sp. strain. Hamzah et al. (2009) describes how they isolated a strain of Penicillium sp. found in Sulaimani City and produced antibiotics using submerged fermentation. For the upstream processes, the organism was obtained, culture conditions were met, and an inoculum was prepared. Penicillium sp. strain was isolated from soil in Sulaimani City and then dried overnight in an oven at 50°C; serial dilutions were man from the sample and were spread on Potato Dextrose Agar (PDA) and Sabouraud’s Agar (SA) plates (Hamzah et al., 2009). The plates were incubated for 3-5 days at 30°C and then microscopical examination was done to detect the genus of fungi as well as register morphological features (Hamzah et al., 2009). Inoculum preparation was done according to Hamzah et al., 2009 and is considered as the starter culture propagation step. Next, submerged fermentation occurred. For submerged fermentation, a nutrient broth (100ml) with a pH of 5.5 in a flask was used; the flask was inoculated with 2ml from the inoculum preparation; the flask was then incubated at 30°C for 7 days (Hamzah et al., 2009). The broth culture would be centrifuged at 5000 rpm for 15 min (Hamzah et al., 2009). This step concludes the upstream processes. For the downstream processes, the product was recovered, purified, and processed. Protease activity and antibiotic production were detected in the supernatant. The protease activity was tested using skim milk agar and blood agar (Hamzah et al., 2009). To detect antibiotics, an antibiotic production assay was used (Chantawannaku et al., 2002). So far, the product has been recovered and purified. Finally, the Penicillium sp. will be processed. This study is significant as it depicts that the growth of the mycelia increased rapidly in the early stages as well as an increase in biomass and antibiotic production when submerged fermentation was used (Hamzah et al., 2009).

References for Extra Credit
Chantawannakul. P.; Oncharoen, A.; Klanbut, K.; Chukeatirote, E.; and Lumyong, S. (2002). Characterization of protease of Bacillus subtilis strain 38 isolated from traditionally fermented soybean in Northern Thailand. Science Asia, 28, 241-245.

Hamzah, H. M., Ali, A. H. L., & Hassan, H. G. (2009). Physiological regulation of protease and antibiotics in penicillium sp. using submerged and solid state fermentation techniques. Journal of Engineering Science and Technology, 4(1), 81-89.

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