Acidogenic fermentation facilitates anaerobic biodegradation of

Subscriber access provided by TULANE UNIVERSITY

Article

Acidogenic fermentation facilitates anaerobic biodegradation of polycyclic aromatic hydrocarbons in waste activated sludge Leiyu Feng, Jianguang Chen, Feng Wang, Yinguang Chen, and Jingyang Luo ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b06425 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on January 31, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Acidogenic fermentation facilitates anaerobic biodegradation of polycyclic aromatic hydrocarbons in waste activated sludge Leiyu Feng a, b, Jianguang Chen a, Feng Wanga, Yinguang Chena, b, *, Jingyang Luoc, d, * a

State Key Laboratory of Pollution Control and Resources Reuse, School of Environmental Science and

Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China b

Shanghai Institute of Pollution Control and Ecological Security, 1239 Siping Road, Shanghai 200092, China

cKey

Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education,

Hohai University, 1 Xikang Road, Nanjing 210098, China dCollege

of Environment, Hohai University, 1 Xikang Road, Nanjing 210098, China

*Corresponding

author

Email: [email protected], [email protected] Tel: 86-21-65981263 Fax: 86-21-65986313

1

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 22

ABSTRACT： The anaerobic biodegradation of phenanthrene (PHE), a typical polycyclic aromatic hydrocarbon (PAH) in waste activated sludge (WAS), during acidogenic fermentation created by alkaline pHs and biosurfactant was examined in this study.

The anaerobic biodegradation efficiency of PHE increased from 17.6% with an

operation time of 8 d in the control to 47.5% at pH 10.0, and to 78.2% under the conditions of pH 10.0 and 0.3 g alkyl polyglucose (APG) per gram of total suspended solids (TSS) with a mineralization rate of 25.1%. Mechanism exploration indicated that the bioavailability of PHE was remarkably improved because of the disruption of sludge structure, and enhancement of transfer and sorption by bacteria and transmembrane transport into microbial cells by alkaline pH and APG.

Under acidogenic conditions, the abundance of key microorganisms,

especially typical acidogenic bacteria that were capable of degrading PHE, activities of key microbial enzymes and quantities of functional genes benefitted PHE biodegradation.

The protein released from WAS facilitated electron

transfer among microorganisms and stimulated the co-metabolism of PHE.

Possible pathways of PHE

biodegradation under acidogenic conditions were presented. Keywords: waste activated sludge; anaerobic fermentation; polycyclic aromatic hydrocarbons; biodegradation; electron transfer Abbreviations: ANOVA: analysis of variance; APG: alkyl polyglucose; BCoA: benzoyl-coenzyme A; BSA: bovine serum albumin; COD: chemical oxygen demand; EPS: extracellular polymeric substances; GC-IRMS: gas chromatography isotope ratio mass spectrometry; GN-PAH: PAH-ring hydroxylating dioxygenase encoding genes from Gram-negative bacteria; GP-PAH: PAH-ring hydroxylating dioxygenase encoding genes from Gram-positive bacteria; HCl: hydrochloric acid; HOCs: hydrophobic organic compounds; iLDH: independent lactate dehydrogenase; MnP: manganese-dependent peroxidase; NADH: nicotinamide adenine dinucleotide; NaOH: sodium hydroxide; NCoA: 1,2-naphthoyl-coenzyme A; PAH-RHD: PAH-ring hydroxylating dioxygenase encoding genes; PAHs: polycyclic aromatic hydrocarbons; PHE: phenanthrene; rpm: revolutions per minute; SCFAs: 2


Page 3 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


short-chain fatty acids; TSS: total suspended solids; VSS: volatile suspended solids; WAS: waste activated sludge; WWTPs: wastewater treatment plants

lNTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) have attracted much attention because of their high toxicity.1-3 PAHs are formed through various pathways and widespread in the natural environment. plants (WWTPs) have been considered as the main final accumulation sites of PAHs.4, 5

Wastewater treatment

Because of the low water

solubility and high hydrophobicity, most PAHs are adsorbed and deposited in waste activated sludge (WAS), a by-product during biological wastewater treatment. as high as 2000 mg/kg dry weight.5–7

Indeed, the content of PAHs in sludge has been reported to be

Polycyclic aromatic hydrocarbons (PAHs) absorbed in sludge would do

great harm to the environment, so it is necessary to remove them efficiently. In principle, microbial degradation is the main way to remove pollutants from the environment because of its convenience and low cost.8, anaerobic conditions.

9

Polycyclic aromatic hydrocarbons can be biodegraded under both aerobic and

The aerobic biodegradation of PAHs has been well studied and many degradation pathways

have been elucidated.10

However, many sites polluted by PAHs are under anaerobic conditions, and anaerobic

microorganisms play a leading role in PAHs removal.

Meanwhile, anaerobic acidogenic treatment has been

regarded a preferred approach to stabilization of WAS and recovery of renewable resources.11-13

In the previous

study, anaerobic biodegradation of a high-molecule-weight PAHs (benz[α]anthracene) has been reported during WAS anaerobic fermentation.14

To date, however, the performance of anaerobic biodegradation of low and

medium-molecular-weight PAHs, which are usually at high levels, during WAS acidogenic fermentation treatment has been seldom documented.

More importantly, the mineralization and biodegradation pathways of PAHs are

vital for understanding their transport, transformation and impacts in WAS acidogenic treatment, but have not never been investigated. 3



Page 4 of 22

Low bioavailability is the main factor limiting the biodegradation of PAHs,15, 16 which is exacerbated in WAS treatment systems because PAHs are tightly wrapped by extracellular polymeric substances (EPS).17

To enhance

their biodegradation during anaerobic fermentation of WAS, PAHs should first be desorbed from EPS, and then their bioavailability was improved by upgrading the solubility in the fermentation liquid. microorganisms also play a critical role in PAHs biodegradation.

Functional

Previous studies have been concentrated on

enhancement of the biodegradation of PAHs by cultivating microorganisms; however, this is not applicable to actual PAHs-contaminated sites because of its unstable capability.18,

19

Various species with multimetabolic

functions are involved in WAS anaerobic treatment, including methanogens, acid-forming bacteria, and sulfate-reducing bacteria, among which methanogens and sulfate-reducing bacteria have been shown to have the ability to degrade PAHs.20–22

However, little information regarding whether acid-forming bacteria can degrade

PAHs under anaerobic conditions is currently available. Therefore, the main aim of this study was to explore the anaerobic biodegradation of PAHs in WAS during acidogenic fermentation, which was the practical treatment solution for WAS.23–25

Because the operational pH

and presence of biosurfactant give obvious effects on WAS acidogenic treatment, the effects of alkaline pHs and alkyl polyglucose (APG) on anaerobic biodegradation of phenanthrene (PHE), which was selected as a typical PAH because it is commonly found at high levels in WAS, were investigated. studied using stable carbon isotope analysis.

Specially, the mineralization of PHE was

The mechanisms involved in the improvement of PHE

biodegradation under the conditions of alkaline pH and biosurfactant were then investigated with respect to the contribution to PHE bioavailability and shifts in microbial community related to PHE biodegradation and their activities.

Moreover, the effects of acid-forming bacteria and the main organic matters from WAS on PHE

biodegradation were explored to further disclose the factors enabling the effective PHE removal during WAS acidogenic treatment.

Finally, possible pathways of PHE metabolism during WAS anaerobic acidogenic 4


Page 5 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


fermentation were proposed.

MATERIALS AND METHODS WAS, Phenanthrene and Alkyl polyglucose.

WAS without PAHs was withdrawn from a municipal WWTP

located at Shanghai, China, and its main characteristics of concentrated WAS were shown in Table 1.

Obviously,

protein was the main component in WAS. Table 1. The main characteristics of WAS Parameter

Value

pH total suspended solids (TSS) (mg/L) volatile suspended solids (VSS) (mg/L) soluble chemical oxygen demand (COD) (mg/L) total COD (mg/L) total protein (mg COD/L) total carbohydrate (mg COD/L) lipid and oil (mg COD/L)

6.8 ± 0.1 14,170 ± 200 9685 ± 185 315 ± 35 13,685 ± 255 7905 ± 185 1045 ± 105 140 ± 15

Phenanthrene with a purity of 98% was purchased from Sigma-Aldrich and dissolved in methanol to 1.0 g/L as stock solution.

APG used in this study was the same with that in the previous study.13

Investigation of PHE Degradation during WAS Anaerobic Acidogenic Fermentation.

pH influence on PHE

degradation was investigated in reactors with 1 L working volume by controlling the pH of WAS at 7.0 –11.0 using 4M alkali (sodium hydroxide) or acid (hydrochloric acid), as well as leaving one reactor without pH regulation as a control.

The effects of APG on PHE degradation were investigated by controlling different dosages of APG,

which were respectively 0, 0.05, 0.1, 0.2, 0.3 and 0.4 g/g TSS, at pH 10.0. PHE concentration was set at 100 mg/kg TSS.

In the WAS fermentation systems, the

The reactors were sealed after the oxygen was eliminated with

nitrogen gas, and then placed in a constant-temperature (35 ± 1°C) shaker.

The fermentation sample was

withdrawn within 2 d to examine the protein and carbohydrate released from sludge and the PHE distribution in solid and aqueous phase.

Anaerobic degradation of PHE was conducted within 8 d, which was sufficient for

production of SCFAs from WAS.24

Abiotic tests indicated that PHE losses throughout non-biological pathways 5



Page 6 of 22

were below 5% (data not shown), which is negligible. Several semi-continuous flow reactors, which were operated respectively without pH control and APG, at pH 10.0 and under the condition of pH 10.0 and 0.3 g APG/g TSS, were carried out for 90 days to examine the activities of enzymes, quantities of functional genes and microbial community during PHE biodegradation. PHE Mineralization Determined by Stable Carbon Isotope.

To determine the mineralization of PHE, three

experiments were conducted using the role WAS, WAS with unlabeled PHE and WAS with 13C labeled PHE at pH 10.0 with 0.3 g APG/g TSS.

The fermentation temperature was 35 ± 1°C and the stirring speed was 130 rpm.

The volume of fermentation liquid in the fermentation reactors was 150 mL.

Each flask was connected

successively to bottles containing ethylene glycol to trap volatile organic compounds and sodium hydroxide (NaOH, 2 M) to trap CO2, and then to a vacuum pump (Supplementary Information, SI, Figure S1).

Fermentation

mixtures were withdrawn from the anaerobic reactors per 2 d to determine the changes in the isotope carbon ratio as well as the production of

13CO

2.

A triplicate set of experiments was conducted to analyze the CO2

concentration. Roles of Acid-forming Microorganisms in PHE Biodegradation.

Proteiniphilum acetatigenes and

Propionibacterium acidipropionici, which have been used in the previous study,26 were applied to explore the roles of acid-forming microorganisms in PHE biodegradation.

The experiments were conducted in the anaerobic

reactors with working volume of 100 mL at 35 ± 1°C and 130 rpm, and the concentration of PHE was controlled at 100 μg/L.

When 2 mL pure culture was inoculated into anaerobic reactors, the tests began and the fermentation

samples were collected per 2d to check the PHE residue Effects of Protein and Carbohydrate from WAS on PHE Biodegradation. dominant types of organic matters from WAS.

Protein and carbohydrate are the

Therefore, it was necessary to explore the influences of protein

and carbohydrate on of PHE biodegradation during WAS anaerobic acidogenic fermentation. 6


Two reactors with

Page 7 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


working volume of 100 mL were fed respectively with bovine serum albumin (BSA, model protein) and dextran (model carbohydrate), and another reactor without protein and carbohydrate was set as the control. concentration of BSA and dextran was 2 g/L.

The fermentation systems were also controlled at 35 ± 1°C and 130

rpm under the conditions of pH 10.0 and 0.3 g APG/g TSS. the content of PHE was 100 μg/L.

The

In each reactor the inoculum was 10 mL WAS and

The fermentation liquid was taken per 2d to examine the PHE residue and

nicotinamide adenine dinucleotide (NADH).

Moreover, the electric impedance of solutions in different reactors

was determined to investigate variations in electron transfer characteristics in the presence of protein. Analytical Methods.

The measurement of protein, carbohydrate, lipid, COD, TSS, VSS, surface tension and zeta

potential were conducted as previously reported.13

The extraction and purification of PHE in sludge were

conducted as described in the previous publication.27

The levels of PHE were determined by high performance

liquid chromatography (Agilent 1260).

Additionally, the electrochemical measurements were carried out

according to the previous method.27 Measurement of NADH, independent lactate dehydrogenase (iLDH) and key enzymes, such as laccase, manganese-dependent peroxidase (MnP), 1,2-naphthoyl-coenzyme A (NCoA) reductase and benzoyl-coenzyme A (BCoA) reductase, were carried out according to the procedures in the SI.

The quantities of genes related to PHE

biodegradation (PAH-ring hydroxylating dioxygenase encoding genes (PAH-RHD), PAH-ring hydroxylating dioxygenase encoding genes from Gram-positive (GP-PAH) and -negative bacteria (GN-PAH) and nidA3B3) were measured as described in the SI.

The microbial community related to PAH biodegradation, stable carbon isotope

ratios by gas chromatography isotope ratio mass spectrometry (GC-IRMS) and PHE metabolites by GC-MS were also conducted as described in the SI.

All the fermentation experiments were conducted in triplicate and an

analysis of variance (ANOVA) at 0.05 level was used to test the significance of results.

7



RESULTS AND DISCUSSION Anaerobic Biodegradation of PHE in WAS under Acidogenic Conditions.

Operational factors, such as pH

and temperature play key roles in organic pollutants biodegradation in the environment. biodegradation was remarkably affected by the fermentation pH.

In the current study, PHE

As shown in Figure 1(A), the PHE

biodegradation efficiency was only 17.6% in the control (pH 6.5) while it increased to 20.6%, 26.7%, 36.0% and 47.5% as pH increased from 7.0 to 10.0.

However, the PHE biodegradation efficiency was reduced to 43.6%

when the fermentation pH further increased to 11.0.

Normally, lots of hydroxyl groups can be generated at pH

11.0, which exert the negative influences on microorganisms and inhibit the metabolism of pollutants.28

In

general, alkaline fermentation pH, especially pH 10.0, was advantageous to anaerobic biodegradation of PHE in WAS. 60

90

A

B

50

80

Degradation efficiency (%)

Degradation efficiency (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 22

40

30

20

10

0

70

60

50

40 Control

7.0

8.0

9.0

10.0

11.0

0

0.05

pH

0.1

0.2

0.3

0.4

APG dosage (g/g TSS)

Figure 1. PHE biodegradation within 8 d at different pHs and APG dosage. Phenanthrene biodegradation was further improved in the presence of APG at pH 10.0 (Figure 1 (B)). maximum biodegradation efficiency increased to 78.2% with 0.3 g APG/g TSS. APG reduced PHE biodegradation to 71.9% with 0.4 g APG/g TSS.

The

However, excessive levels of

Therefore, the combined effects of alkaline

pH and biosurfactant on PHE biodegradation were observed under acidogenic conditions during WAS anaerobic fermentation. PHE Biomineralization during WAS Acidogenic Fermentation.

As shown in Figure S2, the chemical effect of

8


Page 9 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


alkaline pH on PHE degradation can be excluded as negligible changes in PHE concentration were observed in the abiotic tests at different pHs.

Therefore, the efficient removal of PHE was primarily a result of biological effects.

During the biodegradation process, PHE mineralization is vital to the biodegradation process, causing the pollutant to become detoxicated and give less risk to the environment.

In this study, the biomineralization of PHE during

WAS acidogenic fermentation at pH 10.0 and 0.3 g APG /g TSS was traced and monitored by using the stable isotope probing technique.

As shown in Figure 2, the δ13C in the PHE-labeled reactors decreased from -0.27 ‰ to

-15.72 ‰ gradually with an operation time of 8 d, while the were simultaneously produced.

13C-labeled

volatile organics and cumulative

13CO

2

However, the initial δ13C value was -23.89‰ and -24.67‰ in the control and

PHE-unlabeled reactors, respectively, and negligible changes were observed throughout the fermentation processes. These results indicated that changes in δ13C value in the PHE-labeled reactors were a result of anaerobic biodegradation of PHE and that the decrease of δ13C was closely related to mineralization of PHE.

Therefore, the

mineralization rate of PHE was calculated to be 25.1% within 8 d in the anaerobic reactors at pH 10.0 in the presence of APG.

Figure 2. Changes of δ13C values and cumulative of 13CO2 in the PHE degradation reactors. Bioavailability of PHE.

Commonly, the hydrophobic PHE was prone to adsorption and incorporation into

flocculated sludge, which blocked its transfer and uptake by microorganisms for further degradation.29 9


As shown


in Figure 3, the aqueous fraction of PHE was only 6.2% in the control.

However, the alkaline pH and APG were

beneficial for enhancement of the PHE bioavailability as large amounts of PHE were desorbed from solid-state WAS and released into the fermentation liquid.

The proportion of PHE in the aqueous solution was found to be

increased to 65.4% under the conditions of pH 10.0 and 0.3 g APG/g TSS, much higher than that in the control and consistent with PHE biodegradation.

The improved bioavailability of PHE could be attributed to the disruptive

effects on the WAS cemented structure caused by alkaline pH and APG. 100

Solid sludge

Aqueous phase

80

PHE distribution (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 22

60

40

20

0 trol Con

SS SS SS SS SS 10.0 /g T /g T /g T /g T /g T pH APG APG APG APG APG g g g g g 0.4 0.3 0.2 0.1 0.3 and and and and 10.0 10.0 10.0 10.0 pH pH pH pH

Figure 3. PHE distributions in anaerobic reactors within 2d under different fermentation conditions. As shown in Figure S3, the particle size of WAS was remarkably decreased in the fermentation reactors at pH 10.0 with APG.

Meanwhile, large amounts of protein and carbohydrate (Table S4) were released due to the

disruption of WAS, which was beneficial to the mass transfer of hydrophobic organic compounds (HOCs) out of non-aqueous phase liquids and into the surrounding aqueous phase.30

As a biosurfactant, APG can improve the

water solubility of HOCs by the reduction of interfacial tension (Figure S4); therefore, aqueous PHE was further and significantly increased in the reactors with APG. Normally, the bioremediation of PAHs-contaminated sludge consists of several physicochemical interfacial processes as well as microbiological interactions and metabolisms among the sludge-water-microorganism 10


Page 11 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


interfaces.

Under the conditions of pH 10.0 and APG, the zeta potentials in the WAS acidogenic systems

increased remarkably (Figure S4), which facilitated the microbiological interfaces of water-microorganisms and benefitted the sorption of PHE by microbial degraders.

Moreover, transmembrane transport, which is considered

as the rate-limiting process in the intracellular biodegradation of HOCs,31 might be affected by the presence of APG.

Herein, the release of LDH, which can indicate the disruption of cell membrane,28 was ten times higher at

pH 10.0 with APG than in the control, indicating that the intercellular metabolism of PHE was enhanced. It can be seen from the above discussions that the main processes involved in the biodegradation of PHE in WAS, i.e., desorption from sludge structure, transfer and sorption by bacteria and transmembrane transport into microbial cells, were all promoted under the conditions of pH 10.0 and APG.

Thus, the enhanced bioavailability

attributed a lot to the efficient biodegradation of PHE during WAS anaerobic acidogenic fermentation. Shift of Microbial Community under Acidogenic Conditions.

The PHE biodegradation was ultimately

accomplished by microbial degraders in WAS anaerobic fermentation reactors.

Thus, the abundance and activity

of microorganisms that were directly connected with PHE biodegradation were investigated.

In the present study,

the typical GN PHE degraders, such as Dechloromonas, Novosphingobium, Paracoccus, Planctomyces, Pseudomonas, and Sphingomonas, which have the capability of breaking aromatic rings,32–35 were all detected in WAS anaerobic fermentation systems. APG (Seen in Figure 4(A)).

More importantly, they exhibited much higher abundance at pH 10.0 with

Additionally, the abundance of GP PHE degraders, e.g., Rhodococcus and

Mycobacterium,36, 37 was several-fold greater than in the control.

Specifically, the total abundance of PHE-related

microbial degraders was 6.0 % in the fermentation reactor at pH 10.0 with APG, much higher than that of control (1.7%).

Thus, more PHE metabolizer under acidogenic conditions facilitated its biodegradation remarkably.

It should be noted that the WAS anaerobic fermentation reactors contained surfactant and were at alkaline pH, and the predominant microorganisms were acidogenic bacteria (e.g., acetogens sp., propionibacteria sp.), which 11



can efficiently convert organic substances into SCFAs.13, 38

As shown in Figure 4(B), Illumina Misque analysis

demonstrated that more than 50% of anaerobic species were identified as acidogenic bacteria under the conditions of pH 10.0 and APG, which was much higher than in the control.

Meanwhile, as shown in Figure 4(C), in the

presence of Proteiniphilum acetatigenes and Propionibacterium acidipropionici the concentration of PHE could be reduced from 100 to respectively 64.2 and 78.5 μg/L within 8 d, indicating that acidogenic bacteria participated and played important roles in PHE biodegradation.

The higher abundance of acidogenic bacteria was advantageous to

enhancement of PHE biodegradation. (A)

(B)

Control

pH 10.0

0.013%

0.016%

0.020%

pH 10.0

0%

0.022%

0.060%

Control

0.070%

0.085%

0.120%

0.004%

0.014%

0.039%

Acidovorax

pH 10.0 and APG

pH 10.0 & APG

Agrobacterium

Typical acidogenic bacteria Others

Allochromatium Comamonas

Dechloromonas 0.346%

2.325%

0.067%

0.099%

0.106%

0.027%

0.036%

0.037%

0.038%

0.044%

0.074%

0.077%

0.222%

2.641%

(C)

Desulfobulbus GN bacteria

Desulfovibrio Novosphingobium

100

Residual phenanthrene (μg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 22

Paracoccus 0.027%

Planctomyces 0.789%

0.762%

0.080%

0.119%

0.234%

0.132%

0.305%

0.493%

0.047%

0.097%

0.247%

0.029%

0.054%

0.105%

1.578%

Pseudomonas

Sphingomonas

Rhodococcus GP bacteria

80

60

40

Mycobacterium

Initial addition

Abiotic tests

P. acetatigenes P. acidipropionici

Figure 4. Abundance of PHE degrader (A) and acidogenic bacteria (B) in different WAS fermentation reactors; PHE biodegradation by acidogenic bacteria within 8 d (C). The dosage of APG was 0.3 g/g TSS. Microbial Enzymes and Functional Genes Related to PHE Biodegradation.

Microbial metabolism

requires the involvement of enzymes and their activities are closely related to the metabolic function.

Herein,

MnP, laccase, BCoA reductase and NCoA reductase, which have been reported to play important roles during PAHs biodegradation,14, 39–42 were investigated.

As shown in Figure 5, the extracellular enzymes of MnP and

laccase were detected in all reactors for PHE degradation and their activities were remarkably upregulated under 12


Page 13 of 22

the conditions of pH 10.0 and APG, with increases of 1.9- and 1.3-fold being observed at pH 10.0 and 2.7- and 2.0-fold at pH 10.0 with APG of relative to the control.

Similarly, the key intercellular enzymes, BCoA reductase

and NCoA reductase, also showed higher activities under the conditions of pH 10.0 and APG.

The improvement

of PHE bioavailability, which increased the concentration of bioavailable PHE for microbial degraders, could be one of the possible stimulants for the promotion of enzymes activities.

Moreover, surfactants have been

documented to have the ability of improving the contact between substrate and enzyme. 13, 43

Therefore, more

PHE in the current WAS fermentation systems was biodegraded. 300

Relative activities of enzymes (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Control pH 10.0 pH 10.0 and APG

250

200

150

100

50

MnP

laccase

BCoA

NCoA

PAH-degrading enzymes

Figure 5. Key enzymes involved in PHE biodegradation. The dosage of APG was 0.3 g/g TSS. Breakage of aromatic rings, which was regarded as the rate-limiting step of PHE degradation, was initiated by PAH-RHD.

Therefore, the RHD genes that encoded RHDase were investigated by RT-PCR.

The quantities of

PAH-RHD genes were shown to be increased in the anaerobic reactors under the conditions of pH 10.0 and APG when compared with that in the control (Table 2).

The quantities of GN-RHD genes were observed to be 5.37 ×

106 and 6.02 × 106 copies/g TSS at pH 10.0 and under the conditions of pH 10.0 and APG, much higher than that of the control, which further confirm the variation of PAH-RHD.

Evaluation of the GP-RHD genes revealed that

their quantities were much lower than those of the GN-RHD genes, which was consistent with the abundance of corresponding bacteria in the anaerobic fermentation systems.

Moreover, the enhancement of PAH-RHD genes

13



Page 14 of 22

was confirmed by the detection of increased quantities of RnidA3B3, which has been reported to be involved in RHD amplification and transcription.14, 32

The promotion of key enzymes activities and gene quantities related to

PHE biodegradation both revealed the improvement of microbial activities, resulting in the enhanced PHE biodegradation. Table 2. Gene quantities related to PHE biodegradation Gene quantities (copies/g TSS) Control pH 10.0 pH 10.0 and APG

PAH degradation related genes PAH-RHD

GN-RHD

103

105

2.79 × 1.59 × 104 2.67 × 104

GP-RHD 101

8.79 × 5.37 × 106 6.02 × 106

Effects of Organics from WAS on PHE Biodegradation.

4.42 × 2.56 × 102 3.38 × 102

1.05 × 102 2.21 × 102 2.77 × 102

As mentioned above, large amounts of protein and

carbohydrate were released during WAS anaerobic fermentation.

These organics not only enhance the solubility

of PHE, but also benefit microbial metabolism during PHE biodegradation.44 PHE was enhanced by protein and carbohydrate.

RnidA3B3

In this study, the biodegradation of

The biodegradation efficiency of PHE increased from 7.0%

(from 100 to 93.0 μg/L) in the control, to 16.0% and 10.2% in the reactors containing BSA and dextran, respectively (Figure S6).

The positive effects of organic substrates, especially protein, on the promotion of PHE

biodegradation were obvious.

Anaerobic metabolism of organics occurs through complex processes that produce

various metabolites such as succinyl-CoA, fumarate and acetyl-CoA via different metabolic routes.21, 45 metabolites were also the key reactants and intermediates in the PHE degradation pathway.

These

Hence, the higher

concentration of protein and carbohydrate as well as their intermediates under the conditions of pH 10.0 and APG could provide the co-metabolic conditions for PHE degraders to stimulate PHE biodegradation. Electron transfer is one of the most fundamental processes in the biological systems for the biodegradation of organic compounds under anaerobic conditions.46 electron transfer among bacteria.47 microorganisms were investigated.

More importantly, protein has been reported to play a role in

Therefore, the influences of protein on electron transfer among anaerobic As shown in Figure S7, the electrical impedances were reduced remarkably in 14


Page 15 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


aqueous solutions with BSA to only 75% of that of the control, indicating that protein could improve the electron transfer during PHE biodegradation.

Moreover, the content of NADH (Figure S5), which is a key intermediate

related to electron transfer during biological metabolism, was found to be increased, and further confirmed the positive role of protein on electron transfer.

Thus, the higher concentration of protein released from WAS in the

anaerobic reactors under the conditions of pH 10.0 and APG contributed greatly to PHE biodegradation by facilitating the electron transfer among anaerobic microorganisms. Proposed Metabolic Pathway of PHE under Acidogenic Conditions.

In the present study, the metabolic

products of PHE were determined by GC-MS (Figure S8), and the metabolic pathway was proposed in Figure 6. Phenanthrene was initially converted to 1,2-dimethylnaphthalene and further to methylnaphthalene.

The

methylnaphthalene was then dearomatized and reduced to 5,6,7,8-tetrahydro-1-naphthylamine, which may have been catalyzed by NCoA reductase,42, 45 and then to o-xylene.

The o-xylene was subsequently metabolized to

salicylate and phthalate, which can be further degraded into benzoate acid and phenol. intermetabolites were mineralized into CO2 with the participation of BCoA reductase.39, 45

Finally, these

It should be noted that

WAS is a mixture of various microbes, and the diversity of metabolic pathways of these microorganisms makes the routes of PHE biodegradation complicated.

In this study, the proposed metabolic pathway was an integrated

result of all PHE degraders in the anaerobic fermentation system. NH2

Phenanthrene

Methylnaphthalene

1,2-Dimethylnaphthalene

5,6,7,8-Tetrahydro-1-Naphthylamine

COOH

OH COOH

COOH

Phthalic acid

+

CO2

COOH

Benzoic Acid

Phenol

o-Xylene

OH

Salicylic acid

Figure 6. Proposed metabolic pathways of PHE under acidogenic conditions 15



Page 16 of 22

CONCLUSIONS Anaerobic biodegradation of PHE in WAS was enhanced during acidogenic fermentation. The anaerobic biodegradation efficiency of PHE with an operation time of 8 d was 78.2% in the fermentation reactor at pH 10.0 with 0.3 g APG/g TSS, and its mineralization rate was 25.1%.

Both the bioavailability of PHE and the abundance

of key PHE-degrading strains were enahnced under acidogenic conditions.

The key enzymes and functional genes

associated with PHE biodegradation were also enhanced during WAS acidogenic fermentation.

In acidogenic

fermentation systems, the released protein from WAS played important roles in PHE biodegradation.

During

WAS acidogenic fermentation, PHE was initially converted to 1,2-dimethylnaphthalene, and finally mineralized into CO2 with the participation of BCoA reductase.

ASSOCIATED CONTENT Supporting Information The following files are available free of charge: The descriptions of procedures for the measurements of LDH, NADH, enzyme activity, gene quantification, microbial community, PHE metabolites, and 13CO2 production. Table S1. Primer sequences and annealing temperatures of PCR and qPCR assays. Table S2. PCR reaction mixture. Table S3. PCR reaction procedures. Table S4. Concentration of soluble organic matters in the fermentation reactors. Figure S1. Flow-chart for the determination of PHE mineralization. Figure S2. Chemical effect of pH on phenanthrene degradation. Figure S3. Distribution of WAS particle size under different fermentation reactors. Figure S4. Effect of APG on surface tension and Zeta potential. 16


Page 17 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure S5. Relative concentration of NADH from anaerobes in the presence of BSA and dextran. Figure S6. Effects of organic matters on PHE biodegradation. Figure S7. Effects of organic matters on electrical impedance. Figure S8. Mass spectrogram of metabolites during anaerobic biodegradation of phenanthrene.

AUTHOR INFORMATION

Corresponding Author

*Corresponding author-Tel: 86-21-65981263, Fax: 86-21-65986313, E-mail: [email protected], [email protected]

Notes

The authors declare no competing financial interest.

ACKNOWLEDGEMENTS

The work is financially supported by the National Natural Science Foundation of China (51425802, 21777121 and 51708171), State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRK16003 and PCRRF17019), and Fundamental Research Funds for the Central Universities (22120180062).

REFERENCES

(1) Wang, J.; Chen, S. J.; Tian, M.; Zheng, X. B.; Gonzales, L.; Ohura, T.; Mai, B. X.; Simonich, S. L. M. Inhalation cancer risk associated with exposure to complex polycyclic aromatic hydrocarbon mixtures in an electronic waste and urban area in South China. Environ Sci Technol. 2012, 46, 9745-9752, DOI 10.1021/es302272a. (2) Nistico, R.; Franzoso, F.; Cesano, F.; Scarano, D.; Magnacca, G.; Parolo, M. E.; Carlos, L. Chitosan-derived iron oxide systems for magnetically guided and efficient water purification processes from polycyclic aromatic hydrocarbons. ACS Sustain Chem Eng. 2017, 5, 793-801, DOI 10.1021/acssuschemeng.6b02126. 17



Page 18 of 22

(3) Meng, Y.; Liu, X. H.; Lu, S. Y.; Zhang, T.T.; Jin, B. C.; Wang, Q.; Tang, Z. R.; Liu, Y.; Guo, X. C.; Zhou, J. L.; Xi, B. D. A review on occurrence and risk of polycyclic aromatic hydrocarbons (PAHs) in lakes of China. Sci Total Environ. 2019, 651, 2497-2506, DOI 10.1016/j.scitotenv.2018.10.162. (4) Larsen, S. B.; Karakashev, D.; Angelidaki, I.; Schmidt, J. E. Ex-situ bioremediation of polycyclic aromatic hydrocarbons in sewage sludge. J Hazard Mater. 2009, 164, 1568-1572, DOI 10.1016/j.jhazmat.2008.08.067. (5) Meng, X. Z.; Venkatesan, A. K.; Ni, Y. L.; Steele, J. C.; Wu, L. L.; Bignert, A.; Bergman, Å.; Halden, R. U. Organic contaminants in Chinese sewage sludge: a meta-analysis of the literature of the past 30 Years. Environ Sci Technol. 2016, 50, 5454-5466, DOI 10.1021/acs.est.5b05583. (6) Stevens, J. L.; Northcott, G. L.; Stern, G. A.; Tomy, G. T.; Jones, K. C.; PAHs; PCBs; PCNs. Organochlorine pesticides, synthetic musks, and polychlorinated n-alkanes in UK sewage sludge: survey results and implications. Environ Sci Technol. 2003, 37, 462-467, DOI 10.1021/es020161y. (7) Cai, Q. Y.; Mo, C. H.; Wu, Q. T.; Zeng, Q. Y.; Katsoyiannis, A.; Férard, J. F. Bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated sewage sludge by different composting processes. J Hazard Mater. 2007, 142, 535-542, DOI 10.1016/j.jhazmat.2006.08.062. (8) Venosa, A. D.; Campo, P.; Suidan, M. T. Biodegradability of lingering crude oil 19 Years after the Exxon Valdez Oil Spill. Environ Sci Technol. 2010, 44, 7613-7621, DOI 10.1021/es101042h. (9) Mahmoudi, N.; Porter, T. M.; Zimmerman, A. R.; Fulthorpe, R. R.; Kasozi, G. N.; Silliman, B. R.; Slater, G. F. Rapid degradation of deepwater horizon spilled oil by indigenous microbial communities in Louisiana Saltmarsh sediments. Environ Sci Technol. 2013, 47, 13303-13312, DOI 10.1021/es4036072. (10) Peng, R. H.; Xiong, A. S.; Xue, Y.; Fu, X. Y.; Gao, F.; Zhao, W.; Tian, Y. S.; Yao, Q. H. Microbial biodegradation of polyaromatic hydrocarbons. FEMS Microbiol Rev. 2008, 32, 927-955, DOI 10.1111/j.1574-6976.2008.00127.x. (11) Hu, J. W.; Zhao, J. W.; Wang, D. B.; Li, X. M.; Zhang, D.; Xu, Q. X.; Peng, L.; Yang, Q.; Zeng, G. M. Effect of diclofenac on the production of volatile fatty acids from anaerobic fermentation of waste activated sludge. Bioresource Technol. 2018, 254, 7-15, DOI 10.1016/j.biortech.2018.01.059. (12) Yang, J. N.; Liu, X. R.; Wang, D. B.; Xu, Q. X.; Yang, Q.; Zeng, G. M.; Li, X. M.; Liu, Y. W.; Gong, J. L.; Ye, J.; Li, H. L. Mechanisms of peroxymonosulfate pretreatment enhancing production of short-chain fatty acids from waste activated sludge. Water Res. 2019, 105, 239-249, DOI 10.1016/j.watres.2018.10.060. (13) Luo, J. Y.; Feng, L. Y.; Chen, Y. G.; Sun, H.; Shen, Q. T.; Li, X.; Chen, H. Alkyl polyglucose enhancing propionic acid enriched short-chain fatty acids production during anaerobic treatment of waste activated sludge and mechanisms. Water Res. 2015, 73, 332-341, DOI 10.1016/j.watres.2015.01.041. (14) Luo, J. Y.; Wu, L, J; Chen, Y. G.; Feng, L. Y.; Cao, J. S. Integrated approach to enhance the anaerobic biodegradation of benz[α]anthracene: A high-molecule-weight polycyclic aromatic hydrocarbon in sludge by simultaneously improving the bioavailability and microbial activity. J Hazard Mater. 2019, 365, 322-330, DOI 10.1016/j.jhazmat.2018.11.012. 18


Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15) Reichenberg, F.; Karlson, U. G.; Gustafsson, O.; Long, S. M.; Pritchard, P. H.; Mayer, P. Low accessibility and chemical activity of PAHs restrict bioremediation and risk of exposure in a manufactured gas plant soil. Environ Pollut. 2010, 158, 1214-1220, DOI 10.1016/j.envpol.2010.01.031. (16) Bueno-Montes, M.; Springael, D.; Ortega-Calvo, J. J. Effect of a nonionic surfactant on biodegradation of slowly desorbing PAHs in contaminated soils. Environ Sci Technol. 2011, 45, 3019-3026, DOI 10.1021/es1035706. (17) Oh, J. Y.; Choi, S. D.; Kwon, H. O.; Lee, S. E. Leaching of polycyclic aromatic hydrocarbons (PAHs) from industrial

wastewater

sludge

by

ultrasonic

treatment.

Ultrason

Sonochem.

2016,

33,

61-66,

DOI

10.1016/j.ultsonch.2016.04.027. (18) Bacosa, H. P.; Inoue, C. Polycyclic aromatic hydrocarbons (PAHs) biodegradation potential and diversity of microbial consortia enriched from tsunami sediments in Miyagi, Japan. J Hazard Mater. 2015, 283, 689-697, DOI 10.1016/j.jhazmat.2014.09.068. (19) Ma, J.; Xu, L.; Jia, L. Y. Characterization of pyrene degradation by Pseudomonas sp strain Jpyr-1 isolated from active sewage sludge. Bioresource Technol. 2013, 140, 15-21, DOI 10.1016/j.biortech.2013.03.184. (20) Gupta, S.; Pathak, B.; Fulekar, M. H. Molecular approaches for biodegradation of polycyclic aromatic hydrocarbon compounds: a review. Rev Environ Sci Biotechnol. 2015, 14, 241-269, DOI 10.1007/s11157-014-9353-3 (21) Meckenstock, R. U.; Mouttaki, H. Anaerobic degradation of non-substituted aromatic hydrocarbons. Curr Opin Biotechnol. 2011, 22, 406-414, DOI 10.1016/j.copbio.2011.02.009. (22) Nzila, A. Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons under anaerobic conditions: overview of studies, proposed pathways and future perspectives. Environ Pollut. 2018, 239, 788-802, DOI 10.1016/j.envpol.2018.04.074. (23) Zhang, C.; Qin, Y. G.; Xu, Q. X.; Liu, X. R.; Liu, Y. W.; Ni, B. J.; Yang, Q.; Wang, D. B.; Li, X. M.; Wang, Q. L. Free ammonia-based pretreatment promotes short-chain fatty acid production from waste activated sludge. ACS Sustain Chem Eng. 2018, 6, 9120-9129, DOI 10.1021/acssuschemeng.8b01452. (24) Duan, X.; Wang, X.; Xie, J.; Feng, L. Y.; Yan, Y. Y.; Zhou, Q. Effect of nonylphenol on volatile fatty acids accumulation during anaerobic fermentation of waste activated sludge. Water Res. 2016, 105, 209-217, DOI 10.1016/j.watres.2016.08.062. (25) Zhao, J. W.; Liu, Y. W.; Wang, Y. L.; Lian, Y.; Wang, Q. L.; Yang, Q.; Wang, D. B.; Xie, G. J.; Zeng, G. M.; Sun, Y. J.; Li, X. M.; Ni, B. J. Clarifying the role of free ammonia in the production of short-chain fatty acids from waste activated

sludge

anaerobic

fermentation.

ACS

Sustain

Chem

Eng.

2018,

6,

14104-14113,

DOI

10.1021/acssuschemeng.8b02670. (26) Duan, X.; Wang, X.; Xie, J.; Feng, L. Y.; Yan, Y. Y.; Wang, F.; Zhou, Q. Acidogenic bacteria assisted biodegradation of nonylphenol in waste activated sludge during anaerobic fermentation for short-chain fatty acids production. Bioresource Technol. 2018, 268, 692-699, DOI 10.1016/j.biortech.2018.08.053. (27) Luo, J. Y.; Chen, Y. G.; Feng, L. Y. Polycyclic aromatic hydrocarbon affects acetic acid production during 19



Page 20 of 22

anaerobic fermentation of waste activated sludge by altering activity and viability of acetogen. Environ Sci Technol. 2016, 50, 6921-6929, DOI 10.1021/acs.est.6b00003. (28) Xiao, B.; Liu, C.; Liu, J.; Guo, X. Evaluation of the microbial cell structure damages in alkaline pretreatment of waste activated sludge. Bioresource Technol. 2015, 196, 109-115, DOI 10.1016/j.biortech.2015.07.056. (29) Barret, M.; Carrere, H.; Delgadillo, L.; Patureau, D. PAH fate during the anaerobic digestion of contaminated sludge: Do bioavailability and/or cometabolism limit their biodegradation?. Water Res. 2010, 44, 3797-3806, DOI 10.1016/j.watres.2010.04.031. (30) Smith, K. E. C.; Thullner, M.; Wick, L. Y.; Harms, H. Dissolved organic carbon enhances the mass transfer of hydrophobic organic compounds from nonaqueous phase liquids (NAPLs) into the aqueous phase. Environ Sci Technol. 2011, 45, 8741-8747, DOI 10.1021/es202983k. (31) Li, F.; Zhu, L. Z.; Wang, L. W.; Zhan, Y. Gene expression of an arthrobacter in surfactant-enhanced biodegradation of a hydrophobic organic compound. Environ Sci Technol. 2015, 49, 3698-3704, DOI 10.1021/es504673Kj. (32) Hilyard, E. J.; Jones-Meehan, J. M.; Spargo, B. J.; Hill, R. T. Enrichment, isolation, and phylogenetic identification of polycyclic aromatic hydrocarbon-degrading bacteria from Elizabeth River sediments. Appl Environ Microb. 2008, 74, 1176-1182, DOI 10.1128/AEM.01518-07. (33) Teng, Y.; Luo, Y. M.; Sun, M. M.; Liu, Z. J.; Li, Z. G.; Christie, P. Effect of bioaugmentation by Paracoccus sp. strain HPD-2 on the soil microbial community and removal of polycyclic aromatic hydrocarbons from an aged contaminated soil. Bioresource Technol. 2010, 101, 3437-3443, DOI 10.1016/j.biortech.2009.12.088. (34) Salinero, K. K.; Keller, K,; Feil W. S.; Feil H.; Trong S.; Di Bartolo, G.; Lapidus, A. Metabolic analysis of the soil

microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation. BMC Genomics 2009, 10, 351, DOI 10.1186/1471-2164-10-351. (35) Ma, C.; Wang, Y. Q.; Zhuang, L.; Huang, D. Y.; Zhou, S. G.; Li, F. B. Anaerobic degradation of phenanthrene by a newly isolated humus-reducing bacterium, Pseudomonas aeruginosa strain PAH-1. J Soil Sediment. 2011, 11, 923-929, DOI 10.1007/s11368-011-0368-x. (36) Moody, J. D.; Freeman, J. P.; Doerge, D. R.; Cerniglia, C. E. Degradation of phenanthrene and anthracene by cell suspensions

of

Mycobacterium

sp

strain

PYR-1.

Appl

Environ

Microb.

2001,

67,

1476-1483,

DOI

10.1128/AEM.67.4.1476-1483.2001. (37) Leneva, N. A.; Kolomytseva, M. P.; Baskunov, B. P.; Golovleva, L. A. Phenanthrene and anthracene degradation by microorganisms

of

the

genus

Rhodococcus.

Appl

Biochem

Micro+.

2009,

45,

169-175,

DOI

10.1134/S0003683809020094. (38) Zheng, X.; Su, Y.; Li, X.; Xiao, N.; Wang, D.; Chen, Y. Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production. Environ Sci Technol. 2013, 47, 4262-4268, DOI 10.1021/es400210v. (39) Boll, M.; Fuchs, G. Benzoyl‐coenzyme a reductase (dearomatizing), a key enzyme of anaerobic aromatic 20


Page 21 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


metabolism-ATP dependence of the reaction, purification and some properties of the enzyme from Thauera aromatica strain K172. Eur J Biochem. 1995, 234, 921-933, DOI 10.1111/j.1432-1033.1995.921_a.x. (40) Collins, P. J.; Kotterman, M. J. J.; Field, J. A.; Dobson, A. D. W. Oxidation of anthracene and benzo[a]pyrene by laccases from Trametes versicolor. Appl Environ Microb. 1996, 62, 4563-4567, DOI 10.1016/j.enzmictec.2004.04.007, DOI doi:10.1089/oli.1.1996.6.305. (41) Eibes, G.; Moreira, M. T.; Feijoo, G.; Lema, J. M. Enzymatic degradation of low soluble compounds in monophasic water: solvent reactors. Kinetics and modeling of anthracene degradation by MnP. Biotechnol Bioeng. 2008, 100, 619-626, DOI 10.1002/bit.21806. (42) Eberlein, C.; Estelmann, S.; Seifert, J.; von Bergen, M.; Müller, M.; Meckenstock, R. U.; Boll, M. Identification and characterization of 2‐naphthoyl‐coenzyme A reductase, the prototype of a novel class of dearomatizing reductases. Mol Microbiol. 2013, 88, 1032-1039, DOI 10.1111/mmi.12238. (43) Cheng, J. R.; Yu, Y.; Zhu, M. J. Enhanced biodegradation of sugarcane bagasse by Clostridium thermocellum with surfactant addition. Green Chem. 2014, 16, 2689-2695, DOI 10.1039/c3gc42494d. (44) Johnsen, A. R.; Wick, L. Y.; Harms, H. Principles of microbial PAH-degradation in soil. Environ Pollut. 2005, 133, 71-84, DOI 10.1016/j.envpol.2004.04.015. (45) Kleemann, R.; Meckenstock, R. U. Anaerobic naphthalene degradation by Gram-positive, iron-reducing bacteria. FEMS Microbiol Ecol. 2011, 78, 488-496, DOI 10.1111/j.1574-6941.2011.01193.x. (46) Stams, A. J.; Plugge, C. M. Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nat Rev Microbiol. 2009, 7, 568-577, DOI 10.1038/nrmicro2166. (47) Tobin, P. H.; Wilson, C. J. Examining photoinduced energy transfer in Pseudomonas aeruginosa Azurin. J Am Chem Soc. 2014, 136, 1793-1802, DOI 10.1021/ja412308r.

21



TOC Phospholipid

Organic substrates

APG

PHE

Enzyme

WAS

WAS transmembrane transport CO2, H2O

Structure disruption

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 22

Intercellular metabolism

sorption

desorption

Fermentation solution

Synopsis The fate of emerging contaminants (e.g., polycyclic aromatic hydrocarbons) in sludge during anaerobic fermentation directly determines the sustainable engineering application of fermentative products (e.g., SCFAs).

22


Acidogenic fermentation facilitates anaerobic biodegradation of

Recommend Documents