Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
Selection of Osmotolerant Bacteria for Enhanced Phenol
Removal in an Aerobic Sequencing Batch Reactor
Juli Rubin & Lisa Colosi, May 2003
Abstract
The purpose of this paper was to characterize phenol degradation by osmotolerant bacterial
communities in a saline sequencing batch reactor fed simulated wastewater. Application of a
selective pressure via increasingly saline influent led to the successful transition towards an
increasingly osmotolerant reactor community. This was confirmed through MLVSS/TSS/VSS
measurements and culture plating over time. The WWTP showed increasing phenol removal
during the change from non-osmotolerant to osmotolerant communities. Further, phenol
degradation by osmotolerant bacteria was characterized using Michaelis-Menten Kinetics and
the Andrews Inhibition Model. Maximum reaction velocity was found to be 1.67 g/L-s, and the
half-saturation constant (Ks) was found to be 30.9 g/L. Initial specific growth rates were found
to be 0.0192 hr-1 and 0.0187 hr-1 when initial phenol concentrations were 200 and 300 mg/L
respectively; thus confirming phenol’s suspected inhibitory effect on biomass growth rate.
Findings indicate that osmotolerant communities are less effective at degrading phenol than
extreme halophiles, but that phenol degradation increases with salinity.
Introduction & Literature Review
The family of chemicals characterized by the functional phenol group comprise hazardous
wastes when released to the aquatic environment; wastewaters contaminated with phenols or
phenolic resins may only be discharged from local wastewater treatment plants at concentrations
below 50 mg/l.1 Therefore, to be safely released, industrial phenolic wastewaters require in situ
treatment at the plant, where the removal of such organic compounds from wastewater streams is
conventionally achieved via exposure to aerobic bacteria. This treatment can prove less effective
when some characteristic of the wastewater inhibits the metabolic activity of the inoculating
bacteria. In particular, salinity can present a treatment challenge as the salt content can inhibit
the functionality of metabolic pathways or the integrity of cellular membranes of typical
wastewater bacterial communities and result in reduced organic removal efficiency.
The literature bears out significant interest in the physiological capabilities of microorganisms
capable of thriving at extreme conditions including high salinity. Halophiles, organisms which
have evolved to not only thrive in but also require highly saline conditions, can be further
divided into a number of subcategories including osmotolerant, slightly halophilic, moderately
halophilic, or extremely halophilic depending upon the salt concentration of their preferred
growth media. Of particular interest to engineers in search of a means to reliably remove
phenols from wastewater via biological treatment are the osmotolerant bacteria, optimally active
in a range of NaCl concentrations between 5 to 10 mg/L. 2 Following Hidalgo, et al and Peyton,
et al, there is some indication that the application of selective pressure, via increasingly saline
- Page 1 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
influent concentrations, provides one means to enhance a reactor’s biodegradation via transition
to a bacterial community more capable of degrading phenol.
Near-complete removal of phenolic compounds, on the order of 99.5%, has been demonstrated
by Woolard and Irvine via sequencing batch reactors.3 Such impressive removals are particularly
intriguing in light of phenol’s generally bactericidal effect on the majority of wastewater
communities which have not evolved appropriate defense mechanisms. Following a series of
studies with extreme halophiles, Peyton et al were able to characterize phenol degradation
kinetics as zero order with specific growth rates ranging from 0.09 to 0.32 h-1. However, as a
result of their specificity in media, the activity of extreme halophiles is limited to saline
concentrations in excess of 150 mg/L. Presumably, the effectiveness of treatment by cultures of
extreme halophiles will be compromised in the event of periodic exposure to diluted waste
streams with lower salt concentrations, as may occur following a large runoff event.
In light of the limitations of extreme halophiles, it seems prudent to characterize the degradation
of phenol by osmotolerant communities. Having evolved to thrive in media with a much larger
acceptable range of salt concentrations, such organisms will be more reliable if the salinity of the
influent stream is prone to fluctuate. This paper describes the application of selective saline
pressure and qualitatively demonstrates successful selection of an osmotolerant community in
the presence of a baseline phenol concentration. Finally, the paper concludes with a quantitative
characterization of the community’s phenol degradation kinetic parameters including maximum
velocity and half-saturation constant following the classic Michaelis-Menten model and initial
specific growth rate following the Andrews Inhibition Model.4,7
Objectives
Our objectives are designed to validate our hypothesis that selectively grown osmotolerant
bacteria are capable of phenol degradation at a removal efficiency superior to non-osmotolerant
communities. Specifically, they are three-fold as follows.
1. Qualitatively demonstrate successful selection for the osmotolerant members of the
bacterial community in our sludge inoculum via the application of increasingly saline
feed over time and spread-plating culture samples.
2. Characterize the plant’s treatment capabilities via measurement of phenol, TSS and VSS
removal efficiencies over time.
3. Characterize the reaction kinetics of phenol degradation in saline waters after Peyton, et
al.2 These will include maximum velocity, the half velocity coefficient, and the initial
specific growth rate.7
Experimental Materials & Methods
1. Logistical Operation of the Wastewater Treatment Plant (WWTP)
- Page 2 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
A. Plant Configuration
For this study, plants were fed a simulated wastewater, the composition of which was based upon
previous work by Cicek, et al. Plant configuration was modeled after the model put forth in the
CEE 453 Laboratory Research in Environmental Engineering Laboratory Manual.5
B. Plant States, Control Logic, & Timing
The plant operation software automatically cycled operations between five plant states: fill with
waste, fill with water, aerate, settle, and drain. Exit conditions were pre-specified in the control
logic as shown in Table 1 below so that the software could decide when to change to the next
state.
Table 1. Descriptions of Plant States, Objectives, and Exit Condition.
Plant State
Objective
Exit Condition
Fill with waste
Fill with tap water
Aerate
Settle
Drain
Addition of 100 mL waste, 5X.
Addition of 1900 mL water.
Aerobic degradation, mixing.
Clarification of plant effluent.
Removal of plant effluent.
Duration of specified time, pump velocity.
Pressure sensor, fill to total volume of 4 L.
Duration of pre-specified time, 4 hours.
Duration of pre-specified time, 2 hours.
Pressure sensor, reduction in volume to 2 L.
In general, these states represent the basic elements of any operational sequencing batch reactor.
However, certain modifications were made in order to meet our specific objectives while
simultaneously optimizing total suspended solids (TSS) and volatile suspended solids (VSS)
removal with this particular setup. For instance, the “fill” state was subdivided into “fill with
waste” and “fill with tap water” as a means to dilute the waste stream while minimizing the
volume that needed to be refrigerated. Also, following “fill with waste”, “fill with tap water”
allowed a means of purging the lines of organic material and preventing growth that might have
potentially clogged the lines or solenoid valves. Additionally, the aeration duration was
extended to four hours to allow for further removal of phenols, which tend to be somewhat
resistant to treatment as a result of their ring structure.2 Finally, the time allotted for settle was
manipulated in a somewhat subjective manner in the absence of effluent turbidity data.
Collection containers were placed in the drain as a mean to gauge the solids concentration in the
effluent. In light of the extremely good TSS and VSS removals, the time selected seemed to be
sufficient to allow for complete settling.
C. Tank Volume, Plant Flow Rate, & Recycle Flows
The tank held a total volume of 4 L. In general, the plant completed a full cycle of five states in
approximately 6.25 hours. In that time, 2 L of waste + tap water was added and 2 L of effluent
was removed through the use of a standpipe draining by gravity. Thus, the plant’s volumetric
flow rate was roughly 0.32 L/hr (7.68 L/day). Since half the tank volume was drained prior to
the beginning of the next cycle, the gross recycle percentage was 50%. However, sufficient time
- Page 3 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
was provided during the settle state to allow the biomass to settle into a volume less than 2 L
high such that the percentage of biomass recycled was 100%.
Increasingly saline concentrations were applied in an effort to select for osmotolerant organisms.
Sludge wasting was applied regularly as a means to encourage growth of these organisms over
the pre-established communities. In this sense, the recycle ratio was necessarily less than 100%
on a periodic basis whenever biomass was removed. In general, biomass was wasted from the
aeration state weekly, in an effort to maintain the concentration of mixed liquor suspended solids
at its original level of 750 mg/L. Weekly MLVSS levels hovered in the range of 800 to 900
mg/L, so waste volumes ranged from 250 to 670 mL. For these volumes, biomass recycle
percentages were approximately 95% and 85% respectively.
2. Experimental Manipulation of Salinity in the Wastewater Treatment Plant (WWTP)
In order to prevent toxicity due to saline shock, plant salinity was gradually increased via
increased influent salt concentration according to the schedule outlined in Table 2 below.
Originally, it had been intended to adjust salinity each week at a constant rate of increase.
Following an accidental overdose, MLVSS levels and TSS/VSS removals were drastically
reduced, and it became necessary to maintain the same salt concentration for the duration of our
study. As an experimental side note, the solubility of NaCl at refrigeration temperatures (10 ºC)
is only 80 g/L. Following week two, the concentration in the 20X feed stock was far in excess of
this value, so it became necessary to stir the solution continuously from within the refrigerator.6
Table 2: Reactor & Feed Salt Concentrations Over Time
Stage
Reactor [NaCl] (g/L)
NaCL (mM)
100X Feed (g/L)
1
0.59
10
23
2
1.17
20
47
3
5.00
30
200
4
5.00
30
200
5
5.00
50
200
Independent of the salinity manipulation schedule, the analytical schedule was designed to
monitor both the overall health of the biomass as well as its capacity to degrade the aromatic
phenol. Relatively constant levels of MLVSS (via drying and ashing) indicated the salinity and
phenol concentrations had remained within an appropriate, nontoxic range. Similarly, constant
or decreasing effluent VSS concentrations indicated that our altered WWTP was at least as
efficient in organics removal as it had been prior to selection for the osmotolerants. Finally,
measurement of effluent phenol via UV spectrophotometry (wavelength 270 nm) concentrations
was necessary to demonstrate phenol removal efficiency. Please refer to Appendix Table A1 for
the analytical schedule.
3. Microbial Techniques: Validating the Selection of an Osmotolerant Community
- Page 4 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
To confirm the presence of increasingly osmotolerant strains for the degradation of phenol, we
made use of plating techniques and three distinct media types. Twice a week, as indicated in
Table 1A of the appendix, 20 mL samples of the biomass were removed from the tank in aeration
state, placed into a sealed plastic container with 15% (by volume) glycerol, and placed into a
freezer. These remained frozen until the final week of the study, when 1/10 dilutions were plated
on three types of media. The control media was a 1/10 dilution of a commercial TSA growth
media. The second and third plates had the same media supplemented with either 50 mg/L
phenol or 5 g/L NaCl. If our hypotheses were to be borne out, we anticipated the number of
colonies on the control to decrease over time while the number of colonies on the experimental
plates increased over time. In this way, we would be able to qualitatively demonstrate a
transition to osmotolerant communities.
4. Characterizing Plant Treatment Capabilities via Phenol, TSS/VSS Removal Efficiencies
Measurement of TSS and VSS removal efficiencies was used as a rough gauge of the plant’s
overall capacity to degrade waste, where removal efficiency is defined as in Equation2 below.
Equation 2: Definition of Removal Efficiency
Where:
E
X I  X E 
XI
XI is the influent concentration of some species.
XE is the effluent concentration of some species.
Measurements were typically made using effluent discharged to a container placed in the drain
on Wednesdays and Saturdays. Volumes of approximately 50 mL were required to achieve
reproducible values for TSS and VSS using the drying/ashing method as the samples tended to
be rather dilute following treatment. Samples were dried in a Fisher Scientific Isotemp Oven set
at 105 C for 1 hour prior to being ashed in a Fisher Isotemp Muffle Furnace set to 550 C for 20
minutes.
Influent phenol concentrations were held steady for the duration of the plant’s operation. The
reactor’s phenol concentration was held steady at 50 mg/L at the onset of each aeration phase. In
addition, effluent phenol measurements were made twice-weekly through the use of a HP 8452A
Diode Array UV Spectrophotometer ( = 270 nm) to determine the plant’s capacity to remove
phenol. Special care was taken to match the composition of the phenol standards as closely as
possible to the expected effluent composition, but the addition of salt resulted in only a 0.05%
increase in absorption at all phenol concentrations. As such, the same phenol standards,
containing the maximum NaCl concentration diluted in distilled water, were used for the
duration of the study.
5. Determining Reaction Kinetics Parameters of Osmotolerant Phenol Degraders
Reactions kinetic parameters can be determined by utilizing the Michaelis-Menten Equation and
the Lineweaver-Burke plot. Reaction velocity (V) is the change in substrate with time and is
related to the substrate concentration by the Michaelis-Menten equation found below:
- Page 5 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003

Equation 3: Michaelis-Menten Equation
dS Vmax S

dt K s  S
where S is the substrate concentration, Vmax is the maximum rate of substrate utilization, and Ks
is the half velocity coefficient or the substrate concentration at which the reaction is at half of its
maximum velocity.
Kinetic parameters can be determined by linearizing the relationship between reaction velocity
and substrate concentration. Using the inverse of the Michaelis-Menten Equation found below,
Equation 4: Inverse of the Michaelis-Menten Equation
Ks
1
1


V Vmax S Vmax
a linear relationship can be found by plotting 1/V against 1/S where the slope is equal to K s/k
and the y-intercept is equal to 1/Vmax. Once the slope and y-intercept are determined, Ks and
Vmax can be calculated. A plot of 1/V against 1/S is known as a Lineweaver-Burke plot.
Reaction kinetic parameters for phenol degradation by osmotolerant bacteria were determined in
this manner. Two liters of sludge were taken from the reactor during the aeration phase and
subsequently pelleted and washed with distilled water. The pellet was then resuspended in 250
mL of tap water and 40 mL of the resuspension was mixed with 460 mL of tap water to give a
total volume of 500 mL. Three of the cell suspension/tap water mixtures were made and each
fed varying concentrations of phenol (200 mg/L, 300 mg/L, 400 mg/L) while being continuously
stirred. Samples were taken at 15, 45 and 75 seconds for each of the three suspensions and
filtered through Gelman Acrodisc 0.2m syringe filters and kept on ice in order to stop bacterial
degradation. Phenol measurements were then taken using a Hewlett Packard Diode Array
Spectrophotometer and plotted against time using Excel. The reaction velocity for each substrate
concentration was determined by calculating the initial slope of each respective plot. A
Lineweaver-Burke plot was then used to calculate the maximum reaction velocity as well as the
half velocity constant. The inverse of the reaction velocities found was plotted against the
inverse of the corresponding initial substrate concentration fed. Using the Michaelis-Menten
relationship, it was possible to calculate the desired kinetic parameters.
6. Determining Initial Specific Growth Rate
The initial specific growth rate was determined following the methods of Hao et al.6 Sludge
taken from the reactor during aeration was resuspended in 500 mL of tap water to give a cell
concentration of 10mg/L. The 10 mg/L was obtained by taking an MLVSS measurement of the
initial sludge in the reactor and diluting to the desired concentration. Two identical suspensions
were made and one was given a phenol concentration of 200mg/L and the other a concentration
of 300mg/L. Samples were taken every 30 minutes and MLVSS measurements were made.
The MLVSS measurements were plotted against time and the initial specific growth rate was
calculated by finding the slope of the line. The specific growth rate calculated for the
osmotolerant bacteria grown on phenol from our reactor was compared to the specific growth
rate for non-osmotolerant bacteria grown on phenol determined by Hao, et al.6
- Page 6 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
Analysis
Part 1: Oxygen Demand Calculations
Phenol is an aromatic compound; its molecular weight is 94.11 g/mol. Aerobic degradation of
phenol takes the form of Equation 5 below if we make the simplifying, if unjustified, assumption
that degradation is 100% efficient.
Equation 5: Aerobic Degradation of Phenol
C6 H 5OH  7O2  6CO2  3H 2O
At a reactor concentration equal to 50 mg/L, the phenol added an additional 119.0 mg/L
theoretical biological oxygen demand (BOD) to the influent wastewater. The influent feed
concentration had an additional chemical oxygen demand of 6460 mg/L as well as 40.9 mg/L of
nitrogen, which comprised an additional 187.0 mg/L nitrogenous oxygen demand (NOD). The
sum of these components resulted in an overall BOD concentration of roughly 6766 mg/L. Only
100 mL of feed was added during each “fill with waste” phase, so the maximum BOD loading to
the tank at the start of each cycle was on the order of 169 mg. Please refer to Table 3 below for
details of the calculations.
Table 3. Calculation of BOD Addition & Concentration
Parameter
Feed COD (Soluble)
Stock Nitrogen Concentration
Moles Nitrogen
Moles NOD
Mass NOD
Stock BOD Concentration,
(NOD + COD)
Concentration Phenol Added
Molecular Weight Phenol
Moles Phenol Added
Moles BOD Added
Concentration BOD Added
Influent BOD Concentration,
(NOD + COD + Phenol BOD)
Influent BOD Mass, (NOD +
COD + Phenol BOD)
Feed Volume Per Cycle
Total Reactor Volume
Initial Reactor BOD
Value
6460 Mg/L OCD
40.9 Mg/L N
2.92 mmoles N
5.84 mmoles NOD
187 Mg/L NOD
6647 Mg/L
50 Mg/L
94 Mg/mmol
0.53 mmoles/L
3.72 mmoles/L
119 Mg BOD/L
6766 Mg BOD/L
677 Mg BOD
100 mL
4000 mL
169 Mg BOD/L
Based upon this level of influent BOD, it is also possible to calculate the amount of oxygen that
must be delivered to the plant during the aeration phase. Based upon the calculations
summarized in Table 4 below, the plant requires 3.66E-4 mmol O2/s. However, this value
actually represents the average O2 requirement over the whole period since the rate of O2
- Page 7 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
consumption varies non-linearly over the course of the aeration phase.
consumption is greatest upon delivery of feed and decreases with time.
May, 2003
In reality, O2
Part 2: Demonstrating Changes in Dominant Bacterial Communities
Changes in bacterial communities were demonstrated by monitoring variations in reactor
MLVSS and changes in effluent TSS and VSS removal over time. Initially, weekly adjustments
to the influent salt concentration were minimal, and MLVSS measurements remained somewhat
steady. The only source of variation was a result of wasting, which was done weekly to promote
growth of new biomass. During week 3 (4/19) of the plant operation, the influent salt
concentration was significantly increased, resulting in a dramatic reduction in biomass
concentration. This was not surprising; such a rapid transition was bound to be somewhat toxic.
However, the increase in sodium chloride was not immediately followed by a reduction in
MLVSS. Instead, there occurred a lag in MLVSS decline on the order of roughly 10 days. In
order to prevent further disruption to the plant, the influent salt concentration was maintained at
the week 3 concentration for the duration of plant operation. Presumably, the constant
concentration afforded the biomass communities a chance to acclimate to the more saline
environment, as MLVSS measurements after week 3 demonstrated a steady increase over time
until the biomass once again reached a stable concentration. Just as the initial decrease in
MLVSS indicated that the original bacterial community was incapable of surviving in a saline
environment, the eventual recovery and stabilization of MLVSS indicated the growing
dominance of a new osmotolerant population. A graph of MLVSS measurements over time can
be found in Figure 1 below.
- Page 8 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
1.4
1.2
MLVSS (g/L)
1
0.8
0.6
0.4
0.2
0
3/28
4/2
4/7
4/12
4/17
4/22
4/27
5/2
5/7
Date
Figure 1. Biomass Concentrations (MLVSS) O ver Time
Over time, TSS & VSS removal efficiencies demonstrate a trend similar to that of the MLVSS
concentrations. Initial removal was around 100%; however, following the significant increase in
influent salt concentration during week 3, the TSS and VSS removals dropped to approximately
half of their initial values. In a manner similar to the MLVSS recovery, efficiency gradually
increased over time following week 3. Please refer to Figure 2 below, as it indicates growth of a
new salt- tolerant population.
- Page 9 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
100
90
Removal Efficiency (%)
80
70
60
50
40
30
20
10
0
3/30
4/4
4/9
4/14
4/19
4/24
4/29
5/4
5/9
Date
T SS Removal
VSS Removal
Figure 2. Removal Efficiency for TSS, VSS, & Phenol O ver Time
Plating techniques were used as a means to qualitatively confirm the presence of increasingly
osmotolerant strains. The bacterial growth on the 1/10 TSA plates decreased with time while the
growth on the 1/10 TSA plus NaCl-supplemented plates increased with time, thus verifying a
shift to an osmotolerant bacterial community. The level of growth remained relatively constant
over time for the phenol-supplemented plates. Presumably, non-osmotolerant strains of bacteria
are also capable of degrading phenol, and the osmotolerant strains are capable of phenol
degradation even in the absence of salt.
Part 3: Characterization of Phenol Treatment Capabilities
Figure 3 below summarizes removal efficiency for phenol over time.
- Page 10 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
100
90
Phenol Removal Efficiency, %
80
70
60
50
40
30
20
10
0
3/30
4/4
4/9
4/14
4/19
4/24
4/29
5/4
5/9
5/14
Date
Figure 3. Phenol Removal Efficiency O ver Time
The variation in phenol removal efficiencies is very similar to variation in MLVSS and solids
removals. Following the abrupt increase in salt concentration, removal plummets to zero percent
only to increase over time as the biomass acclimates and transitions to become more
osmotolerant.
Characterizing Phenol Degradation Via Determination of Kinetic Parameters
Initial reaction velocities were determined for three different initial substrate concentrations: 200,
300, and 400 mg/L phenol. Please refer to Figure 4 below to view an illustration of substrate
concentration over time or to Table 4 to view the data.
- Page 11 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
450
400
Phenol Concentration (mg/L)
350
300
250
200
150
100
50
0
0
10
20
30
40
50
60
70
80
Time (s)
So = 200 mg/L
So = 300 mg/L
So = 400 mg/L
Figure 4. Phenol Degradation by Osmotolerant Wastewater Biomass
Reaction Velocities for Selected Initial Substrate Concentrations
Table 4: Data Collected for the Determination of Initial Reaction Velocities and MM Parameters
Time (s)
Absorbance
Concentration (mg/L)
0
NA
400
15
0.97
74.36
45
0.73
55.84
75
0.63
48.00
V (mg/L-s)
21.71
Time (s)
Absorbance
Concentration (mg/L)
0
NA
300
15
0.85
64.83
45
0.63
47.42
75
0.46
34.63
V (mg/L-s)
15.68
Time (s)
Absorbance
Concentration (mg/L)
0
NA
200
15
0.51
38.47
45
0.42
31.50
75
0.38
28.40
V (mg/L-s)
10.77
Following the determination of initial reaction velocities, parameters including maximum
velocity and the half-velocity constant were calculated through the use of a Lineweaver-Burke
Plot, Figure 5 below, to linearize the inverse of the Michaelis-Menten (MM) Equation.
- Page 12 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
0.1
0.09
1/V (L-s/mg)
0.08
y = 18.538x + 0.0006
R2 = 0.9974
0.07
0.06
0.05
0.04
0.002
0.0025
0.003
0.0035
0.004
0.0045
0.005
0.0055
1/So (L/mg)
Figure 5. Lineweaver-Burke Plot for Determination of Maximum Velocity and Half-Saturation
Constant
As indicated by the trend line on the graph itself, the best-fit line for the linearized initial reaction
velocities has a slope of 18.538 and a y-intercept of 0.0006. Because the quantities are inverted,
the units are irrelevant; however, the numerical values themselves make possible the calculation
of the maximum velocity, Vmax = 1.67 g/L-s and the half-saturation constant, Ks = 30.9 g/L.
Duplication of the work done by Hao, et al resulted in the formulation of Figure 6 below, the
slope of which is the initial specific growth rate,0, as defined for use with classical Andrews
Inhibition Modeling.4 As indicated by the trend line on the graph, 0 takes a value of 0.0192 hr-1
and 0.0187 hr-1 when the initial concentrations of phenol were 200 and 300 mg/L respectively.
- Page 13 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
0.125
0.105
MLVSS (mg/L)
0.085
0.065
y = 0.0192x + 0.0136
R2 = 0.9606
0.045
y = 0.0187x + 0.0112
R2 = 0.9885
0.025
0.005
0
0.5
1
1.5
Time (hours)
200 mg/L
300 mg/L
Linear (300 mg/L)
Linear (200 mg/L)
Figure 6. Andrews Determination of the Initial Specific Growth Rate
Discussion
1. Evaluation of Plant Operation
The primary difficulties in setting up the WWTP were associated with feed delivery and
drainage. The peristaltic pump wasn’t an accurate means to deliver a pre-specified volume of
waste. Therefore, it was difficult to know exactly how much waste the plant was treating.
Additionally, since draining was driven by gravity, it was critical that all of the biomass settle
below the level of the standpipe prior to the start of the drain phase. For the week that mineral
nutrients were not added to the tap water, the floc settled very poorly and it was difficult to
prevent the loss of sludge. In order to minimize the loss of biomass, effluent solids concentration
was monitored twice-weekly, and the level of the standpipe was adjusted accordingly.
Although enhanced BOD or nitrogen removal were not among the primary goals of this study, it
would have been possible to change the plant configuration to achieve better removal. To
improve BOD removal, the effluent might have been pumped to the top of a trickling filter
column to allow for enhanced removal during travel through the media and biofilm. Similarly,
nitrogen removal could have been achieved through the addition of a separate, methanol-fed
anaerobic tank to allow for denitrification and subsequent sparging of the resultant N2 bubbles
through the secondary clarifier.
2. Evaluation of Results & Comparison with the Literature
- Page 14 of 17 -
2
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
Comparison of the numerical kinetics parameters with those posted to the literature indicates a
high level of discrepancy. Peyton, et al characterized phenol degradation by extreme halophiles
to be strictly zero-order; that is, degradation is independent of substrate concentration. In
contrast, reaction velocities summarized in Figure 4 indicate that phenol degradation may be
first-order for osmotolerant bacteria. Without another paper characterizing Michaelis-Menten
reaction parameters, it is difficult to evaluate the accuracy of Ks and Vmax.
Results from the initial specific growth rate experiment do agree with Hao and Kim’s finding of
growth inhibition at increased phenol concentrations. The specific growth for the 200 mg/L
reactor was larger than the specific growth rate for the 300 mg/L reactor. However, the
magnitude of μ for the osmotolerant strains was almost an order of magnitude smaller than the
value found by Peyton, et al for extreme halophiles suspended in a saline basal media (0.09 hr-1).
The values seem even smaller in comparison to the value found by Hao and Kim, et al for nonosmotolerant species suspended in a non-saline basal media (0.45 hr-1).
Discrepancies between results from this paper and previous papers may be due to a number of
factors. First, this paper analyzed the performance of osmotolerant biomass as opposed to
extreme halophiles as used in Peyton, et al. Given sufficient time, the communities in the reactor
might have become halophilic; but time was a limiting factor in this experiment. Also, these
results are from experiments run with suspensions in tap water. Limited micro-nutrients might
have impeded growth rates. However, since the biomass was able to thrive at all, our results
indicate that very little growth supplements are required for cometabolic phenol degradation in
saline waste streams.
In future studies, it is recommended that the plant be operated for a longer duration.
Additionally, two reactors might be operated at the same time: one saline, one without influent
salt. This would provide a means to make a better comparison between osmotolerant and nonosmotolerant phenol degradation capabilities.
The authors would like to acknowledge Dr. Monroe Weber-Shirk, Mr. Timothy Mattes, and Dr.
Rohit Gupta for their invaluable assistance.☺
- Page 15 of 17 -
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
May, 2003
Appendix
Table A1: Schedule of Analytical Work
Day
Date
Analyses
Feed Adjustment
Plates?*
Wed
Thu
Fri
Sat
Sun
Mon
Tue
4/2
4/3
4/4
4/5
4/6
4/7
4/8
VSS (in/eff), MLVSS _ Baseline
Add Salt Conc.1, Phenol ( 50 mg/L)
Freezer X
Wed
Thu
Fri
Sat
Sun
Mon
Tue
4/9
4/10
4/11
4/12
4/13
4/14
4/15
VSS (in/eff), MLVSS _ Salt @ Conc.1
Wed
Thu
Fri
4/16
4/17
4/18
VSS (in/eff), MLVSS _ Salt @ Conc. 2
Sat
Sun
Mon
Tue
4/19
4/20
4/21
4/22
VSS (in/eff), MLVSS _ Salt @ Conc. 3
Wed
Thu
Fri
Sat
Sun
Mon
Tue
4/23
4/24
4/25
4/26
4/27
4/28
4/29
VSS (in/eff), MLVSS _ Salt @ Conc. 3
Wed
Thu
Fri
Sat
Sun
Mon
Tue
4/30
5/1
5/2
5/3
5/4
5/5
5/6
VSS (in/eff), MLVSS _ Salt @ Conc. 3
VSS (in/eff), MLVSS _ Salt @ Conc.1
Freezer X
Add Salt Conc. 2, Phenol ( 50
mg/L)
VSS (in/eff), MLVSS _ Salt @ Conc. 2
Freezer X
Add Salt Conc. 3, Phenol ( 50
mg/L)
- Page 16 of 17 -
Freezer X
Freezer
(No)
Add Salt Conc. 4, Phenol ( 50
mg/L)
VSS (in/eff), MLVSS _ Salt @ Conc. 3
VSS (in/eff), MLVSS _ Salt @ Conc. 3
Freezer X
Freezer X
Freezer X
Add Salt Conc. 5, Phenol ( 50
mg/L)
Freezer X
Selection of Osmotolerant Bacteria for Enhanced Removal of Phenol in an Aerobic Sequencing Batch Reactor
J. Rubin, L. Colosi
1
2
3
4
5
6
7
May, 2003
Hidalgo, A., Jaureguibeitia, M.B.P., Rodriguez-Fernandes, C., Serra, J.L., Llama, M.J. (2002) Biological
Treatment of Phehnolic Industrial Wastewaters. Enzyme & Microbial Technology, Vol. 31, Issue 3, pp 221-226.
Peyton, B.M., Wilsona, T., Yongeb, D.R. (2002). Kinetics of phenol biodegradation in high salt solutions.
Water Research, Vol. 36, Issue 19 , pp. 4811-4820.
Woolard, C.R., Irvine, R.L. (1995) Treatment of Hypersaline Wastewater in the Sequencing Batch Reactor.
Water Research, Vol. 29, Issue 4, pp. 1159-1168.
Madigan, M.T., Martinko, J.M., Parker, J. Brock Biology of Microorganisms, 10 th Ed., WCB/McGraw-Hill
Publishers © 1999
Weber-Shirk, M., Lion, L.W., Bisogni, J.J. Laboratory Experience in Environmental Engineering, Laboratory
Manual, 7th Edition. Cornell University, © 2003
http://www.chem.purdue.edu/buriak/jmb/CHM112/CHM112_Buriak_Chapter_14b.pdf
Hao, Oliver J., Kim, Michael H., Seagren, Eric A., Kim, Hyunook. (2002). Kinetics of Phenol and Chlorophenol Utilization
by Acinetobacter Species. Chemosphere, Vol.46, Issue 6, pp 797-807.
- Page 17 of 17 -