Accepted Manuscript

Caffeine degradation by methanogenesis: efficiency in anaerobic membrane bi-

oreactor and analysis of kinetic behavior

Rong Chen, Hongyu Jiang, Yu-You Li

PII: S1385-8947(17)31758-8
DOI: https://doi.org/10.1016/j.cej.2017.10.052
Reference: CEJ 17836

To appear in: Chemical Engineering Journal

Received Date: 17 August 2017

Revised Date: 6 October 2017
Accepted Date: 11 October 2017

Please cite this article as: R. Chen, H. Jiang, Y-Y. Li, Caffeine degradation by methanogenesis: efficiency in
anaerobic membrane bioreactor and analysis of kinetic behavior, Chemical Engineering Journal (2017), doi: https://
doi.org/10.1016/j.cej.2017.10.052

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Caffeine degradation by methanogenesis: efficiency in anaerobic membrane

bioreactor and analysis of kinetic behavior

Rong Chen a,b, Hongyu Jiang c, Yu-You Li b,d,*

a
International S&T Cooperation Center for Urban Alternative Water Resources

Development, Key Lab of Environmental Engineering, Shaanxi Province, Xi'an

University of Architecture and Technology, No.13 Yanta Road, Xi'an 710055, PR

China.
b
Department of Civil and Environmental Engineering, Graduate School of

Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku, Sendai,

Miyagi 980-8579, Japan.

c
Department of Fermentation Science, Faculty of Applied Bio-Science, Tokyo

University of Agriculture, 1-1 Sakuragaoka 1-chome, Setagaya-ku, Tokyo 156-8502,

Japan.
d
Graduate School of Environmental Studies, Tohoku University, 6-6-06 Aza-Aoba,

Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, Japan.

Corresponding author

*Yu-You Li, gyokuyu.ri.a5@tohoku.ac.jp

Telephone: +81 22 795 7464

Fax: +81 22 795 7465

1
ABSTRACT

Caffeine, a widely found component in food and pharmaceuticals, is harmful to

ecosystem when discharged into the environment because of its non-eco-friendly

nature and recalcitrant properties. Complete caffeine degradation by methanogenesis

was successful for the first time in a man-made bioreactor. We confirmed the success

through continuously operating a bioreactor, testing the methane potential and

determining the kinetic behaviour. In the continuous operation, an 87.5±5.3% removal

efficiency of caffeine was observed using an anaerobic membrane bioreactor for the

digestion of coffee processing wastewater in the long-term process. In methane

potential tests with caffeine as the sole substrate, the complete conversion from

caffeine to CH4 was achieved in a caffeine-rich environment with a caffeine

concentration up to 2000 mg/L. We have proposed and confirmed a reaction equation

for the caffeine degradation by methanogenesis based on the stoichiometric

relationship between the degraded caffeine and the increased end-products: CH4,

NH4+-N and CO2. In the kinetic behaviour experiment, the regular monitoring the

residual caffeine and the end-products indicated that methanogenic degradation of

caffeine clearly includes two major steps: caffeine converting to intermediates and the

intermediates converting to CH4. The major accumulated intermediates should be

hydrolysis products and the process of hydrolysis products converting to volatile fatty

acids was identified to be the rate-limiting step of caffeine degradation. This work

provided a new thought to microbial caffeine degradation and will be useful in

developing innovative technology for bioenergy harvest at the same time of

decaffeination.

Keywords: Caffeine degradation; Methanogenesis; Anaerobic membrane bioreactor;

Stoichiometry; Kinetic behaviour.

2
Abbreviations: AnMBR, anaerobic membrane bioreactor; TS, total solids; COD, chemical oxygen

demand; HRT, hydraulic retention time; SRT, solid retention time; BMP, biochemical methane

potential; CMP, cumulative methane production; TGA, thermogravimetric analysis; TG-DSC,

thermogravimetric-differential scanning calorimetry; NH4+-N, ammonia nitrogen; VFAs, volatile

fatty acids; VSS, volatile suspended solid.

1. Introduction

Caffeine (1,3,7-trimethylxantine, C8H10N4O2), is a naturally occurring alkaloid

present in many plant species including coffee seeds, citrus fruits, olive oil, tea leaves,

and cocoa beans [1]. As a dietary component widely found in common food and

beverages, the caffeine content of coffee and tea is especially high. Coffee has a

caffeine content ranging from 0.43 to 0.82 mg/mL [2]. Due to its stimulatory nature,

caffeine is also used as a stimulant or adjuvant in psychoactive medicine [3].

According to recent studies, the antioxidant properties of caffeine have a preventive

effect against health disorders associated with oxygenated free radicals [4, 5]. Because

of its wide use in food and pharmaceutical industries, caffeine enters the environment

via liquid effluents and solid waste from processing facilities and domestic waste. The

harmful effects of caffeine when discharged into the environment have been well-

documented. The ingestion of caffeine and its chlorinated by-products has adverse

effect on central nervous system and digestive system of animals [6]. The presence of

caffeine in soil can also affect soil fertility as it inhibits seed germination and growth

of seedlings [7]. Because it is so frequently observed in the aquatic environment,

including wastewater, surface water and ground water [8-10], caffeine can be an

anthropogenic marker for contaminated source water [11] and have been listed as an

emerging organic contaminant [12-14]. It is therefore reasonable to proclaim that

decaffeination is an important matter for health as well as for the general

3
environment.

Decaffeination is typically a physicochemical extraction technique. The solvent

extraction method has to meet stringent environmental restrictions with regard to

disposing of the waste solvent, and the supercritical fluid extraction method is

associated with a high energy demand [2, 15]. Research on microbial decaffeination

in the recent past has consistently presented metabolic challenges because of the

recalcitrant properties of caffeine. This recalcitrance is the most important barrier

impeding the development of low-cost technology via microbial decaffeination. In

addition, several studies included findings that caffeine inhibits the growth of many

bacterial species [16, 17]. Up to now, the process of caffeine degradation has been

reported mostly in a naturally aerobic environment [2, 18]. Some bacterial strains

belonging to genera Serratia and Pseudomonas, and fungal strains belonging to

Aspergillus, Penicillium [19, 20], Stemphylium, Rhizopus and Phanerochaete [21, 22]

have been reported to be able to degrade caffeine, by using caffeine as a source of

nutrient nitrogen. Limited studies related to caffeine degradation in an anaerobic

environment and caffeine-degrading anaerobes have been reported to date. Bradley

studied the potential for in situ biotransformation of caffeine by the sediment in three

rivers which received the effluent from WWTPs in USA, by using 14C-caffeine as

substrate. The results revealed most of the 14C-radioactivity was recovered in 14CO2,

and a small amount was recovered in 14CH4, when the initial caffeine concentrations

was approximately 0.25 μmol/L [23]. A study on the effect of caffeine on anaerobic

digestion of food waste reported low dose caffeine of 100 mg/L can enhanced the

biogas production [24]. Quite limited efficiencies of anaerobic caffeine degradation

were obtained through small-scale batch tests in a few studies [2, 25, 26]. No reports

are available on high-efficient caffeine degradation by long-term anaerobic process in

4
a caffeine-rich environment like coffee processing waste.

Recently, several researchers have reported the successful production of biogas

by implementing the methanogenic degradation of coffee spent, pulp and ground [27,

28]. Because these coffee processing waste/wastewater are reportedly rich in

carbohydrate and protein, anaerobic digestion was considered suitable for bioenergy

production. The behaviour of caffeine in these processes was largely not considered in

those studies: in only a few studies was the enhanced reduction of caffeine as a result

of the pre-anaerobic fermentation of coffee, or the time required for this process,

mentioned [21, 29]. In our experiments, we succeeded in complete caffeine

degradation by methanogenesis in a man-made bioreactor. The performance was

considered by implementing the continuous operation of a bioreactor, carrying out

methane potential batch tests, and determining the kinetic behaviour of the caffeine

degradation. (1) In the continuous operation, an anaerobic membrane bioreactor

(AnMBR) was configured for the methanogenic degradation of coffee processing

wastewater. The removal efficiency was determined by measuring the caffeine

concentration in the influent and effluent. (2) Then the batch tests, using caffeine as

the sole substrate, were carried out to explore the methane potential of caffeine. (3)

Finally, a kinetic experiment was developed to unveil the process characteristics

during caffeine degradation by methanogenesis.

2. Materials and methods

2.1 AnMBR continuous operation

AnMBR system (Fig. 1) consisted of a 6-L completely mixed submerged

AnMBR with a substrate tank and a soluble tank. The flat sheet membrane module

was made of chlorinated polyethylene with a nominal pore size of 0.2 μm, and a total

5
area of 0.116 m2 (Kubota Membrane Cartridge, Japan). Three peristaltic pumps

(Model 7518-10, Cole-Parmer, USA) were individually used to transfer raw substrate

to the soluble tank, feed dissolved substrate into the AnMBR and withdraw permeate

from the membrane. The permeate pump automatically switched on for 5 min and off

for 55 min every one hour to carry out intermittent filtration cycle for the membrane.

The biogas produced was recycled by a constant-speed diaphragm pump (APN-085

LV-1, Iwaki, Japan) to scour the membrane surface for fouling control via a gas

diffuser located below the membrane sheet, with a specific gas flow rate of 2.6 m3/m2

h. A digital pressure meter (AP-V85, Keyence, Japan) was installed between the

membrane module and the permeate pump to record trans-membrane pressure (TMP).

When TMP increased to 30 kPa, the membrane was withdrawn from the reactor for

cleaning to recover its permeability. The cleaning process was detailed in a previous

study [30]. The AnMBR was operated successively under five hydraulic retention

times (HRTs) including 50, 30, 15, 10 and 5 days, resulting in average membrane flux

of 43.1, 71.8, 144, 216, 431 mL/m2·h respectively. Biogas production was measured

according to the volume of biogas collected in a wet tip gas meter (W-NK-0.5B,

Shinagawa, Japan). The operating temperature was kept at 55 °C by means of a water

bath. The seed sludge was from a food waste treatment plant. Excess sludge was

discharged periodically to maintain an average TS concentration of 50 g/L inside the

AnMBR.

The co-substrate fed to the AnMBR was made of coffee processing wastewater,

milk processing wastewater and waste activated sludge with wet weight ratio of

31.8%, 65.4% and 2.8%, respectively. All the raw materials were provided by Tokyo

Gas Co Ltd. Their elementary composition involving the organic elements of C, N, O,

H and P was measured, and then the ratio of each material in the co-substrate was

6
determined with a purpose to maintain an appropriate carbon-to-nitrogen ratio for the

anaerobic digestion [31, 32]. These materials were mixed using a high speed blender

and stored in a substrate tank at 4 oC before use. The characteristics of the co-

substrate were shown in Table 1.

2.2 Batch tests for methane potential of caffeine

In order to explore the methane potential of caffeine, biochemical methane

potential (BMP) tests were implemented using caffeine as the sole substrate for

methane production. BMP tests were carried out in 120 mL serum bottles placed and

incubated in a shaking bath at 125 rpm at 55 °C. A volume of 60 mL sludge taken

from the AnMBR was inoculated. Caffeine powder (Anhydrous caffeine, Wako,

Japan, ≥98.5% purity) was added, resulting in inoculated caffeine concentrations of

500, 1000, 2000, 4000, 8000 and 16000 mg/L in the bottles. The headspace of the

bottle was purged with nitrogen gas for 2 min. When the bottle reached the set

temperature in the water bath, the headspace was vented using a syringe to release the

pressure caused by the thermal expansion. All the analysis was conducted in two

replicates. A blank control without any caffeine feeding was set to provide a reference

for determining the net production of biogas. Biogas production and composition

(percentage of CH4, CO2 and N2) were measured at regular intervals and the

cumulative methane production (CMP) was calculated after every measurement.

When the CMP remained constant for several days, the BMP tests were terminated.

Afterwards, a sludge sample was dried in an oven (DX602, Yamato, Japan) at 105 oC

and then a thermogravimetric analysis (TGA) was carried out to determine the amount

of residual caffeine in the solid. The concentration of ammonia nitrogen (NH4+-N)

was measured immediately following the inoculation of caffeine and when the BMP

tests were terminated.

7
 The efficiencies of caffeine converting to CH4 were calculated by the following

Equation:

affeine on ersion (1)

in- affeine

where CODCMP is the normalized chemical oxygen demand (COD) amount of the

CMP (gCOD), and CODin-caffeine is the normalized COD amount of the inoculated

caffeine (gCOD). The equivalent COD amount of CH4 and caffeine were determined

by the Table S1.

Because the modified Gompertz equation [33] is the most suitable model to

describe the relationship between bacterial growth and metabolic production, it was

used to simulate the results of BMP tests to summarize the performance of

methanogenesis for various inoculated caffeine concentrations.

e
e p -e p -t (2)

where M is caffeine conversion (%), P is the methane conversion potential (%), R is

the methane conversion rate (%/d), is the lag-phase time (days) and e is the

e ponential . The , and were estimated by a non-linear fitting program using

Origin Software.

2.3 Batch experiment for analysis of kinetic behavior

The maximum concentration tolerated for the complete degradation of caffeine

was 2000 mg/L according to the BMP tests. The sludge in the bottles under this

concentration was collected for a kinetics experiment. The kinetics experiment

followed the same process of aforementioned BMP tests. Differently, a 200 mL serum

bottle was used with the total liquid volume ultimately set at 150 mL by adding

deionized water after the sludge and caffeine powder inoculation. A concentration of

2000 mg caffeine/L was obtained by inoculating with 100 mL sludge and a certain

8
amount of caffeine powder. For analysis, a volume of 1 mL liquid was sampled after

every measurement of biogas production and composition. Then the sample was

centrifuged at 15000 rpm at 4 oC for 15 min. The supernatant was collected for

measuring the concentration of NH4+-N, volatile fatty acids (VFAs) and residual

caffeine. Immediately after the inoculation, the concentration of volatile suspended

solid (VSS) in the liquid was measured as per Standard Methods [34]. All the analysis

was conducted in two replicates.

2.4 Analytic methods

The biogas composition including CH4, CO2 and N2 was measured using a gas

chromatograph (Shimadzu, GC-8A, Japan) equipped with a thermal conductivity

detector. All the measurements were normalized to the standard state (0oC, 1atm). The

caffeine concentration was analysed by a capillary electrophoresis (Agilent 7100,

Agilent Technologies, USA). A borax (20mM Na2 B4O7·10H2O, ≥99 purity)

solution made according to the manufacturer's instructions was used as running

buffer. The NH4+-N concentration was also measured by the capillary electrophoresis

using a running buffer (10mM C3H4N2, 5mM (CH3)2C(OH)COOH, 2mM C12H24O6

and 0.2w% CH3COOH, ≥99 purity). All the chemicals were from Wako, Japan.

TGA was implemented for the dried sludge samples using a thermogravimetric-

differential scanning calorimetry (TG-DSC) analyzer (STA449F3, Netzsch,

Germany). The sample was first heated to 105 oC to evaporate any remaining water

and the temperature was then increased with increments of 5 oC per minute till 600 oC

under a nitrogen atmosphere. The mass weight was continuously measured. TGA was

also implemented on a pure caffeine sample to provide a reference for estimating

caffeine accumulation in the dried sludge.

The pH, alkalinity, COD, TS and VS were measured according to the Standard

9
Methods [34]. The content of VFAs, which included valeric acid, butyric acid,

propionic acid, acetic acid, ethanol and methanol, were assayed by a gas

chromatograph (Agilent-6890, Agilent Technologies, USA). The protein content was

determined by the Lowry method [35], the carbohydrate was analyzed by the phenol-

sulfuric acid method [36], and the content of lipid by the chloroform-methanol

extraction method [37].

For mass balance, COD was used as a unified indicator of all the related organic

matter including caffeine, VFAs and CH4, in which their equivalent COD amount was

determined by the Table S1. The results provided a theoretical relationship among

caffeine, CH4 and COD, which can be expressed as: 1 g caffeine=1.07 gCOD, 1 g

COD=0.35 L CH4 and 1 g caffeine=0.37 L CH4. The statistical significance of the

replicates, including those in the batch tests for methane potential and batch

experiment for analysis of kinetic behaviour, was performed using SPSS version 20.0

(IBM Corp., USA) to exam the standard deviations.

3. Results and discussion

3.1 Digestion performance and caffeine removal in the AnMBR

The continuous digestion performance of the AnMBR on the co-substrate under

different HRTs is summarized in Table S2. A higher biogas production rate was

observed with shorter HRTs. High TS and COD removal efficiencies (>90%) were

achieved under all the HRTs. Regarding the three main organic components in the co-

substrate, carbohydrate presented the highest removal efficiency at 98±1%, followed

by lipid (96±4%) and protein (86±3%). The results clearly indicate the desirable

digestion performance of the AnMBR in terms of organic removal and biogas

production when treating the co-substrate. It seems the presence of caffeine imposed

10
almost no inhibition effect on the digestion performance.

The caffeine removal efficiencies during the continuous operation are shown in

Fig. 2. The average caffeine concentration in the co-substrate was 470 mg/L. Around

40 days later since the inoculation, which was deemed to be a acclimation period of

seed sludge to the co-substrate, the concentration of caffeine in the effluent was down

to average 58.7 mg/L and maintained in a range of 32.3 to 78.3 mg/L from the period

of HRT 30d, resulting in a stable and high removal efficiency of 87.5±5.3%.

Moreover, it reached 92.8±0.1% at HRT 5d, and this phenomenon should be derived

from the results of long-term acclimation of caffeine-degrading bacteria. However,

the observed high removal efficiency during the continuous operation was not enough

to verify the high degrading efficiency of caffeine. To confirm this, we investigated

the methanogenic potential of caffeine through BMP tests, using caffeine as the sole

substrate.

3.2 Methane potential from caffeine degradation

The efficiencies of caffeine converting to CH4 were determined by Equation 1

based on the data of the BMP tests, and Fig. 3 shows the results. Because less CH4

production, compared to the blank control sample without caffeine feeding, were

collected under 4000, 8000 and 16000 mg/L, the caffeine conversion efficiencies was

deemed to be negative in those three concentrations. It can be concluded that the

complete conversion (100%) from caffeine to CH4 was obtained in a caffeine-rich

environment with an inoculated caffeine concentration up to 2000 mg/L. The

complete conversion for concentrations of 500, 1000 and 2000 mg caffeine/L required

2.7, 9.1 and 16.8 days respectively. The simulated curves using Equation 2 are also

shown in Fig. 3, regarding the three complete-conversion concentrations of 500,

1000 and 2000 mg/L. The simulated parameters involving the lag-phase time

11
( ), conversion rate (R) and conversion potential (P) are shown in Table 2. An

increase in the caffeine concentration clearly resulted in a significant extension

of . The optimum was obser ed under 500 mg affeine/L on entration, but

the inhibition effect on the R was not obvious when the caffeine concentration

was increased to 2000 mg /L compared to that under 1000 mg /L. The results of

and may suggest that when the affeine on entration is less than 2000

mg/L, the major inhibition effect caused by increased caffeine concentration

mainly lies in the lag-phase time of methane generation rather than in the

reaction rate.

In contrast, when the inoculated caffeine concentration was higher than 4000

mg/L, methanogenesis was inhibited, and a higher caffeine concentration resulted in

greater inhibition effect. The inhibition of caffeine was deemed to be anti-bacterial

effect [38]. However, this effect depends mainly on the caffeine concentration. A

kinetic analysis of cell growth indicated the strain (Pseudomonas sp. GSC 1182)

exhibited shorter lag periods until the caffeine concentration was 2500 mg/L and

longer lag phase was observed at higher concentrations of caffeine. When the initial

caffeine concentration was above 4000 mg/L, considerable decrease in the biomass

was observed [39]. Our findings conform to the previous study very well.

Correspondingly, the caffeine concentration in the co-substrate which was composed

of aforementioned three materials, averaged 470 mg/L, far fom the inhibition

concentration threshold. Even considering the coffee processing wastewater itself, the

caffeine concentration was still less than 1500 mg/L. In a word, the content of

caffeine in coffee processing wastewater imposed little inhibitory effect on the

microbial growth in the AnMBR, except for the effect of extended lag phase with

increased caffeine concentration.

12
 To estimate the fraction of residual caffeine in the sludge, TGA was used

to comparing a pure caffeine sample and a dried sludge sample. This analysis

provides the weight loss of a sample upon heating. As shown in Fig. 4(a),

caffeine was sublimated in a small temperature range from 200 to 278 oC and

can thereby be distinguished from other organic components. The sharp peaks

(Fig. 4b) overlapping that of pure caffeine observed for the sludge samples with

4000, 8000 and 16000 mg caffeine/L concentrations indicate that the residual

caffeine accumulated in the sludge without decomposition. A higher caffeine

concentration suggests more serious accumulation and this would be reflected

by the height of the peak. However, no peak overlapped the peak of pure

caffeine for the sludge samples with 500, 1000 and 2000 mg caffeine/L

concentrations. Hence, the complete degradation of caffeine, through full

methane conversion, for these three concentrations was further verified by these

results. Fig. S1 provided the photos of dried sludge with different caffeine

concentrations taken by a micro lens before TGA. Caffeine particles are easily

observed in the samples under 4000, 8000 and 16000 g caffeine/L, and were

particularly clear in the last two samples.

3.3 Stoichiometric equation of caffeine degradation

Referring to the classical equation for methanogenic digestion proposed by

McCarty in Equation 3 [40], a stoichiometric equation of methanogenic

degradation of caffeine can be hypothesized in Equation 4. Based on this

hypothesis, the stoichiometric relationship among the amount of caffeine

redu tion (labeled as ∆CAF), (labeled as ∆ H 4), cumulative CO 2

produ tion (labeled as ∆ 2) and increased NH 4+-N (labeled as ∆NH 4+-N) can

be e pressed as: ∆ H 4/∆CAF 3.25, ∆ 2/∆CAF 0.75 and ∆NH 4+-

13
N/∆caffeine=4.0

 4n  7b  a  2c   4n  3b  a  2c 
C n H a N b Oc    H 2O   CH 4
 4   8  (3)
 4n  5b  a  2c   
 CO2  bNH 4  bHCO3
 8 

C8 H10 N 4 O 2  11.5H 2 O  3.25CH 4  0.75CO 2  4NH4  4HCO3 (4)

Experimental ∆CH4, ∆ 2 and ∆NH4+-N can be obtained from the results

of the BMP tests under the three complete-degradation concentrations of 500,

1000 and 2000 mg caffeine/L. The stoichiometric relationships between the

reduced caffeine and the increase in end-products are shown in Fig. 5. The

close linear correlation between ∆CH4, ∆NH4+-N and ∆ 2 and the inoculated

caffeine is clear in Fig. 5(a), (b) and (c). The ratio of ∆ H 4/∆CAF at the mole

level was determined as 3.18 (Fig. 5d) and that of ∆NH 4+ -N/∆CAF and

∆CO2/∆CAF was determined as 3.94 (Fig. 5e) and 0.74 (Fig. 5f). In this regard,

the experimental results can match the theoretically stoichiometric relationship

very well. To our knowledge, this is the first proposal and confirmation of the

reaction equation for complete caffeine degradation by methanogenesis.

3.4 Kinetic behavior of caffeine degradation

The results of the kinetics tests to determine the amount of residual

caffeine, VFAs, NH4+-N and CMP are provided in Fig. 6. The results can be

summarized as follows: (1) Residual caffeine (Fig. 6a) presented a sharp

decrease after a 1 day lag-phase, indicating that the inoculated caffeine was

initially consumed in a short time; (2) Only a small amount of acetic acid

(≤3mg/L) was dete ted in VFAs (Fig. 6b), indicating the fast rate of

methanogenesis by using VFAs as substrate resulted in little VFAs

accumulation, and on the other hand, that acetic acid is one of the intermediates

14
of caffeine degradation; and (3) NH 4+-N (Fig. 6c) reached its peak at around the

12th day, 4 days after the peak in the VFAs and 1 day before CMP reached its

potential value (Fig. 6d), indicating a large amount of NH4+-N was released

during VFAs production and accumulated in the liquid.

Fig. 7 shows the mass balance by considering the residual caffeine,

cumulative caffeine loss during sampling and CMP, with the amount of

caffeine and CMP normalized to the COD amount. Because the detected VFAs

were very low, the COD amount of VFAs was ignored in this balance. The total

COD mass in the beginning and in the end matches well, indicating the

inoculated caffeine was completely converted to CH 4 except for a small loss

during sampling. The occurrence of intermediates is clearly observed after the

1st day (lag-phase time). A rapid increase in the amount of intermediates

occurred till the 5th day when the maximum composition, at 53.3% of the total

COD amount, was reached. After that, a stable decrease in the amount of

intermediates was noted due to the increase in CMP. This suggests the

methanogenic degradation of caffeine clearly includes two major steps: caffeine

converting to intermediates and the intermediates converting to CH 4. It was likely

dominated by the production of intermediates and methane in series at different

times. Over the first 5 days, because the rate of intermediates production was

higher than the rate of consumption, the accumulation of intermediates

increased. After that, however, methanogenesis was proceeding at a faster rate

than the production of intermediates due to the rapid consumption of

intermediates for CH 4 production.

Regarding the intermediates, it is difficult to determine with any certainty

which intermediates were active during the process of methanogenic

15
degradation of caffeine since the fate of caffeine catabolism in anaerobic

bioprocess was unknown to date. But we can clarify the rate-limiting step

during the process and identify the major intermediates based on the results of

kinetic analysis. The anaerobic digestion of organic matter to biogas (mainly

methane and carbon dioxide) involves four key steps, these being hydrolysis,

acidogenesis, acetogenesis and methanogenesis [32]. Very few VFAs

(including acetic acid) detection in the whole process shown in Fig. 6b

indicated little accumulation of the products of acidogenesis and acetogenesis.

Therefore, the major accumulated intermediates can be identified to be

hydrolysis products instead of VFAs, and the rate-limiting step was mainly the

step of hydrolysis products converting to VFAs.

Many researches have given information on the pathway of caffeine

degradation in nature. Demethylation and purine catabolism were accepted

widely to be the two key stages for bio-decaffeination [41]. Plants, bacteria and

mammals may induce different fate of demethylation, which was catalysed by

different demethylase enzymes i.e. N-1-demethylase, N-3-demethylase, N-7-

demethylase [2, 15]. Several bacterial strains are capable of utilizing caffeine

via N-1-demethylation [42]. Thus bacterial degradation involves a

demethylation pathway of affeine→theobromine→7-

methyl anthine→ anthine. Then anthine is further degraded by the

conventional purine catabolism pathway via uric acid [18]. Based on above

information, it may be assumed that caffeine-degrading bacteria can thus be

hypothesized to present activity not only in naturally aerobic environment but

also in anaerobic environment. In the process of demethylation, the detached

methyl is supposed to be hydrolysed into methanol which will be further used

16
as substrate by methylotrophic methanogens to produce methane [43]. In the

process of purine catabolism, many anaerobic bacterium breaks down uric acid

to CO2, acetic acid and ammonia [44], among which acetic acid will then be

used as favourable substrate by acetoclastic methanogens for methane

production. However, it is difficult to determine the exact intermediates during

the process of caffeine degradation by methanogenesis and this needs more

efforts in the future work.

3.5 Significance of the findings

Large amount of wastes/by-products are generated during coffee processing from

fruit to cup. As a major by-products, caffeine presents both biomass recalcitrant and

non-eco-friendly properties. Caffeine has been reported to inhibit microbial growth

during coffee waste digestion [25]. But we showed that caffeine is able to be

completely fermented and then be used for methanogenesis, in the continuous

operation of the AnMBR digesting coffee processing wastewater and in the potential

tests by using caffeine as sole substrate. Therefore we should rethink the inhibition

behaviour of caffeine on the anaerobic digestion of coffee waste. Our results indicated

the inhibition effect was slight when the caffeine concentration was less than 2000

mg/L since methanogenesis was recovered after a short period. The methanogenic

activity was inhibited only when the concentration was more than 4000 mg/L.

Accordingly the caffeine content in solid coffee waste is reportedly in the range of

0.5-1.5% (w/w) [45], thus the maximum caffeine concentration is around 1500 mg/L

in the feedstock, assuming 10% solid content, the maximum solid content for ordinary

wet fermentation. This suggests that the caffeine present in coffee waste does not

inhibit the performance of anaerobic digestion, nevertheless, it may have an inhibitory

effect in combination with other components in the waste or other environmental

17
factors during the operation. In addition to this, the presence of caffeine contributes to

providing nitrogen source, to mitigate the problem of high carbon but low nitrogen

(carbon-to-nitrogen ratio>20), a universal property in coffee waste [46]. The lack of

available nitrogen as a nutrient source may limit microbial growth during anaerobic

digestion [31].

Coffee is one of the world's most popular beverages. Global coffee production

has been increasing at an average annual growth rate of 2.4% since 2011, and was

approximately 8.9 million tons in 2016 [47]. The commercial extraction of 1 ton of

roasted beans generates around 1.65 tons of moist coffee waste, which will present

serious environmental and economic problems if it is simply discharged or if it is

disposed of by dumping or by incineration. With high coffee production, it is

imperative to balance this production with the proper utilization of coffee waste. The

use of coffee waste as feedstock for methane production by anaerobic digestion has

been recently highlighted, because of the abundant organic carbon sources including

cellulose, protein, sugars and lipids in coffee waste [45]. The AnMBR applied in this

study integrates anaerobic digestion and membrane technology, and produces high

biomass through separating solid retention time (SRT) from HRT [30]. Solid-liquid

separation by membrane enables the retention of microbe cells to realize a long SRT

in the AnMBR, which contributes to achieve co-cultures of fermentative bacteria and

methanogens through syntrophic interaction [48]. It is therefore reasonable to

assume that caffeine-degrading bacteria are active not only in naturally aerobic

environments but also in anaerobic environments. More importantly, our results

seem to indicate the recalcitrant barrier of caffeine can be addressed very well when

the caffeine-degrading bacteria are associated with the methane-forming archaea.

Efficiently methanogenic digestion of coffee processing wastewater in the presence of

18
caffeine has been achieved by the AnMBR, even at a relatively short HRT of 5d. This

suggests the AnMBR is a promising technology for anaerobic digestion of coffee

waste in a caffeine-rich environment. The obtained results provide a new thought to

decaffeination by introducing bioenergy harvest at the same time, and also of great

help in developing low-cost innovative technology for treating caffeine-containing

wastes. However, considerable future work on the methanogenic degradation of

caffeine is required before it can be industrially implemented. Determining the

degradation pathway and identifying the caffeine-degrading anaerobes are two

important topics.

4. Conclusions

In summary, this study introduces a new process of microbial decaffeination:

complete caffeine degradation by methanogenesis. We confirm the success of this

process by the efficiencies in the AnMBR and the analysis of kinetic behaviour. Up to

92.9% with an average 87.5% in removal efficiency of caffeine was obtained by the

AnMBR treating the co-substrate mainly composed of coffee processing wastewater.

Complete degradation of caffeine was achieved in the methane potential tests using

caffeine as sole substrate, and a stoichiometric reaction equation supporting the

caffeine degradation by methanogenesis was proposed. The rate-limiting step in the

process was identified through a kinetic experiment, and the major intermediates and

possible pathway of the caffeine degradation was suggested. The findings promise to

be of great help in developing innovative technology for harvesting bioenergy at the

same time as carrying out decaffeination.

Acknowledgements

19
 This work was supported by the KAKENHI Grant-in-Aid for Scientific Research

(No. 26289179), JSPS (Japan Society for the Promotion of Science) KAKENHI

Grant-in-Aid for JSPS Fellows (No. 15F15353) and the Shaanxi Program for

Innovative Research Team (No. 2013KCT-13).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org.

References

[1] K. Manoli, G. Nakhla, M. Feng, V.K. Sharma, A.K. Ray, Silica gel-enhanced
oxidation of caffeine by ferrate (VI), Chem Eng J, 330 (2017) 987-994.
[2] S.N. Gummadi, B. Bhavya, N. Ashok, Physiology, biochemistry and possible
applications of microbial caffeine degradation, Appl Microbiol Biot, 93 (2012) 545-
554.
[3] A. Nehlig, J.L. Daval, G. Debry, Caffeine And the Central-Nervous-System -
Mechanisms Of Action, Biochemical, Metabolic And Psychostimulant Effects, Brain
Res Rev, 17 (1992) 139-169.
[4] J.A. Vignoli, D.G. Bassoli, M.T. Benassi, Antioxidant activity, polyphenols,
caffeine and melanoidins in soluble coffee: The influence of processing conditions
and raw material, Food Chem, 124 (2011) 863-868.
[5] I. Inkielewicz-Stepniak, W. Czarnowski, Oxidative stress parameters in rats
exposed to fluoride and caffeine, Food Chem Toxicol, 48 (2010) 1607-1611.
[6] P. Mazzafera, Degradation of caffeine by microorganisms and potential use of
decaffeinated coffee husk and pulp in animal feeding, Scientia Agricola, 59 (2002)
815-821.
[7] S. Nanjundaiah, S. Mutturi, P. Bhatt, Modeling of caffeine degradation kinetics
during cultivation of Fusarium solani using sucrose as co-substrate, Biochem Eng J,
(2017).
[8] Q. Sui, J. Huang, S.B. Deng, G. Yu, Q. Fan, Occurrence and removal of
pharmaceuticals, caffeine and DEET in wastewater treatment plants of Beijing, China,
Water Res, 44 (2010) 417-426.
[9] R.L. Seiler, S.D. Zaugg, J.M. Thomas, D.L. Howcroft, Caffeine and
pharmaceuticals as indicators of waste water contamination in wells, Ground Water,
37 (1999) 405-410.
[10] F. Qi, W. Chu, B.B. Xu, Modeling the heterogeneous peroxymonosulfate/Co-
MCM41 process for the degradation of caffeine and the study of influence of cobalt
sources, Chem Eng J, 235 (2014) 10-18.
[11] I.J. Buerge, T. Poiger, M.D. Muller, H.R. Buser, Caffeine, an anthropogenic
marker for wastewater contamination of surface waters, Environ Sci Technol, 37
(2003) 691-700.

20
[12] M.K. Arfanis, P. Adamou, N.G. Moustakas, T.M. Triantis, A.G. Kontos, P.
Falaras, Photocatalytic degradation of salicylic acid and caffeine emerging
contaminants using titania nanotubes, Chem Eng J, 310 (2017) 525-536.
[13] O.M. Rodriguez-Narvaez, J.M. Peralta-Hernandez, A. Goonetilleke, E.R.
Bandala, Treatment technologies for emerging contaminants in water: A review,
Chem Eng J, 323 (2017) 361-380.
[14] T. Deblonde, C. Cossu-Leguille, P. Hartemann, Emerging pollutants in
wastewater: A review of the literature, Int J Hyg Envir Heal, 214 (2011) 442-448.
[15] S. Gokulakrishnan, K. Chandraraj, S.N. Gummadi, Microbial and enzymatic
methods for the removal of caffeine, Enzyme Microb Tech, 37 (2005) 225-232.
[16] M.A. Isla, R.N. Comelli, L.G. Seluy, Wastewater from the soft drinks industry as
a source for bioethanol production, Bioresource Technol, 136 (2013) 140-147.
[17] N.H. Tran, T. Urase, H.H. Ngo, J.Y. Hu, S.L. Ong, Insight into metabolic and
cometabolic activities of autotrophic and heterotrophic microorganisms in the
biodegradation of emerging trace organic contaminants, Bioresource Technol, 146
(2013) 721-731.
[18] H. Ashihara, A. Crozier, Caffeine: a well known but little mentioned compound
in plant science, Trends Plant Sci, 6 (2001) 407-413.
[19] Y. Asano, T. Komeda, H. Yamada, Enzymes involved in theobromine production
from caffeine by Pseudomonas putida No. 352, Bioscience, biotechnology, and
biochemistry, 58 (1994) 2303-2304.
[20] P. Mazzafera, O. Olsson, G. Sandberg, Degradation of caffeine and related
methylxanthines bySerratia marcescens isolated from soil under coffee cultivation,
Microbial ecology, 31 (1996) 199-207.
[21] M. Hakil, S. Denis, G. Viniegra-Gonzalez, C. Augur, Degradation and product
analysis of caffeine and related dimethylxanthines by filamentous fungi, Enzyme
Microb Tech, 22 (1998) 355-359.
[22] B.R. Mohapatra, N. Harris, R. Nordin, A. Mazumder, Purification and
characterization of a novel caffeine oxidase from Alcaligenes species, J Biotechnol,
125 (2006) 319-327.
[23] P.M. Bradley, L.B. Barber, D.W. Kolpin, P.B. McMahon, F.H. Chapelle,
Biotransformation of caffeine, cotinine, and nicotine in stream sediments:
Implications for use as wastewater indicators, Environ Toxicol Chem, 26 (2007)
1116-1121.
[24] V. Prabhudessai, A. Ganguly, S. Mutnuri, Effect of caffeine and saponin on
anaerobic digestion of food waste, Ann Microbiol, 59 (2009) 643-648.
[25] V.R.S. Babu, S. Patra, M.S. Thakur, N.G. Karanth, M.C. Varadaraj, Degradation
of caffeine by Pseudomonas alcaligenes CFR 1708, Enzyme Microb Tech, 37 (2005)
617-624.
[26] N.G. Azhar, D.C. Stuckey, The Influence Of Chemical-Structure on the
Anaerobic Catabolism Of Refractory Compounds - a Case-Study Of Instant Coffee
Wastes, Water Sci Technol, 30 (1994) 223-232.
[27] Q. Li, Y.Y. Li, W. Qiao, X.C. Wang, K. Takayanagi, Sulfate addition as an
effective method to improve methane fermentation performance and propionate
degradation in thermophilic anaerobic co-digestion of coffee grounds, milk and waste
activated sludge with AnMBR, Bioresource Technol, 185 (2015) 308-315.
[28] W. Qiao, K. Takayanagi, M. Shofie, Q.G. Niu, H.Q. Yu, Y.Y. Li, Thermophilic
anaerobic digestion of coffee grounds with and without waste activated sludge as co-
substrate using a submerged AnMBR: System amendments and membrane
performance, Bioresource Technol, 150 (2013) 249-258.

21
[29] M. Hakil, F. Voisinet, G. Viniegra-Gonzalez, C. Augur, Caffeine degradation in
solid state fermentation by Aspergillus tamarii: effects of additional nitrogen sources,
Process Biochem, 35 (1999) 103-109.
[30] R. Chen, Y. Nie, Y. Hu, R. Miao, T. Utashiro, Q. Li, M. Xu, Y.-Y. Li, Fouling
behaviour of soluble microbial products and extracellular polymeric substances in a
submerged anaerobic membrane bioreactor treating low-strength wastewater at room
temperature, J Membrane Sci, 531 (2017) 1-9.
[31] C.L. Mao, Y.Z. Feng, X.J. Wang, G.X. Ren, Review on research achievements of
biogas from anaerobic digestion, Renew Sust Energ Rev, 45 (2015) 540-555.
[32] M. Kamali, T. Gameiro, M.E.V. Costa, I. Capela, Anaerobic digestion of pulp
and paper mill wastes - An overview of the developments and improvement
opportunities, Chem Eng J, 298 (2016) 162-182.
[33] C. Huilinir, A. Quintriqueo, C. Antileo, S. Montalvo, Methane production from
secondary paper and pulp sludge: Effect of natural zeolite and modeling, Chem Eng J,
257 (2014) 131-137.
[34] APHA, Standard Methods for the Examination of Water and Wastewater, 21st
ed. American Water Works Association and Water Environment Federation
Washington, DC, USA, (2005).
[35] J.H. Waterborg, The Lowry method for protein quantitation, The protein
protocols handbook, (2002) 7-9.
[36] S.S. Nielsen, Phenol-sulfuric acid method for total carbohydrates, in: Food
Analysis Laboratory Manual, Springer, 2010, pp. 47-53.
[37] E.G. Bligh, W.J. Dyer, A Rapid Method Of Total Lipid Extraction And
Purification, Can J Biochem Phys, 37 (1959) 911-917.
[38] A. amana ičienė, V. osto ojus, I. Ba hmato a, A. amana ičius, Anti-
bacterial effect of caffeine on Escherichia coli and Pseudomonas fluorescens, Acta
Medica Lituanica, 10 (2003) 185-188.
[39] S. Gokulakrishnan, S.N. Gummadi, Kinetics of cell growth and caffeine
utilization by Pseudomonas sp GSC 1182, Process Biochem, 41 (2006) 1417-1421.
[40] P.L. McCarty, Anaerobic waste treatment fundamentals, Public works, 95 (1964)
107-112.
[41] H. Ashihara, A. Crozier, Biosynthesis and catabolism of caffeine in low-caffeine-
containing species of Coffea, J Agr Food Chem, 47 (1999) 3425-3431.
[42] R.M. Summers, T.M. Louie, C.L. Yu, M. Subramanian, Characterization of a
broad-specificity non-haem iron N-demethylase from Pseudomonas putida CBB5
capable of utilizing several purine alkaloids as sole carbon and nitrogen source,
Microbiol-Sgm, 157 (2011) 583-592.
[43] P.L. Paulo, A.J.M. Stams, J.A. Field, C. Dijkema, J.B. van Lier, G. Lettinga,
Pathways of methanol conversion in a thermophilic anaerobic (55 degrees C) sludge
consortium, Appl Microbiol Biot, 63 (2003) 307-314.
[44] R.M. Summers, S.K. Mohanty, S. Gopishetty, M. Subramanian, Genetic
characterization of caffeine degradation by bacteria and its potential applications,
Microb Biotechnol, 8 (2015) 369-378.
[45] P.S. Murthy, M.M. Naidu, Sustainable management of coffee industry by-
products and value addition-A review, Resour Conserv Recy, 66 (2012) 45-58.
[46] D. Pujol, C. Liu, J. Gominho, M.A. Olivella, N. Fiol, I. Villaescusa, H. Pereira,
The chemical composition of exhausted coffee waste, Ind Crop Prod, 50 (2013) 423-
429.

22
[47] J. Kim, H. Kim, G. Baek, C. Lee, Anaerobic co-digestion of spent coffee grounds
with different waste feedstocks for biogas production, Waste Manage, 60 (2017) 322-
328.
[48] R. Chen, Y. Nie, N. Tanaka, Q. Niu, Q. Li, Y.-Y. Li, Enhanced methanogenic
degradation of cellulose-containing sewage via fungi-methanogens syntrophic
association in an anaerobic membrane bioreactor, Bioresource Technol, (2017).

23
 Figure Captions

Fig. 1. Schematic diagram of the used AnMBR.

Fig. 2. Caffeine removal efficiencies during the AnMBR continuous operation.

Fig. 3. Caffeine conversion efficiencies under different inoculated caffeine

concentrations in the BMP tests.

Fig. 4. Relative mass decrease with continuous increasing temperature in the

thermogravimetric analysis: (a) pure caffeine, and (b) dried sludge samples under

different inoculated caffeine concentrations in the BMP tests.

Fig. 5. Linear correlation of reduced caffeine with CH4 production, increased NH4+-N

and CO2 produ tion: (a) ∆ H4, (b) ∆NH4+-N and ( ) ∆ 2 under the three complete-

degradation on entrations, and (d) ∆ H4/∆ AF, (e) ∆NH4+-N/∆ AF and (f)

∆CO2/∆ AF ratio at mole le el.

Fig. 6. Results of residual caffeine (a), VFAs (b), NH4+-N (c) and CMP (d) in the

kinetic experiment.

Fig. 7. COD mass balance based on residual caffeine, caffeine loss during sampling

and CMP.

Table 1. Characteristics of co-substrate used in this study.

Table 2. Simulated lag-phase time ( ), methane conversion rate (R), and methane

conversion potential (P) of caffeine degradation under the three complete-conversion

inoculated concentrations.

24
 Gas meter
Biogas
Biogas

Timer Pressure Permeate

pump P Ti P pump sensor pump
P Effluent
Mixer Gas Ti
pump Timer
Ｍ
P
recirculation

Mixer
Ｍ Membrane
4oC module
Biogas

Water
bath
55C

Substrate tank Dissolution tank AnMBR

Fig. 1. Schematic diagram of the used AnMBR.

25
 50d 30d 15d 10d 5d
HRT
700 100
Caffeine concentration (mg/L)

600

Removal efficiency (%)

80
500
400 60

300 Caffeine in substrate Caffeine in effluent 40

Removal efficiency
200
20
100
0 0
20 30 40 50 60 70 80 90 100 110
Time (days)

Fig. 2. Caffeine removal efficiencies during the AnMBR continuous operation.

26
 120
100
Caffeine conversion (%)

80
500 mg/L
60 1000 mg/L
40 2000 mg/L
4000 mg/L
20 8000 mg/L
16000 mg/L
0
-20
-40
-60
0 2 4 6 8 10 12 14 16 18
Time (days)

Fig. 3. Caffeine conversion efficiencies under different inoculated caffeine

concentrations in the BMP tests.

27
 18 3.5
(a) (b) 16000 mg/L
d(relative decrease)/dt (%/min)

d(relative decrease)/dt (%/min)

16
3 8000 mg/L
14 4000 mg/L
Caffeine 2.5 2000 mg/L
12 1000 mg/L
10 2 500 mg/L

8 1.5
6
1
4
2 0.5

0 0
100 150 200 250 300 350 400 100 150 200 250 300 350 400
Temperature (oC) Temperature (oC)

Fig. 4. Relative mass decrease with continuous increasing temperature in the

thermogravimetric analysis: (a) pure caffeine, and (b) dried sludge samples under

different inoculated caffeine concentrations in the BMP tests.

28
 (a) (b) (c)
50 750 12

∆NH4+-N (mg/L)
40 600 10

∆CO2 (mL)
∆CH4 (mL)

30 450 8
6
20 300
4
10 150
2
0 0 0
500 1000 2000 500 1000 2000 500 1000 2000
CAF concentration (mg/L) CAF concentration (mg/L) CAF concentration (mg/L)

(d) (e) (f)

2.5 3 0.6
∆ H4= 3.1815∆ AF ∆NH4+-N= 3.9402∆ AF ∆CO2= 0.7417∆ AF
2 R² = 0.999 2.5 R² = 0.97 0.5 R² = 0.999
∆NH4+-N (mmol)
∆ H4 (mmol)

2000mg/L 2000mg/L
∆CO2 (mmol)
2 0.4 2000mg/L
1.5
1.5 0.3
1 1000mg/L
1000mg/L 1 0.2 1000mg/L
0.5 0.5 0.1
500mg/L 500mg/L 500mg/L
0 0 0
0 0.3 0.6 0.9 0 0.3 0.6 0.9 0 0.3 0.6 0.9
∆CAF (mmol) ∆CAF (mmol) ∆CAF (mmol)

Fig. 5. Linear correlation of reduced caffeine with CH4 production, increased NH4+-N

and CO2 produ tion: (a) ∆ H4, (b) ∆NH4+-N and ( ) ∆ 2 under the three complete-

degradation on entrations, and (d) ∆ H4/∆ AF, (e) ∆NH4+-N/∆ AF and (f)

∆CO2/∆ AF ratio at mole le el.

29
 2500
Residual caffeine (mg/L)

(a)
2000

1500

1000

500

0
(b)
9
VFAs (mg/L)

0
(c)
NH4+-N (mg/L)

600

400

200

0
100
(d)
80
CMP (mL)

60
40
20
0
0 1 2 3 4 5 6 7 8 9 101112131415
Time (days)

Fig. 6. Results of residual caffeine (a), VFAs (b), NH4+-N (c) and CMP (d) in the

kinetic experiment.

30
 Residual caffeine Cumulative caffeine loss during sampling CMP
100%
Intermediates:
COD composition

80%
53.3% of COD
60%

40%

20%

0%
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Time (days)

Fig. 7. COD mass balance based on residual caffeine, caffeine loss during sampling

and CMP.

31
Table 1 Characteristics of co-substrate used in this study

Parameters Concentrations

Total solid (TS) 41.8±5.6 g/L

Volatile solid (VS) 33.7±6.9 g/L

Total COD (TCOD) 42.4±9.9 g/L

Soluble COD (SCOD) 28.7±10.3 g/L

Protein 8.92±1.73 g/L

Carbohydrate 12.6±5.1 g/L

Lipid 3.91±1.91 g/L

pH 4.83±0.76

Alkalinity 270±130 mg-CaCO3/L

Caffeine 470±100 mg/L

32
Table 2. Simulated lag-phase time ( ), methane conversion rate (R), and methane

conversion potential (P) of caffeine degradation under the three complete-conversion

inoculated concentrations.

Caffeine concentration R P

(mg/L) (d) (%/d) (%)

500 1.02 98.1 99.9

1000 4.10 26.2 99.6

2000 7.05 21.2 100

33
 Graphic Abstract

2000 Caffeine reduction

Caffeine reduction 100

Residual caffeine (mg/L)

CH4

CH4 production (mL)

CH₄4 production
CH production
80
1500
O
CH3 60
H3C
N 1000
N1 7
40
Caffeine 3
O N N 500
20
CH3
0 0
0 1 2 3 4 5 6 7 8 9 1011121314
Discharged

Time (days)

Anaerobic
First success in complete
membrane
caffeine degradation by
bioreactor methanogenesis
Refractory and
in a bioreactor
non-eco-friendly

34
 Highlights

 Caffeine degradation by methanogenesis was first succeeded in a man-made

bioprocess

 Caffeine removal efficiencies were 87.5±5.3% in an anaerobic MBR

 Complete bioconversion of caffeine to CH4 was first confirmed

 Stoichiometry and kinetic behavior of caffeine degradation was identified

35