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Article

Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (Anammox) Activities

1
Department of Environmental Engineering, Faculty of Engineering, Kasetsart University, No. 50, Phaholyothin Road, Ladyao Subdistrict, Jatujak District, Bangkok 10900, Thailand
2
Department of Water Resources and Environmental Engineering, Tamkang University, 151 Yingzhuan Road, Tamsui District, New Taipei City 25137, Taiwan
3
Department of Chemistry, Faculty of Science, Kasetsart University, No. 50, Phaholyothin Road, Ladyao Subdistrict, Jatujak District, Bangkok 10900, Thailand
*
Author to whom correspondence should be addressed.
Water 2022, 14(14), 2238; https://doi.org/10.3390/w14142238
Submission received: 1 June 2022 / Revised: 8 July 2022 / Accepted: 14 July 2022 / Published: 16 July 2022

Abstract

:
An anaerobic ammonium oxidation (anammox) process was employed to remove nitrogen from wastewater generated from a coffee brewing facility. The effects of caffeine and chemical oxygen demand (COD) in coffee wastewater on anammox activity were investigated. The anammox activity was inhibited in synthetic wastewater with a caffeine concentration greater than 350 mg/L. Daily additions of caffeine at 2.5 mg/L for 28 days to the same substrate did not inhibit anammox activity. However, daily additions of coffee wastewater with COD of ≥387 mg/L and caffeine at 2.5 mg/L significantly inhibited anammox activity. Because the pH was increased in the system, resulting in an increase in free ammonia (FA) concentration, one could postulate that FA is an inhibitor of anammox activity. Quantitative polymerase chain reaction (qPCR) analysis was employed to determine the populations of anammox and denitrifying bacteria. Coffee wastewater with bacterial COD to total nitrogen (bCOD:TN) ratios of 0.3–0.6:1 did not have any effect on the abundances of anammox and denitrifying bacteria. The results from this work suggest that biodegradable COD (bCOD) rather than total COD (TCOD) should be used for calculating the COD:TN ratio during the study of the effects of nitrogen removal from real wastewaters using the anammox process. A not-competitive model could fit the anammox inhibition with caffeine concentrations at 50–500 mg/L with maximum specific anammox activity (SAAmax) of 0.594 mg-N/mg-volatile suspended solids (VSS)/d and inhibitory constant (Ki) of 480.97 mg/L.

1. Introduction

Coffee is one of the most popular drinks in the world with about one third of the world’s population consuming it. Businesses related to the production and serving of coffee are booming [1]. Wastewaters generated from coffee production contain high organic matter as total chemical oxygen demand (TCOD) as high as 35,600 mg/L, soluble COD (SCOD) of 12,800 mg/L), high nitrogen content (as total Kjeldahl Nitrogen or TKN) of 560 mg N/L, and high caffeine (>1000 mg/L) [1,2,3]. Due to its high COD, an anaerobic treatment system is strongly recommended to treat this type of wastewater. Although the anaerobic treatment process could significantly remove organic matter in terms of COD, nitrogen removal has been reported to not be very efficient [2,4,5,6]. Thus, a subsequent nitrogen removal process should be used for treating the anaerobic-treated effluent. Due to a low COD:TN (total nitrogen) ratio of this treated wastewater, conventional biological treatment (nitrification and denitrification processes) is not an ideal choice as several disadvantages are associated with this process, specifically high oxygen concentration demand, requirement for additional carbon source for the denitrification process, high sludge production, and significantly increased nitrous gas (N2O) emission [7,8,9].
In this study, the anammox process was suggested as an alternative approach to treat coffee wastewater for biological nitrogen removal (BNR). Unlike conventional nitrification and denitrification processes, the anammox process converts nitrite (NO2) to nitrogen gas (N2) with ammonium (NH4+) as the electron donor with the formation of nitrate (NO3) as indicated in the following reaction.
NH4+ + 1.32NO2 + 0.066HCO3 + 0.13H+ → 1.02N2 + 0.26NO3 + 0.066CH2O0.5N0.5 + 2.03H2O
Many advantages, such as low energy cost, no additional organic carbon requirement, lower sludge production, and lower N2O gas emissions, are frequently cited for anammox process [10,11]. Furthermore, the anammox process is a recommended process to remove nitrogen from wastewaters with high ammonium–nitrogen but low carbon concentrations, that is, low COD:TN ratio.
Many researchers [12,13,14,15] have applied the anammox process for treating wastewaters with low carbon and high NH4+ concentrations (COD:TN ratio < 1), such as supernatant from the anaerobic treatment of sludge from domestic wastewater, pharmaceutical wastewater, livestock wastewater, and landfill leachate. So far, no study has applied the anammox process to treat coffee wastewater. Several studies [16,17,18] have reported that caffeine is an antimicrobial agent. Ramanavičienė et al. [16] indicated that caffeine is an effective antimicrobial agent against E. coli through the damage of DNA and inhibition of protein synthesis. It is possible that the presence of caffeine with antibiotics (penicillin, amoxicillin, ampicillin, and benzylpenicillin) could enhance the inhibition of microbial activities, producing an even greater inhibition than that reported for caffeine on Staphylococcus aureus [19] or furazolidone against Vibrio [20]. Additional work could better define the suitability of the anammox process in the presence of various inhibitors.
The main goal of this study was to investigate the effect of caffeine and COD from coffee wastewater on the anammox process for both acute and long-term periods by observing specific anammox activity (SAA) in a suspended growth system. A simple Monod-based model is used to distinguish the mechanisms for caffeine inhibition on anammox activity. The results from this work could lay a foundation for application of the anammox process for nitrogen removal from coffee wastewater.

2. Material and Methods

2.1. Experimental Setup and Preparation of Wastewaters

Enriched anammox cultures were harvested from anaerobic sequencing batch reactors (ASBRs) maintained in the Environmental Engineering’s laboratory of Faculty of Engineering, Kasetsart University, Bangkok. The cultures were immediately transferred to each experimental reactor that was used throughout this study. The initial biomass concentration in each suspended growth reactor was around 0.8–1.2 g/L. To ensure an oxygen free environment, an inert gas mixture (95% Ar and 5% CO2) was bubbled into the reactors to expel dissolved oxygen (DO). All reactors were shielded from light and operated at constant room temperature (25–30 °C). All samples for acute and long-term experiments were obtained by using a needle joint with a syringe and filtered through a 0.45-μm nylon membrane filter before measurement. Impeller speed by magnetic stirrer is fixed only 40 rpm through experimental work because high share stress could affect the size of anammox granule.

2.1.1. Preparation of Synthetic Wastewater

A synthetic wastewater was prepared to contain ammonium–nitrogen concentration of 15 mM and nitrite-nitrogen of 19.8 mM, replicating the 1:1.32 ammonia–nitrogen to nitrite-nitrogen ratio used by Egli et al. [21] and Van Dongen et al. [22]. These concentrations were achieved by dissolving 990 mg (NH4)2SO4 and 1346 mg NaNO2 per liter synthetic wastewater. (210 mg NH4+-N/L and 273 mg NO2-N/L). Other minor ingredients in the synthetic wastewater per liter were 937.3 mg KHCO3, 7.5 mg FeSO4·7H2O, 28.77 mg Na2EDTA·2H2O, 18.75 mg KH2PO4, 150 mg MgSO4·7H2O, 394.68 mg CaCl2·2H2O, 0.06 mg Na2O3Se·5H2O, 0.165 mg MoNa2O4·2H2O, 0.187 g CuSO4·5H2O, 0.322 mg ZnSO4·7H2O, 0.742 mg MnCl2·4H2O, 0.18 mg CoCl2·6H2O and 0.142 mg NiCl2·6H2O. The characteristics of the synthetic wastewater are shown in Table 1.

2.1.2. Coffee Wastewater

The coffee sample was collected from an espresso prepared at Café Amazon, Bangkok, Thailand. Prior to its use, the coffee sample was stored in the refrigerator. Just before the experiment, it was allowed to warm to room temperature. A 1 mL portion of the coffee sample was diluted to 1 L with the synthetic wastewater described above to produce the coffee wastewater for the study. The characteristics of the coffee wastewater after mixing with synthetic wastewater are given in Table 2.

2.1.3. Acute Effects of Caffeine Dosing on Anammox Activity

To investigate the acute effects of caffeine dosing on anammox activity, glass vials with 100 mL maximum volume (working volume 80 mL) were used as reactors. After filling with the mixtures to be tested and sparging with inert gas, the vials were sealed. Constant mixing was carried out with an orbital shaker set at 120 rpm.

2.1.4. Long-Term Effect of Caffeine Dosing on Anammox Activity

To investigate the long-term effect of caffeine dosing on anammox activity, a cylindrical acrylic vessel with 1.8 L maximum volume (working volume 1 L) was used as a reactor and operated in an oxygen free environment. Initially, a 500 mL portion of anammox culture was mixed with 500 mL synthetic wastewater containing 5 mg/L caffeine in the reaction vessel. This test was operated as a sequencing batch reactor (SBR) system as follows: fill (11 min), reaction time with mixing (23.5 h), settle (10 min) and decant (9 min). All reactors were mixed with magnetic bar on a magnetic stirrer. For each cycle, 50% of supernatant was decanted and a new caffeine solution was added.

2.1.5. Effect of Cod from Coffee Wastewater on Anammox Activity. Recovery of Anammox Cultures after Stopping the Addition of Coffee Wastewater

To investigate the effect of COD from coffee wastewater on anammox activity, a SBR was used as described in the previous section. At the start, a 500 mL portion of anammox culture was mixed with 500 mL prepared coffee wastewater in the SBR. After purging DO with inert gas sparging, the SBR followed this sequence: fill (20 min), reaction time with mixing (23 h), settle (25 min), and decant (15 min). For each sequence, 500 mL of supernatant was decanted and fresh coffee wastewater was added.

2.2. Experimental Tests

2.2.1. Acute Effects of Caffeine Dosing on Anammox Activity

Caffeine (99% purity, Glentham Life Science, Corsham, UK), in quantities of 0, 100, 500, 700 and 1000 mg were diluted to 1 L with the prepared synthetic wastewater. Aliquots of 40 mL of the caffeine solutions were transferred into test reactors (vials). After mixing with 40 mL anammox culture, the caffeine concentrations in each experimental vial were 0, 50, 250, 350 and 500 mg/L, respectively. The NH4+ and NO2 concentrations were determined from the beginning of the experiment to a reaction time of 7 h. Specific anammox activity (SAA) was calculated based on the removal rate of NH4+ and NO2; see Section 2.3.

2.2.2. Long-Term Effect of Caffeine Dosing on Anammox Activity

The long-term effect of caffeine on anammox activity was studied by feeding a caffeine solution daily to the anammox system. The daily additions of 500 mL of caffeine solution (5 mg caffeine/L in the synthetic wastewater) maintained a 2.5 mg/L initial caffeine concentration in the reactor. The reactor was operated for 4 weeks. Each week the anammox activities were determined versus time by taking hourly samples of 20 mL for 7 h in order to measure NH4+ and NO2 concentrations and biomass concentration after feeding

2.2.3. Effect of Cod from Coffee Wastewater on Anammox Activity and Recovery of Anammox Cultures after Stopping the Addition of Coffee Wastewater

The SBR was operated for 28 days during which daily additions of 500 mL coffee wastewater were fed after decantation. The reactor contents after feeding coffee wastewater were found to contain 154 mg/L COD and 74 mg/L bCOD. After the SBR system was operated in the presence of coffee wastewater for 28 days, the system was fed with synthetic wastewater only for another 28 days to observe the reversibility of anammox inhibition.

2.3. Specific Anammox Activity (SAA)

Specific anammox activity (SAA) was calculated using the removal rate of NH4+-N and NO2-N by analyzing hourly samples from the reactor contents. The amount of total nitrogen (TN) (NH4+-N plus NO2-N) removed was plotted against reaction time, and the slope of line was normalized with the biomass of anammox bacteria in terms of volatile suspended solids (VSS). The SAA was expressed in the terms of mg N mg−1 VSS d−1, as shown in Equation (2). From this term, the percent of specific anammox activity in the presence of the coffee wastewater was calculated as Equation in (3).
SAA   ( mg-N / mg-VSS / d ) = slope   of   NH 4 + - N + slope   of   NO 2 - N MLVSS day × 24   h
Equation (3) was used to evaluate the effect of caffeine on the SSA.
SAA ,   % = SAA SAA i × 100
SAAi is the maximum specific activity on the control assay (no presence of caffeine) and SAA is the maximum specific activity observed with various caffeine concentrations.

2.4. Free Ammonia Calculation

The concentration of free ammonia (FA) is calculated using Equation (4)
FA = 17 × NH 4 + × 10 pH 14 × ( e 6344 273 + T + 10 pH )
where FA is the calculated concentration of free ammonia in mg N/L, NH 4 is the concentration of ammonium in mg N/L determined by analysis, pH is for using of this formula is solution of SBR during reaction time, and T is the temperature of water in °C.

2.5. Analytical Methods

2.5.1. Chemical Analysis

All chemical analysis in this work was conducted according to the Standard Methods of APHA [23]. Ammonium concentration was determined by the titrimetric method (4500-NH3). Nitrite concentration was determined based on the colorimetric method using a spectrophotometer (U-2800, Hitachi, Tokyo, Japan), at wavelength of 534 nm and a light path of 1 cm (4500-NO2). BOD concentration was determined following the Azide Modification of Iodometric Method (5210B). COD concentration was determined by Titrimetric method (5220C). Suspended and volatile solids concentrations were determined by drying at 103–105 °C and 550 °C, respectively.

2.5.2. Solid-Phase Extraction Procedure for Caffeine Extraction

C18 SPE cartridge (VertiPakTM C18 SPE, Vertical Chromatography, Thailand) was used for caffeine extraction. Before sample loading, the solid-phase adsorbent was pre-conditioned with 3 mL of methanol and 3 mL of water. 10-mL of sample was loaded into a syringe, connected to the cartridge with PTEE tubes, pushed through the cartridge slowly. The caffeine-loaded solid phase was washed with 3 mL of water and eluted with 3 mL of methanol. Caffeine concentration in the eluted sample was then determined.

2.5.3. Determination of Caffeine Concentration

A High Performance Liquid Chromatography (HPLC, LC-20A, Shimadzu Corporation Tokyo, Japan) was used to measure the concentration of caffeine following the method of Motora and Beyene [24]. A reverse phase column (ODS 250 × 4.6 mm) was used for the separation of caffeine and column temperature was set at 25 °C. Water and methanol at the volume ratio of 65 to 35 were used as the mobile phase and the flow rate of mobile phase was 1 mL/min. The photodiode array detector was set at 272 nm. The injection volume was 10 µL. The limit of detection of caffeine analysis by using HPLC in this work was 1 mg/L.

2.5.4. Statistical Analysis

Statistical analyses (one-way ANOVA) were used to compare the presentation of all effluent samples during experimental works and including performance of the different biomasses (total bacteria, AOB, NOB, and DNB). Statistical significance tested at p-values ≤ 0.05 were applied by using Excel program.

2.5.5. Quantitative Polymerase Chain Reaction (qPCR)

Biomass samples were collected for the molecular analysis. The biomass samples were extracted using phenol/chloroform extraction protocol adapted from Zhou et al. [25]. DNA concentrations were measured using a NanoPhotometer® N60 (Implen, California, USA). The abundance of total bacteria, denitrifying bacteria (DNB), and anammox bacteria were quantified using quantitative polymerase chain reaction (qPCR). Table 3 shows the target genes for total bacteria, DNB, and anammox bacteria and their corresponding primers. The qPCR analysis was carried out using a CFX96 Touch Real-Time PCR Detection System (BioRad Laboratories, California, USA). Each 20 µL of PCR mixture contained 8.2 µL of quantiNova SYBR Green Master MIX (2X) (New England Biolabs, Massachusetts, USA), 0.4 µL of forward and reverse primer (20 µM), 1 µL of template DNA, and 10 µL of dH2O. Thermal cycling conditions were denaturation at 95 °C for 3 min, followed by 95 °C for 10 s, 53–59 °C for 20 s, and 72 °C for 10 s. Standard curves were generated by 5-fold serial dilutions of plasmid DNA containing specific target gene inserts.

3. Results and Discussion

3.1. Acute Effects of Caffeine Dosing on Anammox Activity

Figure 1 shows the removal efficiencies for total nitrogen (NH4+-N plus NO2-N) under the caffeine concentrations of 0, 50, 250, 350, and 500 mg/L. The total nitrogen removal efficiencies decreased from 83% to 55% with increased caffeine concentrations from 50 to 500 mg/L. For caffeine concentrations of 0, 50, 250, 350, and 500 mg/L, the specific anammox activities (SAA) were 0.663, 0.527, 0.436, 0.311, and 0.300 mg-N/mg-VSS/d, respectively, as indicated in Figure 2. For the same caffeine concentrations, the values for SAA inhibition were 0%, 0%, 10%, 32%, and 33%, respectively, shown in Figure 2. There was no significant inhibition of anammox activity with caffeine concentrations less than 250 mg/L. The anammox activity was severely compromised (> 32%) with the caffeine concentration >350 mg/L. Ramanavičienė et al. [16] reported that a caffeine concentration of 1% by volume, with nutrient broth medium containing 0.12% glucose could damage DNA of E. coli and inhibit protein synthesis. Banerjee and Chatterjee [20] showed that a 500 mg/L caffeine concentration in the presence of an antibiotic (furazolidone concentrations of 0.15, 0.40, 0.50, 0.7, and 0.8 mg/L) would decrease the survival of Vibrio bacteria. Moreover, Esimone et al. [19] reported that caffeine concentrations of 5 and 10 mg/mL, mixed with an antibiotic (amoxicillin concentrations of 7.81, 15, 15.23, 31.25, 125, 250, and 500 mg/mL) could increase the inhibition of the activity of Staphylococcus aureus. In this research work, it was found that anammox bacteria are more sensitive to caffeine concentration than other microorganisms (E. coli, Staphylococcus aureus, and Vibrio).
The Monod-base not-competitive model was used to describe caffeine inhibition. As indicated in Figure 3, the not-competitive model could fit the data with SAAmax of 0.594 mg-N/mg-VSS/d and Ki of 480.97 mg/L.
Chen et al. [29] studied effect of quinoline on anammox activity. Quinoline is one type of nitrogen heterocyclic compound that are found in refractory coking wastewater. They found that only 13.1 mg/L of quinoline concentration would affect anammox activity. A non-competitive model was able to be used in this case. By comparison, quinoline is much more toxic than caffeine on anammox activity. Li et al. [30] investigated the effect of pyridine (that is also found in refractory coking wastewater) on anammox activity. They used a non-competitive model to explain anammox activity and reported the Ki of pyridine at 135.19 mg/L.
Since the not-competitive model could not distinguish between a non-competitive model (expectation is that caffeine is able to bind free enzyme and substrate from the binding site) and an un-competitive model (expectation is that caffeine is able to bind site away from the substrate binding site), the mechanism for caffeine inhibition on anammox activity could not be described clearly. Further investigation on the mechanism for caffeine inhibition is needed.

3.2. Long-Term Effect of Caffeine Dosing on Anammox Activity

To study the long-term effect of caffeine dosing on anammox activity the initial total nitrogen was 252 mg N/L. The reactor was fed a synthetic wastewater containing 2.5 mg/L caffeine. Although caffeine concentrations in treated effluents from anaerobic wastewater treatment plants from a soft drink company are around 7–300 μg/L [1], the higher concentration used in this research is considered reasonable. The higher concentration should expedite the inhibition process. In addition, the HPLC detection limit for caffeine is 1 mg/L, as used in this work.
For the control system, i.e., no caffeine in the feed solution, the nitrogen removal efficiency averaged around 96% and the SAA range was from 1.35 to 0.87 mg-N/mg-VSS/d. See Figure 4 and Figure 5. With the experimental system being fed with caffeine, the nitrogen removal efficiency was decreased slightly to 94% for the first day, slightly decreased to 81% after a week of operation, and then stabilized around 90–94% thereafter as shown in Figure 4. A comparison of SAA inhibition for the control and the experimental systems reveals a slight inhibition of SAA by caffeine, as shown in Figure 5.

3.3. Treatment of Coffee Wastewater Using Anammox Process and the Recovery of Anammox Cultures after Discontinuance of Coffee Wastewater Additions

The experimental control SBR was started with total nitrogen (TN) concentration (NH4+ plus NO2) of 215 mg N/L. The nitrogen removal efficiency was found to be 82% with SAA at 0.867 mg-N/mg-VSS/d. Daily additions of coffee wastewater resulted in the accumulation of COD in the reactor. As indicated in Figure 6, COD in the treated effluent increased from 154 mg/L at day 1 to 508 mg/L at day 28. Meanwhile, the TN concentration increased from 286 mg N/L at day 1 to 391 mg N/L at the end of the 28 day experimental period.
Anammox activity was inhibited after daily additions of coffee wastewater as indicated in Figure 7. As the caffeine concentration was quite low (2.5 mg/L), caffeine alone would not have caused the inhibition of SSA. It has been reported that free ammonia (FA) concentrations between 13 and 90 mg N/L could cause significant inhibition of anammox activity [31,32]. In the current study, it was found that FA concentrations increased from 0.06 at hour 1 to 0.79 mg N/L after 7 h of coffee wastewater additions. Meanwhile, FA concentration continually increased to 11.01 mg N/L on day 21. The increasing FA concentration could be the reason for the inhibition of anammox activity. FA concentration would be increased due to the high pH. For this reason, the ammonium–nitrogen removal rate was significantly decreased. Because anammox activity was inhibited, nitrite concentrations increased from 140.8 to 224.3 mg N/L on day 28. Therefore, the nitrogen removal efficiency was decreased. Several studies [21,33,34] have confirmed that high nitrite concentration could be another inhibitor of anammox activity. A previous study [35] reported that nitrite accumulation in an anammox system caused flotation events, which led to biomass washout that affected the removal efficiency.
The accumulation of COD in the reactor contents from 154 mg/L on day 1 to 508 mg/L on day 28 was very similar to the findings of Chamchoi et al. [36]. They reported that when the concentration of COD was >300 mg/L, anammox activity was markedly suppressed.
Figure 8 shows the result of bacteria abundance (total bacteria, anammox bacteria, and denitrifying bacteria (DNB)) in SBR that was analyzed by the qPCR technique. The dominant anammox species in this work were Candidatus Kuenenia stuttgartiensis and Candidatus Brocadia fulgida. However, the total chemical oxygen demand (TCOD) concentrations from 154 to 508 mg/L from coffee wastewater did not significantly change the abundances of anammox bacteria (ranging from 6.82 × 109 to 1.04 × 1010 copies/g-sludge) and DNB (ranging from 8.4 × 106 to 1.2 × 107 copies/g-sludge) in the system. Leal et al. [37], He et al. [38], and Wang et al. [39] reported that the abundance of DNB was increased when higher COD concentrations were fed to the system. The abundance of DNB found in this work was quite similar to the results from all the cited references [37,38,39]. The abundance of DNB from the work of the three researchers did not change significantly when COD ranged between 106–251 mg/L.
Although the abundance of DNB from this work was similar to the abundances of DNB from the three referenced studies, one must point out two experimental differences. First, in all of the reference experiments, a synthetic substance (glucose) was used as the COD source. For this reason, TCOD and BOD values are quite similar. However, a real substrate (coffee wastewater) was used in this work as the COD source. Thus, TCOD and BOD values are not the same value. For this reason, when using coffee wastewater, the bCOD should be used instead of TCOD for calculation of the bCOD:TN ratio. The bacterial degradation of total COD (154 mg/L) was equivalent to the bCOD (74 mg/L), or only 48% the value of TCOD. Second, the total nitrogen and organic matter concentrations from the three referenced studies and this work were quite different. For example, in the research of Wang et al. [39], there were three total nitrogen concentrations: 200 mg N/L, 600 mg N/L, and 1000 mg N/L, and the COD:TN ratio was 0.5. In this work, the calculated bCOD:TN ratios were 0.3 at day 1 and 0.4 at day 7, 0.5 at day 14, and 0.6 at days 21 and 28.
Following the 28 days of long-term dosing with caffeine, the system was then fed synthetic wastewater only (no COD from coffee wastewater). The ammonium–nitrogen concentration in the synthetic wastewater was gradually increased from 3 mM to 15 mM. It was shown that the anammox activity slowly recovered over a period of 36 days, at which point the nitrogen removal efficiency has returned to 96%. See Figure 9.
However, after using the biological treatment process to remove both organic matter and nitrogen from coffee wastewater, the treated effluent of coffee might not be directly discharged to the environment because of color remaining in coffee wastewater. A chemical treatment (adsorption process) using activated carbon might be indicated to complete the process.

4. Conclusions

  • The addition of caffeine in concentrations greater than 350 mg/L caffeine significantly inhibits anammox activity.
  • There is no long-term effect with low caffeine concentration (2.5 mg/L) on anammox activity.
  • The mechanism for caffeine inhibition on anammox bacteria could not be explained clearly because the not-competitive inhibition model could not distinguish between non- and un-competitive inhibition.
  • The presence of ≥387 mg/L COD from coffee wastewater could significantly inhibit the anammox activity. However, this inhibition effect is reversible. After the discontinuance of coffee wastewater additions to a batch reactor, the anammox activity could recover in 28 days.
  • In using an anammox treatment with real wastewater (coffee wastewater) as substrate, bCOD:TN should be used rather than TCOD:TN as the control parameter because bCOD and TCOD are not equal.

Author Contributions

T.W.: performing research, analyzing data, and writing the first draft; T.S.: advise analysis and methodology; P.L.N.: initial idea for project including funding acquisition, designing research, troubleshooting, and analyzing data; P.L.N. and C.-W.L.: writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research work was supported by the Graduate School, Kasetsart University (KU), the Kasetsart University Research and Development Institute (KURDI) and Faculty of Engineering, Kasetsart University, Bangkok, Thailand.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to thank the Graduate School, Kasetsart University (KU), the Kasetsart University Research and Development Institute (KURDI) and Faculty of Engineering, Kasetsart University, Bangkok, Thailand for grants that thoroughly supported in this research. The authors also would like to thank John Elliott (Golden, Colorado) for valuable assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Campos, R.C.; Pinto, V.R.A.; Melo, L.F.; da Rocha, S.J.S.S.; Coimbra, J.S. New sustainable perspectives for “Coffee Wastewater” and other by-products: A critical review. Future Foods 2021, 4, 100058. [Google Scholar] [CrossRef]
  2. Von Enden, J.C.; Calvert, K.C.; Sanh, K.; Hoa, H.; Tri, Q.; Vietnam, S.; Consulting, C. Review of coffee waste water characteristics and approaches to treatment. In PPP Project, Improvement of Coffee Quality and Sustainability of Coffee Production in Vietnam; German Technical Cooperation Agency (GTZ): Bonn, Germany, 2002; pp. 1–10. [Google Scholar]
  3. Rattan, S.; Parande, A.; Nagaraju, V.; Ghiwari, G.K. A comprehensive review on utilization of wastewater from coffee processing. Environ. Sci. Pollut. Res. 2015, 22, 6461–6472. [Google Scholar] [CrossRef] [PubMed]
  4. Smith, A.L.; Stadler, L.B.; Love, N.G.; Skerlos, S.J.; Raskin, L. Perspectives on anaerobic membrane bioreactor treatment of domestic wastewater: A critical review. Bioresour. Technol. 2012, 122, 149–159. [Google Scholar] [CrossRef] [PubMed]
  5. Delgado Vela, J.; Stadler, L.B.; Martin, K.J.; Raskin, L.; Bott, C.B.; Love, N.G. Prospects for biological nitrogen removal from anaerobic effluents during mainstream wastewater treatment. Environ. Sci. Technol. Lett. 2015, 2, 234–244. [Google Scholar] [CrossRef]
  6. Ijanu, E.; Kamaruddin, M.; Norashiddin, F. Coffee processing wastewater treatment: A critical review on current treatment technologies with a proposed alternative. Appl. Water Sci. 2020, 10, 11. [Google Scholar] [CrossRef] [Green Version]
  7. Yang, Q.; Peng, Y.; Liu, X.; Zeng, W.; Mino, T.; Satoh, H. Nitrogen removal via nitrite from municipal wastewater at low temperatures using real-time control to optimize nitrifying communities. Environ. Sci. Technol. 2007, 41, 8159–8164. [Google Scholar] [CrossRef]
  8. Law, Y.; Ye, L.; Pan, Y.; Yuan, Z. Nitrous oxide emissions from wastewater treatment processes. Philos. Trans. R. Soc. B Biol. Sci. 2012, 367, 1265–1277. [Google Scholar] [CrossRef] [Green Version]
  9. Liu, T.; Hu, S.; Guo, J. Enhancing mainstream nitrogen removal by employing nitrate/nitrite-dependent anaerobic methane oxidation processes. Crit. Rev. Biotechnol. 2019, 39, 732–745. [Google Scholar] [CrossRef]
  10. Jetten, M.S.; Wagner, M.; Fuerst, J.; van Loosdrecht, M.; Kuenen, G.; Strous, M. Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Curr. Opin. Biotechnol. 2001, 12, 283–288. [Google Scholar] [CrossRef]
  11. Joss, A.; Salzgeber, D.; Eugster, J.; König, R.; Rottermann, K.; Burger, S.; Fabijan, P.; Leumann, S.; Mohn, J.; Siegrist, H. Full-scale nitrogen removal from digester liquid with partial nitritation and anammox in one SBR. Environ. Sci. Technol. 2009, 43, 5301–5306. [Google Scholar] [CrossRef]
  12. Cao, S.; Du, R.; Peng, Y.; Li, B.; Wang, S. Novel two stage partial denitrification (PD)-Anammox process for tertiary nitrogen removal from low carbon/nitrogen (C/N) municipal sewage. Chem. Eng. J. 2019, 362, 107–115. [Google Scholar] [CrossRef]
  13. Kuenen, J.G. Anammox bacteria: From discovery to application. Nat. Rev. Microbiol. 2008, 6, 320–326. [Google Scholar] [CrossRef] [PubMed]
  14. Wang, C.-C.; Lee, P.-H.; Kumar, M.; Huang, Y.-T.; Sung, S.; Lin, J.-G. Simultaneous partial nitrification, anaerobic ammonium oxidation and denitrification (SNAD) in a full-scale landfill-leachate treatment plant. J. Hazard. Mater. 2010, 175, 622–628. [Google Scholar] [CrossRef] [PubMed]
  15. Meng, H.; Yang, Y.-C.; Lin, J.-G.; Denecke, M.; Gu, J.-D. Occurrence of anammox bacteria in a traditional full-scale wastewater treatment plant and successful inoculation for new establishment. Int. Biodeterior. Biodegrad. 2017, 120, 224–231. [Google Scholar] [CrossRef]
  16. Ramanavičienė, A.; Mostovojus, V.; Bachmatova, I.; Ramanavičius, A. Antibacterial effect of caffeine on Escherichia coli and Pseudomonas fluorescens. Acta Med. Litu. 2003, 10, 185–188. [Google Scholar]
  17. Dash, S.S.; Gummadi, S.N. Inhibitory effect of caffeine on growth of various bacterial strains. Res. J. Microbiol. 2008, 3, 457–465. [Google Scholar]
  18. Norizan, S.N.M.; Yin, W.-F.; Chan, K.-G. Caffeine as a potential quorum sensing inhibitor. Sensors 2013, 13, 5117–5129. [Google Scholar] [CrossRef]
  19. Esimone, C.; Okoye, F.; Nworu, C.; Agubata, C. In vitro interaction between caffeine and some penicillin antibiotics against Staphylococcus aureus. Trop. J. Pharm. Res. 2008, 7, 969–974. [Google Scholar] [CrossRef] [Green Version]
  20. Banerjee, S.; Chatterjee, S. Radiomimetic property of furazolidone and the caffeine enhancement of its lethal action on the vibrios. Chem.-Biol. Interact. 1981, 37, 321–335. [Google Scholar] [CrossRef]
  21. Egli, K.; Fanger, U.; Alvarez, P.J.; Siegrist, H.; van der Meer, J.R.; Zehnder, A.J. Enrichment and characterization of an anammox bacterium from a rotating biological contactor treating ammonium-rich leachate. Arch. Microbiol. 2001, 175, 198–207. [Google Scholar] [CrossRef]
  22. Van Dongen, U.; Jetten, M.S.; Van Loosdrecht, M. The SHARON®-Anammox® process for treatment of ammonium rich wastewater. Water Sci. Technol. 2001, 44, 153–160. [Google Scholar] [CrossRef] [PubMed]
  23. APHA. Standard Methods for the Examination of Water and Wastewater, 23rd ed.; American Public Health Association: Washington, DC, USA, 2005. [Google Scholar]
  24. Motora, K.G.; Beyene, T.T. Determination of caffeine in raw and roasted coffee beans of ilu abba bora zone, South West Ethiopia. Indo Am. J. Pharm. Res. 2017, 7, 463–470. [Google Scholar]
  25. Zhou, J.; Bruns, M.A.; Tiedje, J.M. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 1996, 62, 316–322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Muyzer, G.; De Waal, E.C.; Uitterlinden, A.G. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 1993, 59, 695–700. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  27. Schmid, M.C.; Hooper, A.B.; Klotz, M.G.; Woebken, D.; Lam, P.; Kuypers, M.M.; Pommerening-Roeser, A.; Op Den Camp, H.J.; Jetten, M.S. Environmental detection of octahaem cytochrome c hydroxylamine/hydrazine oxidoreductase genes of aerobic and anaerobic ammonium-oxidizing bacteria. Environ. Microbiol. 2008, 10, 3140–3149. [Google Scholar] [CrossRef] [PubMed]
  28. Throbäck, I.N.; Enwall, K.; Jarvis, Å.; Hallin, S. Reassessing PCR primers targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with DGGE. FEMS Microbiol. Ecol. 2004, 49, 401–417. [Google Scholar] [CrossRef] [PubMed]
  29. Chen, Q.Q.; Zhang, Z.Z.; Sun, F.Q.; Shi, Z.J.; Huang, B.C.; Fan, N.S.; Jin, R.C. Insight into the short-and long-term effects of quinoline on anammox granules: Inhibition and acclimatization. Sci. Total Environ. 2019, 651, 1294–1301. [Google Scholar] [CrossRef]
  30. Li, S.; Zhou, X.; Cao, X.; Chen, J. Insights into simultaneous anammox and denitrification system with short-term pyridine exposure: Process capability, inhibition kinetics and metabolic pathways. Front. Environ. Sci. Eng. 2021, 15, 1–14. [Google Scholar] [CrossRef]
  31. Waki, M.; Tokutomi, T.; Yokoyama, H.; Tanaka, Y. Nitrogen removal from animal waste treatment water by anammox enrichment. Bioresour. Technol. 2007, 98, 2775–2780. [Google Scholar] [CrossRef]
  32. Fernández, I.; Dosta, J.; Fajardo, C.; Campos, J.; Mosquera-Corral, A.; Méndez, R. Short-and long-term effects of ammonium and nitrite on the Anammox process. J. Environ. Manag. 2012, 95, S170–S174. [Google Scholar] [CrossRef]
  33. Bettazzi, E.; Caffaz, S.; Vannini, C.; Lubello, C. Nitrite inhibition and intermediates effects on Anammox bacteria: A batch-scale experimental study. Process Biochem. 2010, 45, 573–580. [Google Scholar] [CrossRef]
  34. Kimura, Y.; Isaka, K.; Kazama, F.; Sumino, T. Effects of nitrite inhibition on anaerobic ammonium oxidation. Appl. Microbiol. Biotechnol. 2010, 86, 359–365. [Google Scholar] [CrossRef] [PubMed]
  35. Dapena-Mora, A.; Campos, J.; Mosquera-Corral, A.; Jetten, M.; Méndez, R. Stability of the ANAMMOX process in a gas-lift reactor and a SBR. J. Biotechnol. 2004, 110, 159–170. [Google Scholar] [CrossRef] [PubMed]
  36. Chamchoi, N.; Nitisoravut, S.; Schmidt, J.E. Inactivation of ANAMMOX communities under concurrent operation of anaerobic ammonium oxidation (ANAMMOX) and denitrification. Bioresour. Technol. 2008, 99, 3331–3336. [Google Scholar] [CrossRef]
  37. Leal, C.D.; Pereira, A.D.; Nunes, F.T.; Ferreira, L.O.; Coelho, A.C.C.; Bicalho, S.K.; Mac Conell, E.F.A.; Ribeiro, T.B.; de Lemos Chernicharo, C.A.; de Araújo, J.C. Anammox for nitrogen removal from anaerobically pre-treated municipal wastewater: Effect of COD/N ratios on process performance and bacterial community structure. Bioresour. Technol. 2016, 211, 257–266. [Google Scholar] [CrossRef]
  38. He, S.; Yang, W.; Qin, M.; Mao, Z.; Niu, Q.; Han, M. Performance and microbial community of anammox in presence of micro-molecule carbon source. Chemosphere 2018, 205, 545–552. [Google Scholar] [CrossRef]
  39. Wang, X.; Yang, R.; Guo, Y.; Zhang, Z.; Kao, C.M.; Chen, S. Investigation of COD and COD/N ratio for the dominance of anammox pathway for nitrogen removal via isotope labelling technique and the relevant bacteria. J. Hazard. Mater. 2019, 366, 606–614. [Google Scholar] [CrossRef]
Figure 1. Percentage of total nitrogen removal efficiency at the end of 7 h as a function caffeine dosing concentration (0, 50, 250, 350, and 500 mg/L).
Figure 1. Percentage of total nitrogen removal efficiency at the end of 7 h as a function caffeine dosing concentration (0, 50, 250, 350, and 500 mg/L).
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Figure 2. Specific anammox activity (SAA) and percent inhibition versus the acute caffeine dosing concentration.
Figure 2. Specific anammox activity (SAA) and percent inhibition versus the acute caffeine dosing concentration.
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Figure 3. Specific anammox activity vs. caffeine concentration. The line represents the best fit of non- or an un-competitive model.
Figure 3. Specific anammox activity vs. caffeine concentration. The line represents the best fit of non- or an un-competitive model.
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Figure 4. Nitrogen removal efficiency during long-term caffeine dosing.
Figure 4. Nitrogen removal efficiency during long-term caffeine dosing.
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Figure 5. Specific anammox activity (SAA) of control and caffeine and inhibition (%) during the long-term caffeine dosing.
Figure 5. Specific anammox activity (SAA) of control and caffeine and inhibition (%) during the long-term caffeine dosing.
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Figure 6. Total nitrogen removal efficiency and COD concentration for the anammox system to treat coffee wastewater.
Figure 6. Total nitrogen removal efficiency and COD concentration for the anammox system to treat coffee wastewater.
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Figure 7. Specific anammox activity (SAA) and free ammonia (FA) concentration during the long-term caffeine dosing.
Figure 7. Specific anammox activity (SAA) and free ammonia (FA) concentration during the long-term caffeine dosing.
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Figure 8. qPCR analysis of microbial abundance of (A) anammox and DNB, control, day 1, 21, and 28 during daily addition of COD from coffee wastewater (B) anammox and DNB, control day 35 and 65 during recovery.
Figure 8. qPCR analysis of microbial abundance of (A) anammox and DNB, control, day 1, 21, and 28 during daily addition of COD from coffee wastewater (B) anammox and DNB, control day 35 and 65 during recovery.
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Figure 9. Percentage of total nitrogen removal and synthetic wastewater concentration during long-term coffee dosing.
Figure 9. Percentage of total nitrogen removal and synthetic wastewater concentration during long-term coffee dosing.
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Table 1. Characteristics of synthetic wastewater in this study.
Table 1. Characteristics of synthetic wastewater in this study.
ParameterValue
pH7.75 ± 0.11
Total Nitrogen (mg N/L)471.67 ± 3.94
Ammonium (mg N/L)209.44 ± 6.16
Nitrite (mg N/L)262.23 ± 5.06
Table 2. Characteristics of coffee wastewater (after mixing with synthetic wastewater) in this study.
Table 2. Characteristics of coffee wastewater (after mixing with synthetic wastewater) in this study.
ParameterValue
pH4.21
COD (mg/L)387.05
bCOD (mg/L)120.23
Caffeine (mg/L)3.12
Note: Coffee wastewater was used in this experiment. (1 mL portion of the coffee sample was diluted to 1 L with the synthetic.).
Table 3. List of PCR primers for the amplification.
Table 3. List of PCR primers for the amplification.
Target GenePrimerSequence (5′-3′)References
EUB
(Total bacteria)
338F
518R
ACTCCTACGGGAGGCAGCAG
ATTACCGCGGCTGCTGG
Muyzer et al. [26]
hzo
(Anammox bacteria)
hzocl1F1
hzocl1R2
TGYAAGACYTGYCAYTGG
ACTCCAGATRTGCTGACC
Schmid et al. [27]
nirS
(Denitrifying bacteria, DNB)
Cd3aF
R3cd
GTSAACGTSAAGGARACSGG
GASTTCGGRTGSGTCTTGA
Throback et al. [28]
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Wongphoom, T.; Saleepochn, T.; Noophan, P.L.; Li, C.-W. Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (Anammox) Activities. Water 2022, 14, 2238. https://doi.org/10.3390/w14142238

AMA Style

Wongphoom T, Saleepochn T, Noophan PL, Li C-W. Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (Anammox) Activities. Water. 2022; 14(14):2238. https://doi.org/10.3390/w14142238

Chicago/Turabian Style

Wongphoom, Titima, Tharinee Saleepochn, Pongsak Lek Noophan, and Chi-Wang Li. 2022. "Effects of Caffeine and COD from Coffee Wastewater on Anaerobic Ammonium Oxidation (Anammox) Activities" Water 14, no. 14: 2238. https://doi.org/10.3390/w14142238

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