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and outdated drugs.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 co-
metabolism 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 co-
metabolic 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 multi-
substrate, 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
𝑴𝑻𝑪 = 𝑪𝒎𝒊𝒏 × 𝒅 𝑷𝒐 (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).