Including genomic dna extraction pcr system rt-pcr

	COMSATS University Islamabad Synopsis for MS ✓Ph.D.☐

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time polymerase chain reaction (RT-PCR), and polymerase chain reaction (PCR) processes include traditional plating techniques, bioluminescence methods, immunoassays, flow cytometry, and a number of DNA-based approaches. The aim of current study is to investigate the potential Antimicrobial resistant bacteria and determinants, that arise during the process bioethanol production at pretreatment stage. The physico-chemical Total dissolved solids (TDS) and total suspended solids (TSS), pH, biochemical oxygen demand (BOD), and chemical oxygen demand (COD) measurements will be utilized to identify the chemical parameters in the analysis of the biomass. Antimicrobial resistance (AMR) will be assessed using a variety of methodological techniques, such as genomic DNA extraction, polymerase chain reaction (PCR), and real-time PCR system (RT-PCR). The phenol-chloroform extraction procedure will be followed in order to obtain genomic DNA.

energy. As a result, the country’s future energy requirements have become a policy priority in recent years (Yatim et al., 2016). Scientists have looked into options for switching from gasoline to biofuels (Moreira and Goldemberg, 1999). Brazil has an extensive and intriguing history of ethanol manufacturing. In order to reduce oil imports, the Brazilian Alcohol Program (PROALCOOL) was established in 1975. Its goal is to produce ethanol from sugarcane (Muthaiyan et al., 2011). The Brazilian Federal Government mandated that gasoline must contain anhydrous ethanol in the late 1970s. Additionally, they pushed automakers to create engines that could run entirely on hydrated ethanol. The program of ethanol manufacture, that was prompted by brazil. It was first designed to curtail the rate of oilimports, but in few time it became evident that it also had major economic and environmental benefits, making it the world's largest biomass energy program. Later on other countries like America, and European UNION went a step further by locating national targets for the use of bioethanol instead of gasoline (Hira and Oliveira, 2009). As a result, intensive research efforts are currently concentrated on ethanol manufacturing and its co-products. Typically, enzymatic hydrolysis of carbohydrates is the first stage in the production of ethanol, followed by fermentation processes that convert the resultant sugars into ethanol (Muthaiyan et al., 2011). Yet, commercial ethanol production is a non-sterile process, contaminants can reduce industrial output and result in large financial losses. The bioethanol industry currently controls the pollutants in the fermenters

using a variety of antimicrobials, including antibiotics (Brexó and Sant’Ana, 2017). Alternative antimicrobials are required to keep bioethanol production at a cost effective level due to the growth of antibiotic resistance among contaminating bacteria in bioethanol fermenters (Muthaiyan et al., 2011). Additionally, an increasing number of ethanol manufacturer are working to develop distiller grains that can be marked as free of antibiotics in order to sell them

To provide a new research direction for the quest for novel, unusual antimicrobial
mediators to address the impurity problem in the lubricant bioethanol sectors, specific focus has been placed on natural antibacterial compounds generated from plant sources (Chaturvedi et al., 2021).

1.2. Contamination Problems in Ethanol Production

Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

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Acid Treatment
One of the few methods developed to lessen bacterial contaminations throughout the ethanol production process is acid washing. This practice involves removing yeast cells from the fermentation broth, adjusting the pH of the cell paste to 2.0 with sulfuric acid, and then digesting the cells for two hours at the same pH before returning them to the fermenter (Simpson and Hammond, 1989). The practice of washing pitching yeast with bactericidal

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1.6. Antibiotic Resistance

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1.6.2. Antibiotic Resistance Mechanisms
A prior investigation into lactobacilli strains, originating from milk products, animal plants and human sources, revealed a notable occurrence of spontaneous mutations leading to antibiotic resistance. It highlighted that high impulsive alteration rates are common among lactobacilli strains. Furthermore, antibiotic resistance plasmids associated with tetracycline, erythromycin, and chloramphenicol resistance were frequently identified in lactobacilli strains, alongside spontaneous mutations (Salminen et al., 1998). Bacteria should be prepared to obtain antibiotic resistance inheritable factor from other microbes, if they are to live in an environment where antibiotics are present. The antibiotic resistance plasmids discovered in lactobacilli are noteworthy due to their capacity for conjugative transfer to various types species, and even genera, potentially extending to human or animal pathogens (Salminen et al., 1998). A number of drug efflux proteins, including an ABC transporter and a proton motive force-dependent drug transporter, have also been identified in Lactobacillus lactis. Although the natural substrates are unknown, both are in charge of the resistance to ethidium bromide at high concentrations (Van Veen and Konings,1998). Furthermore, a novel vancomycin resistance mechanism was identified in L. rhamnosus. Vancomycin is a glycopeptide antibiotic that hinders peptidoglycan formation, which is the primary structural component of bacterial cell walls. As a result, vancomycin therapy can be particularly harmful to Gram-positive bacteria. Probiotic L. rhamnosus GG was the subject of significant research by Tynkkynen et al., and

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2. Literature Review
Microbial contamination poses a significant challenge to the ethanol industry due to the risk of reduced output and the production of unwanted organic acids. In one of a study by Kotrba et al. (2012), they examined various input costs for propagation, such as labor, dry yeast, energy, antibiotics, nutrients and sugars. Their model research suggests that propagation costs for a 100 MMgy (million gallons per year) ethanol plant can vary between $404,000 and $540,000 annually (Lingle et al., 2012).

Oil liquor factories, some of which run with lean profit precincts, would suffer financially fro m yield losses of even 1% of present output levels (Abbott et al., 2004). As a result, industry is very interested in minimizing yield losses from ethanol. There is a roughly 10% reduction in ethanol production for every milliliter of mash containing 10 million lactobacilli cells (Manitchotpisit et al., 2013). To assess how pollution may affect ethanol production consider a distillery that processes 1.8 million liters (475,000 gallons) of beer mash yearly, as described by Deuringer and Fleischer. Less contamination will likely result in an increase in ethanol output of 20 million liters, bringing the ethanol content in the beer mash from 10% to 11%.

contributes to these losses (Abbott and Ingledew, 2005). These undesirable bacteria produce inhibitory byproducts like acetic and lactic acids, competing with Saccharomyces cerevisiae for essential nutrients (Bayrock and Ingledew, 2001). In specific environments, such acids have been demonstrated to lead to prolonged lag periods, reduced growth rates, lower biomass yields, and even the death of Saccharomyces cerevisiae (Graves et al., 2006). The presence of acetic or lactic acid in the medium during fermentation has both been observed to result in decreased ethanol production rates and yields (Dien et al., 2000).

2.1. Potential Contamination Sources
Contamination remains a persistent challenge in commercial fermentation cultures, particularly in non-aseptic settings, such as fuel ethanol fermentations where the fermentation mashes are not sterile. Presterilizing the fermentation medium is unfeasible because high sterilization temperatures would adversely affect the enzymes required for grain starch saccharification (Chin and Ingledew, 1994). Potential sources of contamination, both direct and indirect, have been identified in large-scale yeast ethanol production. Direct sources include components introduced into the fermenter, such as pretreatment corn, inocula, enzyme additives, corn steep liquor, and process-related aerosols. Indirect sources encompass transfer lines and the water used for pump and agitator seals (Schell et al., 2007). Generally, containers and transmission lines are sanitized using condensation or a 3% concentration hot caustic soda solution before use. Between batch operations, the equipment in smaller fermentation tanks is sanitized with a solution of chlorine or iodine. However, sterilization may not be sufficient since, as was previously indicated, it cannot control the germs emerging from direct sources of contamination (Schell et al., 2007). In conjunction with fermenters based on starch and sugar, fermenters based on lignocellulose feedstock are frequently linked to bacterial contamination. Schell et al. investigated the presence, recognition, and management of bacterial contamination in a pilot-scale investigation of the conversion process of maize fiber to ethanol. They showed that contaminated Lactobacillus bacterial strains could quickly absorb arabinose, a sugar that the yeast cannot use, and produce acetic and lactic acids that were bad for fermentation efficiency. This indicates that maintaining a yeast monoculture can be extremely challenging in the presence of any microorganisms capable of consuming biomass-derived sugars, even if these sugars are not utilized by the primary fermenting microorganism (Schell et al., 2007). Most commercial alcohol fermentations are conducted without pre-sterilization due to the high cost of substrate cooling. This approach fosters the growth of bacteria, wild S. cerevisiae strains, and non-S. cerevisiae contaminants, leading to reduced productivity and operational

2.2. Wild Yeast Contamination
Apart from S. cerevisiae, unintentionally introduced wild yeasts can disrupt the fermentation process. These wild yeasts, due to their well-known biology, have been a persistent challenge for brewers and winemakers, particularly in beverage fermentations throughout history. Consequently, they have been the subject of numerous investigations. "Wild yeasts" are yeast strains found in snifter, cocktail or other brewery materials that, through their activities, not only fail to enhance ethanol production but often significantly disrupt the production process, resulting in an unsatisfactory final product in terms of taste and quality (De Barros Pita et al., 2012). Historically, distinguishing them from cultured yeasts has been difficult grounded on structural and physiological characteristics, but recent molecular advancements have enabled the identification and differentiation of the majority of potential contaminated wild yeast strains (Guillamón et al., 1998).

In contrast to bacterial impurity during fermentation, for which antibiotics and acid treatment can be used, currently there is no antibiotic therapy accessible to prevent the growth of wild yeast. Therefore, the complete replacement of the fermenter's yeast population is the treatment for yeast contamination. Early detection of contaminated yeasts, followed by the addition of a new batch of the desired yeast, is the only way to mitigate the potential economic losses caused by yeast contamination. Consequently, vigilant observation for wild yeast contamination is a critical aspect of managing the industrial fuel ethanol production process (Guillamón et al., 1998). Nevertheless, these organisms can also have occupied from batch fermentation plants. They are more likely to pose problems in ethanol facilities employing continuous processes. According to Baslio et al., 24 yeast species were isolated and identified from bioethanol distilleries in Northeast Brazil that utilized sugarcane juice or cane molasses for ethanol fermentation. Their findings revealed that Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, and various C. intermedia strains were linked with severe pollutions in fermenters (Basílio et al., 2008).

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significant group responsible for adverse effects on fermentation processes. They transform carbohydrates into lactic acid, which not only reduces ethanol production but also hampers yeast fermentation as the acid levels increase. This detrimental impact has been observed in simulated malt whiskey fermentation involving Lactobacillus brevis and Lactobacillus plantarum, resulting in decreased pH, diminished ethanol output, and hindered yeast growth (Makanjuola et al., 1992).

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Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

4. Objectives:

1.To determine the potential antimicrobial resistant bacteria (AMR) during various phases of bioethanol production.

Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

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6.3. Evaluation of Antimicrobial Resistance (AMR) and Antimicrobial Resistance Bacteria (ARB)

Different methodological approaches, including genomic DNA extraction,real-time PCR system (RT-PCR), and polymerase chain reaction (PCR), will be used to evaluate antimicrobial resistance (AMR). While nutrient agar media and LB broth will be used to evaluate antimicrobial resistance bacteria (ARB).

Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

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This experiment will be conducted at COMSATS University Islamabad, Abbottabad campus, Pakistan.

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Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

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Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

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Student’s Name: Tehniat Afa
Enrollment Number: CIIT/FA22-RES-023

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