HPLC results for microbial activity analysis
Sodium lactate and sodium acetate can be used as favorable carbon
source during microbial incubations. On the other hand, the change of
their concentration can be monitored to analyze microcosm activity in the
liquid medium.
The first part of the experiment to study on pharmaceutical degradation
kinetics lasted for 17 days . From Fig 1 and 2, concentration levels of
sodium lactate and sodium acetate in the abiotic setup remained
constantly at the initial value, approximately 1.7 mM. This demonstrated
that no microbial activity, which can consume the two salts as carbon
sources were undergoing in the abiotic control (containing no sediment)
during the entire experimental process.
Figure 1. Concentration of Sodium Acetate over time.
Figure 2. Concentration of Sodium Lactate over time.
In two microbial active controls, the depletions of the two salts were
undergoing expeditiously with similar rates. By calculation, the time of
50% disappearance (DT50) and 90% disappearance (DT90) of sodium
acetate were 1.1 days and 2.0 days. The DT50 and DT90 of sodium lactate
were 1.1 days and 1.9 days. 4 days later, the carbon source concentration
in the two active controls was out of analytical detection limit. The rapid
salts consumption indicated that the microcosms from the sediment had a
high activity.
Carbon source decrease also happened in sorptive controls, however, in
lower rates compared to the microbial active control samples. In sorptive
control 1, the DT50 and DT90 of sodium acetate were 4.9 days and 8.9
days; the DT50 and DT90 of sodium lactate were 3.3 days and 5.8 days. It
took 8 - 10 days to use up all the additional carbon sources, whereas in
sorptive control 2, the DT50 and DT90 of sodium acetate were 5.2 days
and 14.2 days; the DT50 and DT90 of sodium lactate were 4.3 days and
16.0 days. The probable reason caused the carbon sources depletion
would be the sediment existence in the sorptive control bottles. Autoclave
procedures could not sterilize the sediment completely, residual
microorganisms consumed the lactate and acetate over the experiment.
Monovalent organic acids, including lactate acid and acetate acid, are
weakly adsorbed to soil or sediment. Hence, sorptive depletion from the
two salts can be neglected. In spite of the sorption behavior, another
possible reason caused the decreases of lactate and acetate may be the
complex formation between the two anions and mineral cations in
sediments, such as iron (Fe). However, previous expermental results
exhibited the iron amount from Steinlach sediment was below 1µmol/g
soil. Therefore, the complex formation did not play a significant role for
the salts decrease as well. On the other hand, during the successive
sampling procedure, alien microbial pollution could also be led into the
setup bottles. Thus, residual microactivities in the Schott bottles may be
the major reason caused the carbon source depletion.
IC results
1 Chloride
In the experimental duration, chloride concentration was monitored as a
conservative tracer. Over the incubation period of 17 days, in all the
microbial and sterilized setups, chloride concentration stayed consistent
as expected (Fig 3).
Figure 1. Concentration of Chloride over time.
The slight increase happened at the third data point appeared in all the
setup results. It was probably due to the inaccuracy caused by the dilution
in the individual measurement, or instrumental differences after
maintenance.
2 Nitrate
From Fig 4, no conspicuous addition or depletion on nitrate concentration
was observed in abiotic control curve. This result corresponds to its
sodium lactate and acetate results very well. It confirmed that no potential
microbial activities were involved in the abiotic control setup. For the two
sorptive controls, sorptive control 2 kept a constant nitrate decrease rate,
whereas sorptive control 1 consumed over 75% nitrate within first two
days. However, they both achieved complete nitrate depletion on the 6th
day. A possible hypothesis toward this phenomenon is the existence of
microbial groups in setup bottles. They could either be the residual
microorganisms from Steinlach sediment, or accidentally introduced
during the sampling processes in experiment. While with the lack of
oxygen, denitrification predominated and thus the nitrate concentration
went down. No evidence showed the increase of nitrate concentration
level in sorptive control setups after the 6th day. It cannot be concluded as
the microbial residues were cleaned up in sterilizing procedure.
Figure 2. Concentration of Nitrate over time.
Nitrate in microbial setups and active control setups were used up in the
1st day. A possible explanation is the inadequate contact among sediment,
medium and oxygen at the experimental beginning. In this depletion,
nitrate was reduced into nitrite and further possibly nitrogen with the
effect of denitrifying bacteria in sediment. Denitrification occurred when
the oxygen level was lower than needed, or even more, under anoxic
condition. Afterwards, five setups without autoclaving experienced nitrate
accumulations. It was slow in first 7 days. As long as the microbial
communities obtained plenty oxygen, the nitrate amount grew more rapid,
especially in active controls. In the nitrate boost, with the appearance of
oxygen, nitrifying bacteria went more active. Ammonium in the
sedimental biomass could be offered as nitrogen source. Nitrate was
produced eventually via the nitrification process. Another nitrate
concentration depletion happened in both active controls on 8 th day
measurement. This may also caused by the lack of oxygen in the Schott
bottles. Generally, nitrate concentration level in active control setups was
higher than the microbial samples except for microbial setup 3. This may
due to the adverse effects of pharmaceutical compounds towards the
microcosms.
4.2.3 Nitrite
The initial nitrite concentration for the mimic Steinlach river medium was
zero. From Fig 5, all respective curves showed no concentration increase
happened excluding the two sorptive controls, which implied the aerobic
circumstance was constantly maintained in microbial setups over the
experiment duration. Slight nitrite increases were observed in both
sorptive controls. However, it was gone in the next measurement. As the
product of denitrification in anaerobic respiration, the occurrence of
nitrite could be considered as a proof of the microorganisms’ existence in
autoclaved sorptive controls. It also indicated that within the first day,
denitrification process played a crucial role in sorptive controls.
Noticeably, from the result displayed in Fig 5, the microbial activity in
the sorptive controls should be less than other five active samples. In
these five samples, corresponding to the nitrate decrease, no nitrite
accumulation was exhibited, which means further nitrification reactions
happened more rapidly than in the two sorptive controls.
Figure 3. Concentration of Nitrite over time.
4 Sulfate
Fig 6 presents all respective curves of sulfate concentration change over
time. A trifling diminution of sulfate concentration happened to every
setup in the first two sampling time points. This change was mainly
caused by the fluctuation between two measurement batches. Over the
experiment duration, sulfate level in abiotic control persisted at the
original value. Slight sulfate concentration increases happened in both
sorptive controls. Compared to them, the sulfate accumulation in all
microbial setups and active control setups was more distinct. The sulfate
rise in non-autoclaved samples can be considered as an indication of the
aerobic condition maintenance. Within the natural sulfur cycle, the
transformation between sulfide and sulfate depends on the participation
of oxygen. Under anoxic circumstance, anaerobic respiration plays a
major role in the microcosms, and sulfate in the medium could be
reduced to sulfide. On the contrast, with adequate oxygen, sulfide from
the sedimental mineral could be oxidized into sulfate by lithotrophic
bacteria. Therefore, the sulfate increase displayed in sorptive controls
indicated the existence of microbial residue as well. On the other hand,
considering the additional carbon source load and pharmaceutical load
contained no sulfur element, the eventual sulfate increase could be from
the contamination over the experiment process. In the experiment, to
maintain the aerobic condition and air exchange in the non-autoclaved
bottles, the screwing lids were not tightened. Thus, there could be
contaminations which introduced sulfate into samples and caused the
sulfate increase.
Figure 4. Concentration of Sulfate over time.