Biotech Report

1.0

Research Purposes

The purpose of this study is to focus on biological methods as an eco-friendly alternative for the removal of mixed pharmaceuticals which are Diclofenac, Ibuprofen and Sulfamethoxazole using a bacteria consortium. Pharmaceutical waste can be generated through partially used or unused dosage forms, patient’s personal medications and outdated drugs. The expired drugs may accumulate due to in appropriate donations or insufficient stock management and distribution. This results in environmental pollution caused by the pharmaceutical compounds. Despite being detected in a low concentration, pharmaceutical compound possesses treat to the environment as they are designed to stimulate specific biological responses even at low concentration. In addition, the emergence of the pharmaceuticals might be due to the partial metabolism from human body which are excreted through urine or feces as part of detoxification as well as improper disposal of pharmaceutical waste. Hence, the study of degrading pharmaceutical compounds by using biological methods had gain the interest of researches. The aim of this study is to develop a bacterial consortium isolated from different origins, to evaluate the ability of such consortium to remove a mixture of pharmaceuticals in batch systems at lab scale and to assess the bacteria consortium’s resistance to the other micropollutants present in the environment. The biodegradation experiment will be conducted by the observing the biodegrading of the drug mixture by the mixed bacteria in a closed bottle test. The resistance of the bacteria towards pharmaceutical compound will be performed using commercial antibiotic discs.

2.0

Methodology Prior to the determining the ability of the mixed bacteria to degrade the drug

mixture (DCF, IBU and SMX), antagonism test was conducted to identified whether the bacterial strain (D15, D16, S2 and S4) can grow successfully with various antibiotics disc applied to the agar plate. Next, biodegradation experiments will be proceeded in a closed bottle test for the assessment of ready biodegradability of chemicals. The enriched mix culture is also spiked with glucose as an additional carbon source to test the efficiency of biodegradation of the drug mixtures under the presence of another energy source. In order to assess the abiotic losses or the adsorption of drugs onto the cells, controls were included in all assays. This assessment is important to become the base value of the biodegradation of drugs under controlled environment. The controlled condition of without inoculum and with autoclave biomass (5%) can signify the biodegradability of drugs on its own without the presence of targeted bacteria or any other germs. HPLC analysis was conducted to determine the quantitative data (concentration) for drugs mixture present in the medium after 24hours of incubation by the measurement of peak area. This HPLC step is a huge advancement for the biotechnology research world because in the past, the biodegradation process only used observation method on the growth of bacterial strain on the agar plate with the preferred substrates. Lastly, this study also focusing on the susceptibility of the bacterial strains to antibiotics and heavy metals to inspect the strains ability to tolerate various antibiotics and heavy metals. The particular study is generally important to determine which antibiotics that can be tolerated by the strains or toxic to the strains. The resistance towards various antibiotics and heavy metals will conclude that the bacterial consortium was able to survive in the highly contaminated ecosystem or wastewater treatment plant (WWTP). Therefore, the potential of bioremediation to treat pharmaceuticals pollutant in the conventional WWTP can be considered for the integration of biotechnology and engineering aspects.

3.0

Research Outcome The most striking evidence from this research was the antagonistic test had shown

positive outcome for the bacterial strain growth with various antibiotics which includes Colistin sulfate CT50, Sulphonamide compound S30, and Amoxycillin AML25 and shows the resistance towards heavy metals such as lead and mercury as no inhibition zones was observed around the isolates. This indicated that strains D15, D16, S2 and S4 can grow simultaneously within a complementary association, probably by developing a metabolic cooperation. This will allow better optimization of the pharmaceutical’s biodegradation in the mixed cultures. The research study on microbial degradation of mixed pharmaceuticals (DCF, IBU and SMX) had also shown a striking outcome in which the mixed micro-pollutant was removed by bacterial consortium in higher rate in comparison to removal rate by single strain bacteria. It is an evidence of degradation of pharmaceuticals can be achieved by co-metabolic system of bacterial consortium. The use of co-metabolism is to initiate a conversion reaction of the persistent compound to intermediates that are probably more biodegradable and therefore can join the central metabolic pathway for further biotransformation. The bioremediation study is majorly focused on a known single strain of bacteria to degrade the pollutants; however, the results shows a slower degradation rate due to the adaptation to xenobiotics substrate only by the single strain without any cometabolism application. Bioremediation process is naturally dependent on the cooperation of metabolic activities and synergistic effect of the mixed microbes. For example, some species can remove the toxic metabolites of the preceding species and others can degrade compounds which the first species are able to partially degrade, promoting a process known as cometabolic process. Several studies reported that the use of microbial consortium can increase the biodegradation rate of xenobiotics. Reis et al. revealed that the mixed bacteria exhibited a higher biodegradation rate compared to single microbial strain. According to the obtained results, the elimination rate of the pharmaceutical in the presence of glucose as an additional carbon and energy source was higher than that

shown in drug mixtures only in the medium. This is because the simultaneous utilization of supplement carbon source with a compound that is less energetic may sustain cell growth and act as an electron donor to facilitate the biodegradation process. However, the results shown in both metabolic and co-metabolic conditions, removal of SMX was not observed. This is due to the bacterial populations used in the biodegradation tests might have developed resistance to it. Albuquerque et al. reported that in the presence of multisubstrate, each population uses its preferred substrate while the other carbon sources are left available for other microbial groups.

4.0

Conclusion and Review

The articles had mentioned that a study conducted by Backhaus T. (2014), the ecotoxicity of pharmaceuticals mixture is usually above the effect of a single compound. It does not provoke the significant toxic effect when it is detected at low concentrations and acting individually on the exposed organisms. However, this study does not account the study of toxicity effect of the target pharmaceuticals (DCF, IBU and SMX). In our opinion, toxicity test can be a major factor for pharmaceuticals studies to inform and scientifically explained to the society regarding the effects of these pharmaceutical pollutant’s emergence in the environment and potentially extended study can be conducted by researchers. The assess the acute toxicity of DCF, IBU and SMX, the method of Microbial Assay for Risk Assessment (MARA) can be performed with various microorganism. Generally, the assay will be applied by using at least 10 bacterial strain and 1 strain of yeast as the reference or experimental subjects conducted in 96-well in 3 independent trials. Image analysis will be subjected to the plates after 18 hours of incubation in 30C. Reduction of the tetrazolium salt method which precipitate at the bottom at the bottom of the wells can be applied for the determination of microbial growth after the exposure to the concentration gradient of DCF, IBU and SMX. The outcome can be expressed as Microbial Toxic Concentration (MTC) according to equation 1 for each microorganism and the whole experiment.

𝑷 ( 𝒕𝒐𝒕 )−𝟏 𝑷𝒐

𝑴𝑻𝑪 = 𝑪𝒎𝒊𝒏 × 𝒅

(𝟏)

For the biodegradation test, the study does not analyze the intermediate or metabolites produced as the results of microbial degradation. In our opinion, it is essential for degradation study of any pollutants to identify the intermediates formed from the parent compound. This is due to the possibility of the smaller intermediates to possess more harmful effect to the organisms. Besides, the presence of the metabolites can be guidance for research studies to hypothesize the metabolic pathway of the drugs degradation, therefore, identified the enzyme that is responsible for the degradation and potentially commercialized for implementation in our conventional wastewater treatment plants (WWTPs).