Chapter- 5

Aerobic and anaerobic microorganism for the removal of micropollutants

Most of the conventional wastewater treatment plants (WWTPs) are not well-equipped to entirely
remove organic micropollutants at low concentrations. In that chapter we discuss about aerobic and
anaerobic system for removal of micropollutants.

Aerobic system for removal of micropollutants.

1. Removal of micropollutants by Aerobic Activated Sludge: The conventional activated sludge

(CAS) process is the mostly widely used wastewater treatment system in the world; therefore, the
study of the removal organic micropollutants (OMPs) by CAS is of vital importance.[1, p. 118]
2. Adsorption and Biodegradation of Organic Micropollutants by Non-acclimated Aerobic
Activated Sludge: Organic micropollutants (OMPs) with different chemical and physical
properties will be removed by using batch reactors.[1, p. 119]
3. The Adaptation of Removal of Organic Micropollutants by Aerobic Activated Sludge: for some
OMPs, the removal had been enhanced during the adaptation, which showed an OMPs
concentration decrease in the effluent water; for most of the OMPs, the OMPS concentration
stayed the same, which could be explained by quick adaptation or the culturing time is too short
for obvious adaptation.[2, p. 39]
4. Aerobic Nanofiltration Membrane Bioreactor: Membrane bioreactor (MBR), which is combined
of biological treatment and membrane separation, has got a rapid growth in both domestic and
industrial wastewater treatment and reuse in recent years due to its significant advantages over
conventional activated sludge process such as better effluent quality and less footprint. However,
the microfiltration (MF) and ultrafiltration (UF) membrane widely used in membrane bioreactor
cannot reject OMPs due to their much higher pore size or molecular weight cut-off (MWCO) than
OMPs.[2, p. 57]

Anaerobic system for removal of micropollutants:

Anaerobic digestion is the standard process used around the world in STPs to stabilize primary
and secondary sewage sludge in anaerobic digesters (ADs), but it is also applied to treat
mainstream wastewater through up flow anaerobic sludge blanket (UASB) reactors and anaerobic
membrane bioreactors (AnMBRs). These technologies allow the recovery of energy by
effectively degrading organic carbon into biogas; besides, they have demonstrated the capacity to
remove OMPs. However, the anaerobic removal of OMPs is quite variable: some compounds can
be completely removed while others persist unaltered after this process, as occurs in other
biological treatments.[3]
Microbiological Aspects of Micropollutant Degradation:

Microbial degradation of micropollutants is associated with catabolic activity of microbes, micropollutants

get assimilated as growth substrates. Microbial growth on micropollutants depends on various operating
conditions such as optimum temperature, pH, doubling time, light requirement, and agitation. which
indirectly affects the pollutant degradation. Pollutant properties such as surface properties, aqueous
solubility, and charge are some important factors that determine the treatment efficiency.

Microorganisms capable of organic micropollutant degradation:

Microorganisms Micropollutants Concentration Degradation Refer

efficiency ence
(%)

Bacterial strains
Gordonia sp. Dop5 DnOP 750 mg/L 100 [4]
Acinetobacter sp. HS-B1 DBP 300 mg/L 100 [5]
Arthrobacter sp. C21
Bacillus thuringiensis Ibuprofen 25 mg/L 46.56 [6]
Naproxen 6 mg/L 100
Rhodococcus sp. JX-2 17β-Estradiol 30 mg/L 94 [7]
Pseudomonas putida Estrone 5 mg/L 100 [8]
Estradiol 94.86
17α-Ethinylestradiol 94.90
Estriol 94.56
Bisphenol A 96.56
Ochrobactrum sp. Erythromycin A 100 mg/L 97 [9]
Acinetobacter sp. Sulfamethoxazole 240 mg/L 100 [10]
Rhodococcus sp. BCH2 Atrazine 1000 mg/L > 90 [11]
Bacillus sp. PDK1
Bacillus sp. PDK2
Ochrobactrum sp. Jdc-41 DBP 500–1000 mg/L 99 [12]
Klebsiella sp. Diclofenac 70 mg/L > 90 [13]

5.1.1 Aerobic microorganisms:

Microorganism that are capable of living in the presence of molecular oxygen. In general, these
microorganisms require oxygen for their growth and metabolism although some of them are capable of
using oxygen when present, and carry out aerobic respiration. Others can switch to anaerobic methods in
absence of oxygen

All human beings are aerobes, this kind of metabolism requires special adaptations is not intuitively
obvious. However, oxygen produces negatively (-VE) charged ions when metabolized and, unless
organisms have developed antioxidant pathways to counter these, using oxygen is dangerous. These O2
radicals are toxic, and only organisms which express radical-scavenging enzymes (catalase and superoxide
dismutase) can survive their presence.[14]

Examples of aerobic microorganisms:

1. Nocardia sp.,
2. Psuedomonas aeruginosa,
3. Mycobacterium tuberculosis,
4. Bacillus, Nocardia and Pseudomonas aeruginosa
5. Staphylococcus species
6. Streptcoccus species
7. Enterobacteriacae species

Aerobic microorganisms are divided into the following groups:

• Obligate aerobic microorganisms: It strictly depend on energy production through aerobic

respiration and unable to survive for more than a few minutes in the absence of oxygen.
• facultative aerobic microorganisms: It can use oxygen, but are able to survive in its absence,
through e.g., fermentation.
• Microaerophiles aerobic microorganisms: They are organisms that may use oxygen, but only at
low concentration.
• Aerotolerant aerobic microorganisms: It can survive in presence of oxygen, but it does not use it
as electron acceptor.

5.1.2 Anaerobic microorganisms:

Anaerobic microorganisms do not require energy oxygen for metabolism. As such, they are different from
other types of microorganisms that need oxygen for their energy needs.

Compared to aerobic microorganisms that need oxygen to grow, anaerobic microorganisms are capable
of using various other substances during metabolism.[15]

Examples of anaerobic microorganisms include:

• Actinomyces
• Clostridium
• Propionibacterium
• Bifidobacterium
• Bacteroides
• Fusobacterium
• Prevotella
Based on metabolic characteristics, anaerobic microorganisms are divided into the following
groups:

• Facultative anaerobes: which can grow without oxygen. But it uses oxygen if it is present.
• Aerotolerant bacteria: which cannot use oxygen for growth, but tolerate its presence.
• Obligate anaerobes: which are harmed in presence of oxygen.

Aerobic and anaerobic microorganisms can be identified by growing them in test tubes of
thioglycolate broth:

1. aerobic bacteria gather at the top of the test tube in order to absorb maximal amount of oxygen.
2. anaerobic bacteria are poisoned by oxygen, so they gather at the bottom to avoid oxygen.
3. Facultative bacteria gather mostly at the top. It can grow with or without oxygen because they
can metabolize energy aerobically or anaerobically. but as lack of oxygen molecule does not hurt
them, they can be found all along the test tube.
4. Microaerophiles gather at the upper part of the test tube but not at the top. They require oxygen,
but at a very lower concentration.
5. Aerotolerant bacteria are not affected at all by oxygen, and they are evenly spread along the test
tube. they are not poisoned by oxygen. They can be found evenly spread throughout the test
tube.[15]
5.2 Factors effecting the feasibility of microorganism selection:
To maximize the removal efficiency of OMPs in aerobic and anaerobic and also in any other biological
system, it is essential to understand the factors driving and limiting the biotransformation pathways,
bearing in mind that enzymes are ultimately responsible for the biotransformation of OMPs if it occurs via
either metabolism or co-metabolism.

5.2.1 Structure of compound

OMP chemical structure has a direct effect on biotransformation; only compounds chemically compatible
with the active site of the enzymes present in the reactor could be transformed. Furthermore, the
chemical structure of OMPs determines their physicochemical properties, like hydrophobicity and
bioavailability, which are intrinsically related to their fate in biological treatments.

Some prevalent reactions under anaerobic conditions are reductive dehalogenation (e.g., iopromide and
diatrizoate) and cleavage of ether bonds (e.g., triclosan and venlafaxine). Moreover, it has been
demonstrated that the presence of electron-donating (e.g., hydroxyl and amine) or electron-withdrawing
(e.g., halogen and amide) groups governs the fate of OMPs. According to Wijekoon et al. compounds
bearing electron-donating groups in their structure are effectively biotransformed under anaerobic
conditions, as might be the case with sulfamethoxazole (–NH2, –CH3, –NHR), naproxen (–OCH3), and
trimethoprim (–OCH3). However, electron-withdrawing groups might have a negative effect on the
biotransformation of OMPs, such as carbamazepine (–CONH2), ibuprofen, and diclofenac (–COOH).
However, this criterion is not valid to predict the biotransformation of all OMPs. For instance, under
anaerobic conditions, the amide group (–CONH2, electron withdrawing) of atenolol is hydrolyzed, while
the electron-donating groups (–OH, –NHR) remain unaltered. Since most OMPs have complex molecules
with several functional groups that might undergo different enzymatic reactions, it is very difficult to
theoretically predict the biotransformation pathways of these pollutants.

Some structure of OMP is given below:

Trimethoprim Gemfibrozil

Diphenhydramine TDCPP

Dilantin TCPP
 Diclofenac TCEP

Sulfamethoxazole TCEP

Clofibric acid Sucralose

Carbamazepine Naproxen

Propylparaben Atenolol

Oxybenzone Bisphenol A

Primidone Ibuprofen

Methyparaben Acesulfame

5.2.2 Operating Condition

Conventional activated sludge (CAS) process:

The conventional activated sludge (CAS) process is the mostly widely used wastewater treatment system
in the world; therefore, the study of the removal organic micropollutants (OMPs) by CAS is of vital
importance. In this study, conventional aerobic activated sludge wastewater treatment was chosen for
the sludge operation, and followed a classical sequencing batch reactor mode. In order to get the non-
acclimated activated sludge, synthetic wastewater without any OMPs content was used as influent. After
2 weeks of operation, the effluent water reached equilibrium, and the reactor was kept operating for
about 6 months. All of the parameters of the effluent reached steady state after two weeks. The sludge
could be used for the studying of OMPs removal by non-acclimated aerobic activated sludge.[2]
Operational parameters of the bioreactor:

The operation was kept the same for about 6 months, and the bioreactor effluent was stable during the
process.

MLSS Temperature Influent COD Stirring rate DO pH

(mg/L) (℃) (mg/L) (rpm) %
1220 22±1 390-410 200 75-81 6.8-7.1

Aerobic Nanofiltration Membrane Bioreactor:

Microbial growth on micropollutants depends on various operating conditions such as optimum
temperature, pH, doubling time, light requirement, and agitation. which indirectly affects the pollutant
degradation. Pollutant properties such as surface properties, aqueous solubility, and charge are some
important factors that determine the treatment efficiency.

The operation conditions of the aerobic and the anaerobic MBRs are summarized in Table 2. The MBRs
have been operated for 266 days, with varying loading rates. The results from day 100 to day 266 are
presented in this paper. The experiments consisted of 5 runs, and each run had a different HRT. The HRT
ranged from 0.5 days to 2.0 days for both MBRs. The permeate flux for both MBRs was manually controlled
by giving the backpressure on the recirculation loop to adjust HRT. The excess volume is returned to the
bioreactor.[2]

Operation conditions for the aerobic and the anaerobic MBRs.[16]

Parameter Run1 Run2 Run3 Run4 Run5
Operation time (Days) 100- 128-164 165-200 201-236 237-266
127
Recycle Flowrate (L/d) 10 2000 2000 3000 3000
HRT (D) 2 1 0.67 0.5 1
Average transmembrane pressure (psi)
Aerobic MBR <0.1 6 10 15 8
Anaerobic MBR <0.1 5 5 10 6

5.2.3 Process variable

Both aerobic and anaerobic MBRs were capable of soluble-COD removal from municipal wastewater.
Aerobic MBR removed 63 to 78% of the primary-effluent-soluble COD at HRT values ranging from 0.5 to
2 days, whereas the anaerobic MBR removed between 55 and 68% of the primary-effluent-soluble COD
over the same range of HRT. It is possible that the same removals could be obtained at much lower HRTs,
based on the trends seen in COD removal as a function of HRT in both aerobic and anaerobic MBRs. The
average primary-effluent-soluble COD of 84 mg/L was reduced to 14 to 31 mg/L by the aerobic MBR and
to 24 to 38 mg/L by the anaerobic MBR. These results suggest that an anaerobic MBR is nearly equal to
an aerobic MBR, in terms of soluble COD removal at the same operating HRT. However, in the case of
anaerobic MBR, no aeration costs are involved, as oxygen is not needed for anaerobic metabolism. No
significant anaerobic gas production was observed, and no odors were detected from the anaerobic MBR.
Changes in HRT did not result in significant deterioration of the performance of the aerobic and the
anaerobic MBRs.

Temperature: Anaerobic processes strongly depend on the operation temperature. At low temperatures,
most biological reactions pertinent to anaerobic digestion, including hydrolysis and various fermentation
processes, are less energetically favorable. Hydrolysis of particle organics is considered as the rate-limiting
step in anaerobic digestion and is of great importance in DWW treatment, especially when a large fraction
particulate organic is presented. Generally, hydrolysis rates decline with the decrease of temperature,
making longer SRTs demand for hydrolysis at psychrophilic temperatures.

5.3 Application of aerobic and anaerobic system for micropollutants removal.

Micropollutants is a serious environmental problem with several negative outcomes for human health
and ecosystems. Many efforts have been made to remove OMP using a variety of physical, chemical and
biological methods. By far, the most attention has been taken for wastewater treatment in order to
obtain high-quality effluents, recover algal biomass for fertilizers, protein-rich feed, biofuel, and put
them to other practical use.[17]

Fig: Application for micropollutants removal in wastewater.

Micropollutant treatment efficiencies and main removal mechanisms performed by
different microalgae species.
Main removal microorganism Micropollutants Removal References
mechanism species (%)

Biodegradation Phaeodactylum Oxytetracycline 97 [18]

tricornutum
Selenastrum 17b-estradiol, 60-100 [19]
capricornutum 17a-
Chlamydomonas ethinylestradiol 100
reinhardtii
Scenedesmus Diclofenac 99 [20]
obliquus,
Chlorella 71
vulgaris,
Chlorella 67
sorokiniana
Chlorella Paracetamol 99 [21]
sorokiniana
Algal-bacterial Naproxen 28-52 L
L´
consortia o
p
Inoculum from Acetaminophen 99 e
z
lake water -
S
Algae pond Estrone, 17b- 52-56 [22] e
r
estradiol, 17a- n
a
Bioadsorption Microalgal- ethinylestradiol
Triston 85–100 [23] e
17a- t
bacterial a
ethinylestradiol l
consortia .
(
Propylparaben 87–100 2
0
1
9
Selenastrum 17b-estradiol 42 [19] )´
o
capricornutu p
e
Chlamydomonas 54 z
-
reinhardtii S
e
r
Photodegradation Chlorella Diclofenac 40–60 [21] n
sorokiniana a
e
t
a
l
.
(
2
0
1
9
)
L´
o
p
e
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