The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment

Bioresource Technology xxx (2016) xxx–xxx

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Short Communication

The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment Yanyan Su a,⇑, Artur Mennerich b, Brigitte Urban c a

Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg, Denmark Faculty of Civil and Environmental Engineering, Ostfalia University of Applied Sciences, Suderburg, 29556, Germany c Institute of Ecology, Leuphana University of Lüneburg, Lüneburg 21335, Germany b

h i g h l i g h t s  The effect of unintended wall attached biofilm on nutrient removal was studied.  Reactor wall attached biofilm enhanced the phosphorus removal performance.  Reactor wall attached biofilm enhanced the total nitrogen removal capability.  Reactor wall attached biofilm had no influence on biomass generation.

a r t i c l e

i n f o

Article history: Received 13 May 2016 Received in revised form 20 June 2016 Accepted 24 June 2016 Available online xxxx Keywords: Wall attached biofilm Long-term operation Microalgae Wastewater treatment Nutrient removal

a b s t r a c t The influence of the reactor wall attached biofilm on the nutrient removal performance was investigated in an open photobioreactor during long-term operation. Total nitrogen and phosphorus removal efficiencies were statistically similar between reactor with (reactor A) and without (reactor B) biofilm at the Hydraulic Retention Time (HRT) of 18, 13.5 and 9 days. When the HRT reduced to 8 days, total nitrogen and phosphorus removal efficiencies in the reactor A were 42.95 ± 5.11% and 97.97 ± 1.12%, respectively, while significant lower removal efficiencies (38.06 ± 5.80% for total nitrogen and 83.14 ± 8.16% for phosphorus) were obtained in the reactor B. The VSS concentrations throughout the test were statistically similar for the two reactors, with a mean value of 0.63 ± 0.25 g/l for reactor A and 0.69 ± 0.20 g/l for reactor B. This study indicated that the reactor wall attached biofilm supported high phosphorus and nitrogen removal, which may provide insight into the practical implementation of microalgae-based wastewater treatment. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Microalgae can be dominant primary producers in all aquatic habitats and contribute approximately 40–50% of the oxygen in atmosphere (Andersen, 2005). Based on their nutrients requirements, microalgae-based system has been proposed as a promising biological method for both secondary and tertiary wastewater treatment (Laliberte et al., 1994; Oswald, 1990). It is costeffective and efficient compared with the conventional nutrients removal processes, such as nitrification-denitrification and biological phosphorus removal (Khin and Annachhatre, 2004; Oehmen et al., 2007). Moreover, algae-based system could store wastewater nutrients and light energy into the algal biomass which is a valueadded bioresource and substrate for biofuel (e.g., biodiesel and ⇑ Corresponding author. E-mail address: [email protected] (Y. Su).

bioethanol) or bioenergy (e.g., biogas) production (Krirolia et al., 2013; Oswald, 2003). Generally, two cultivation systems are used, namely open ponds and enclosed photobioreactors (Gupta et al., 2015). Open system is easier and cheaper to construct and operate (Arbib et al., 2013). By contrast, enclosed system offers a low risk of contaminations and provides a better control on culture conditions (Gupta et al., 2015). In both types of reactors, microalgae can either grow in suspension or in biofilms (Boelee et al., 2014). The algal biofilm systems show unique advantages over the conventional suspended cultivation systems, such as high nutrients removal rate and low costs on biomass harvesting (Gross et al., 2015). In most of the studies, the algal biofilm mainly growing on certain specific carriers or matrices are investigated (Gao et al., 2015; Munoz et al., 2009). However, microalgae may also grow on the wall of the reactor unintentionally and thus form the biofilm during their cultivation. The impact of this unintentional biofilm on wastewater

http://dx.doi.org/10.1016/j.biortech.2016.06.099 0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Su, Y., et al. The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.06.099

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Y. Su et al. / Bioresource Technology xxx (2016) xxx–xxx

treatment, which is the important information for better understanding the technology, has never been investigated. In this study, for the first time, the effects of the microalgal biofilm growing on the bioreactor wall on the nutrient removal and the biomass productivity during long-term operation were investigated.

Table 1 The Hydraulic Retention Time (HRT) during the operation period for the reactor A (with biofilm) and the reactor B (without biofilm). Period

Duration in different period

Exchange volume (ml) per day

HRT (days)

1 2 3 4

0–46 46–81 81–107 107–157

278 370 550 625

18 13.5 9 8

2. Methods 2.1. Microalgal cultivation

2.3. Analytical methods

Two unicellular microalgae (Scenedesmus rubescens and Chlorella vulgaris) with high-potentials for wastewater treatment and good settleability were chosen in this study (Su et al., 2012a). S. rubescens were purchased from the Institut für Getreideverarbeitung GmbH (Germany) and grown on 1/2 Tamiya media at room temperature. C. vulgaris was obtained from Scandinavian Culture Collection of Algae & Protozoa (Denmark) and grown on a modified MWC media at room temperature (Su et al., 2012b). The above two algal species were cultivated individually before the test. The algal inoculum was mixed with S. rubescens and C. vulgaris with 1:1 (dry weight). The initial total suspended solids (TSS) and volatile suspended solids (VSS) of the mixed algal culture were 1.07 g and 0.385 g, respectively. Two stainless steel pots (Switzerland) were used as photo-bioreactors with the total volume of 5 L (10 cm in depth). The mixed algal culture was cultivated under laboratory conditions from 9th Sep, 2011. Two compact fluorescent lamps (Sylvania, F20W/860/E27) were used to irradiate the reactors from the top in a light: dark cycle of 12:12 h (illumination started from 10 am to 10 pm), to mimic natural solar day-night cycle, with around 7000 lux (measured with TES-1335 Digital light meter, Germany). The mixing procedure was provided during daytime starting from 10 am to 10 pm (same as the irradiation cycle).

The dissolved oxygen (DO) and pH were measured near the midway of the reactors every day by using a digital multi parameter meter (Multi 3430 WTW, Germany) coupled with an O2 sensor (CellOx 325, WTW, Germany) and a pH electrode (pH SenTix 940, Germany). TKN was analyzed according to DIN EN 25663-H11 (DEV, 2002). VSS were analyzed according to DIN ISO 11465 (DEV, 2002). NO3 and NO2 were determined using an Ion Chromatograph (Dionex DX-100, USA) according to DIN EN ISO 10304-1 (DEV, 2002). NH+4 and PO34 were determined according to DIN 38406-E5-1 and DIN EN ISO 6878-D11 (DEV, 2002) using an UV/Vis Spectrometer (Perkin Elmer, Lambda 40, USA). Before analysis of the above parameters in liquid, the samples were membrane filtered (0.45 lm). All the experiments were performed in duplicate. Analysis of variance (ANOVA) test was employed to examine any significant difference (at 0.05 level) among the data.

2.2. Experiment operation After 17 days’ acclimatization, the bioreactors were operated in a semi-continuous mode from 26th Sep, 2011 to 1st March, 2012 (157 days). In order to investigate the influence of bioreactor wall attached algal biofilm on the performance of nutrient removal, the wall of the reactor A was undisturbed throughout the test. While, the wall of the reactor B was cleaned everyday as following: before starting the mixing process, the suspension of reactor B was transferred into a clean beaker and the reactor B was cleaned with a brush to remove the biofilm on the wall every day. Afterwards, the suspension was transferred from the beaker into the reactor B. The characterizations of the wastewater from the second clarifier in wastewater treatment plant of Holthusen (Germany) used here were: total COD: 30.20 ± 2.50 (mg O2/l), total kjeldahl nitrogen (TKN): 26.40 ± 0.70 (mg N/l), NH+4-N: 25.20 ± 0.30 (mg/l), PO34 -P: 1.74 ± 0.12 (mg/l), NO3 -N: 0.75 ± 0.06 (mg/l) and NO2 -N: 0.10 ± 0.06 (mg/l). The aforementioned wastewater added with NH+4 and PO34 to reach the final concentration of 28.79 ± 2.65 mg N/l and 4.48 ± 0.67 mg P/l, was used as influent. Before the mixing process, certain volume of supernatant was withdrawn from the reactors and the same volume of wastewater was added into the reactors every morning to keep the HRT 18, 13.5, 9 and 8 days for the two reactors (Table 1). 250 ml samples were collected from the exchanged supernatant twice a week (Monday and Thursday) and analyzed for VSS, Total Kjeldahl Nitrogen (TKN), NH+4, NO3 , NO2 and dissolved phosphorus (PO34 ). The water evaporative loss of both reactors was replenished to 5 L every morning in order to reduce the error.

3. Results and discussion 3.1. Phosphorus removal Based on the nutrient removal performance, the HRT was decreased from 18 days to 8 days gradually for the reactor with biofilm (reactor A) and without biofilm (reactor B). There are four different operation periods named 1, 2, 3 and 4 for both reactors (Table 1). The time course of P-PO34 is shown in Fig. 1A. In period 1, the phosphorus removal efficiency of both reactors rose gradually to above 99% at an HRT of 18 days with the average influent total phosphorus concentration of 4.70 ± 0.21 mg P/l. When the HRT was reduced to 13.5 and then to 9 days, the phosphorus removal efficiency was stable at above 99% for both reactors. When the HRT was further decreased to 8 days, the high phosphorus removal efficiency (around 98%) was only observed in reactor A, while the significant lower (p < 0.05, ANOVA analysis) P-PO34 removal efficiencies (83.14 ± 8.16%) were recorded in reactor B. The above results indicated that the wall attached biofilm enhanced the phosphorus removal performance. Biotic assimilation by biomass and abiotic orthophosphate precipitation at high pH (9–11) are two main phosphorus removal mechanisms in algae-based system (Nurdogan and Oswald, 1995). However, the pH below 8.5 in this study was not sufficient to promote abiotic removal mechanism (Fig. S1A). 3.2. Nitrogen removal NH+4 was not completely depleted in both reactors throughout the test (Fig. 1B). In period 1, the N-NH+4 removal efficiency was 94.20 ± 4.90% and 99.20 ± 0.50% for the reactor A and B, respectively, corresponding to the inlet NH+4 concentration of 26.95 ± 1.27 mg N/l. N-NH+4 removal efficiencies were decreased in the following two periods: 64.70 ± 16.00% and 70.18 ± 17.60% for reactor A and B (statistically similar, p > 0.05) in period 2, respectively; 57.67 ± 5.91% and 60.73 ± 5.58% (statistically similar,

Please cite this article in press as: Su, Y., et al. The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.06.099

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A

PO43- -P (mg P/l)

7

40

reactor A with biofilm reactor B without biofilm inlet

6 5 4 3

2

1

2

3

4

30 25 20

4

3

2

1

15 10 5

1

0

0 0

18

20

40

60

80 Date

100

120

0

140

14

C

16

12

reactor B without biofilm inlet

NO2--N (mg N/l)

1

2

8

40

60

80 Date

6

100

120

140

reactor A with biofilm reactor B without biofilm inlet

10

12 10

20

D

reactor A with biofilm

14 NO3--N (mg N/l)

reactor A with biofilm reactor B without biofilm inlet

B

35 NH4+-N (mg N/l)

8

8 6

1

4

4

3

2

4

4

3

2

2

0

0 0

20

40

60

80 Date

100

120

140

0

20

40

60

80 Date

100

120

140

Fig. 1. Time course of the inlet and outlet PO34 (A), NH+4 (B), NO3 (C) and NO2 (D) in the reactor A (with biofilm) and the reactor B (without biofilm).

(Godos et al., 2009). Moreover, there are few reports on the occurrence of denitrification in high rate algal ponds probably due to the high dissolved oxygen (de Godos et al., 2009). Besides, the denitrification activity was decreased as the organic matter contents in the influent was too low (total COD 30.20 ± 2.50 (mg O2/l)) to promote the denitrification (Gonzalez-Fernandez et al., 2011). Therefore, nitrification stimulated by microalgal oxygen production and assimilation into biomass are responsible for the nitrogen removal in this system. 3.3. Biomass generation during the operation As shown in Fig. 2, the VSS increased during the operation. The mean VSS was increased from 0.34 ± 0.10 g/l in period 1 to

1.2

reactor A with biofilm 1

reactor B without biofilm

0.8 VSS (g/l)

p > 0.05) for reactor A and B in period 3, respectively. However, at an HRT of 8 days, the N-NH+4 removal efficiency in reactor A (73.20 ± 4.22%) was significant higher (p < 0.05) than that of reactor B (66.35 ± 4.90%). N-NO3 was always detected in the effluent of both reactors throughout the test due to the nitrification role (Fig. 1C). Nitrification is a common process that takes place when the dissolved oxygen is high in the medium (Gonzalez-Fernandez et al., 2011). There are two processes in nitrification, namely nitritation (NH+4 is oxidized into NO2 ) and nitratation (NO2 is further oxidized into NO3 ). Microalgal oxygen production stimulates the nitrifier ammonium oxidation (Gonzalez-Fernandez et al., 2011), which is consistent with the trend of the DO that the DO increased gradually since the beginning of the operation and became stable till period 2 when the intensive nitrification was occurred correspondingly. However, with the decreases in the HRT, less nitritation and more nitratation were occurred and no nitrite was detected since period 3 in both reactors. The total nitrogen removal efficiency was lower in reactor A (56.99 ± 11.15%) than that of the reactor B (63.83 ± 5.70%) in period 1. No significant differences were found in total nitrogen removal between reactor A and B in both period 2 and period 3. However, total nitrogen removal efficiency in reactor A (42.95 ± 5.11%) presented significant higher at an HRT of 8 days (period 4) compared with that of reactor B (38.06 ± 5.80%). Four possible mechanisms, including ammonia volatilization, nitrification, denitrification and assimilation into biomass, are involved in the nitrogen removal in algae-based system. Ammonia volatilization is the main nitrogen removal mechanism at high temperature and pH (pH above 10). However, the contribution of ammonia volatilization throughout the experiment can be considered negligible as relatively low pH (<8.5) is present in both reactors (Fig. S1A) throughout the operation. Besides, no denitrification was occurred in both reactors because the DO concentration was too high (above 4 mg/l, Fig. S1B) for denitrification process

0.6 0.4 0.2 0

2

1 0

20

40

60

4

3 80 Date

100

120

140

Fig. 2. Time course of volatile suspended solids (VSS) in the reactor A (with biofilm) and the reactor B (without biofilm).

Please cite this article in press as: Su, Y., et al. The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.06.099

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Y. Su et al. / Bioresource Technology xxx (2016) xxx–xxx

0.90 ± 0.04 g/l in period 4 for the reactor A and from 0.46 ± 0.13 g/l in period 1 to 0.82 ± 0.13 g/l for the reactor B. No significant differences in VSS concentration were found between reactor A and B in period 1, 3 and 4. However, at an HRT of 13.5 days (in period 2), the VSS concentration in the reactor B was significant higher (p < 0.05) than that of the reactor A, with an average value of 0.57 ± 0.12 g/l and 0.73 ± 0.03 g/l for reactor A and B, respectively. The mean biomass concentration throughout the test of the reactor A (0.63 ± 0.25 g/l) was slightly lower than that of the reactor B (0.69 ± 0.20 g/l). ANOVA analysis showed that the biomass concentrations in these two reactors during the whole operation were statistically similar (P > 0.05). If the biofilm get too thick, the interior layer would get dark due to light attenuation and suffer the nutrient depletion caused by the less transport of nutrients across the film (Kesaano and Sims, 2014; Schnurr et al., 2013). In this study, the wall attached biofilm did not slough from their substratum during the operation. The physiochemical properties of the material and the surface roughness play important role in the algal attachment (Gross et al., 2015). 4. Conclusions When the HRT was reduced from 18 to 8 days, the reactor with wall attached biofilm showed significant higher total nitrogen and phosphorus removal capability compared with the reactor without biofilm. While, the wall attached biofilm had no influence on biomass generation during wastewater treatment as no significant differences were found in biomass concentration between these two reactors. This study supplements new information on the unintended reactor wall attached biofilm in suspended algaebased wastewater treatment system. Acknowledgements This work was supported by German Academic Exchange Service (DAAD) and Europäischer Fonds für regionale Entwicklung (EFRE). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2016.06. 099.

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Please cite this article in press as: Su, Y., et al. The long-term effects of wall attached microalgal biofilm on algae-based wastewater treatment. Bioresour. Technol. (2016), http://dx.doi.org/10.1016/j.biortech.2016.06.099