Investigation Into the Novel Microalgae Membrane Bioreactor with Internal Circulating Fluidized Bed for Marine Aquaculture Wastewater Treatment

membranes

Article Investigation into the Novel Microalgae Membrane Bioreactor with Internal Circulating Fluidized Bed for Marine Aquaculture Wastewater Treatment

Yi Ding 1, Zhansheng Guo 1, Junxue Mei 1, Zhenlin Liang 1, Zhipeng Li 2,* and Xuguang Hou 1,*

1 Marine College, Shandong University, Weihai 264209, China; [email protected] (Y.D.); [email protected] (Z.G.); [email protected] (J.M.); [email protected] (Z.L.) 2 State Key Laboratory of Urban Water Resources and Water Environment, School of Marine Science and Technology, Harbin Institute of Technology at Weihai, Weihai 264200, China * Correspondence: [email protected] (Z.L.); [email protected] (X.H.)

Received: 25 October 2020; Accepted: 17 November 2020; Published: 18 November 2020

Abstract: A microalgae membrane bioreactor (MMBR) with internal circulating fluidized bed (ICFB) was constructed at room temperature to study the removal efficiency of marine aquaculture wastewater pollutants and continuously monitor the biomass of microalgae. Within 40 days of + operation, the removal efficiency of NO3−–N and NH4 –N in the ICFB-MMBR reached 52% and 85%, respectively, and the removal amount of total nitrogen (TN) reached 16.2 mg/(L d). In addition, · 3 the reactor demonstrated a strong phosphorus removal capacity. The removal efficiency of PO4 −–P reached 80%. With the strengthening of internal circulation, the microalgae could be distributed evenly and enriched quickly. The maximum growth rate and biomass concentration reached 60 mg/(L d) · and 1.4 g/L, respectively. The harvesting of microalgae did not significantly affect the nitrogen and phosphorus removal efficiency of ICFB-MMBR. The membrane fouling of the reactor was investigated by monitoring transmembrane pressure difference (TMP). Overall, the membrane fouling cycle of ICFB-MMBR system was more than 40 days.

Keywords: microalgae; membrane bioreactor; internal circulating ﬂuidized bed; marine aquaculture wastewater; nitrogen and phosphorus removal

1. Introduction With the industrialization and intensive development of aquaculture, the density and scale of aquaculture are expanding, and the water demand for aquaculture is also increasing. However, a large amount of aquaculture wastewater will be produced in the process of aquaculture [1]. The wastewater mainly contains inorganic nitrogen, phosphorus, organic pollutants and other pollutants [2]. If directly discharged into the water, it will lead to eutrophication. In addition, ammonia and nitrite also have strong toxic effects on aquatic organisms. These above problems have restricted the sustainable development of marine aquaculture. In order to remove the pollutants in wastewater, physical and chemical methods can obtain a high nutrient removal efficiency, but there are some shortcomings such as high energy consumption and secondary pollution [3]. In contrast, biochemical methods have the advantages of low cost, low energy consumption and low secondary pollution. Therefore, biochemical methods are favored by many researchers. At present, many kinds of nitrogen and phosphorus removal processes have been reported, but there will be problems such as high sludge yield, high-energy consumption of aeration and poor phosphorus removal efficiency. Microalgae, as a kind of bioenergy, can effectively absorb nitrogen and phosphorus in the environment during the growth process [4,5]. Meanwhile, microalgae can synthesize

Membranes 2020, 10, 353; doi:10.3390/membranes10110353 www.mdpi.com/journal/membranes Membranes 2020, 10, 353 2 of 10 carbohydrates from light energy, and produce proteins, oils and other substances through further biochemical reactions [6]. Thus, microalgae are widely used in aquaculture, food, biodiesel, agricultural fertilizer and other fields [7–10]. At present, there is much research on the removal of nitrogen and phosphorus in wastewater by microalgae. However, most of the research objects are mainly aimed at low salinity wastewater [11,12]. On the other hand, most of these studies are mainly based on sequencing batch test. Microalgae metabolites cannot be excreted in the sequencing batch reactor, which affects the nitrogen and phosphorus removal efficiency of microalgae reactor. Membrane bioreactor (MBR) has been considered to be one of the most effective technologies to treat urban and industrial wastewater in the world. There are many advantages of MBR, such as good effluent quality, high organic load, low sludge yield and small occupation area [13]. For this reason, MBR has been widely used in wastewater treatment field. In addition, the pollutants could be degraded efficiently in MBR, and the biomass could be effectively retained inside the reactor. Therefore, the combination of microalgae and MBR process can be considered. Recently, some researchers have used microalgae membrane bioreactor to treat domestic sewage and aquaculture wastewater [14,15], and achieved remarkable removal efficiency. However, most microalgae membrane bioreactors have a single structure and poor fluidity of algal fluid in the reactor [16]. Enhanced flow of microalgae in the reactor cannot only improve the efficiency of mass transfer, but also effectively alleviate the precipitation of microalgae. Therefore, based on the absorption of nitrogen and phosphorus by microalgae, a novel microalgae membrane bioreactor (MMBR) with internal circulating fluidized bed (ICFB) was constructed to efficiently degrade pollutants and enrich microalgae in order to realize the reuse of marine aquaculture wastewater. At the same time, the membrane fouling was continuously monitored in the reactor, which would provide a reference for the treatment of marine aquaculture wastewater and the utilization of microalgae.

2. Materials and Methods

2.1. Membrane Bioreactor Setup In this experiment, the ICFB-MMBR was made of plexiglass with a working volume of 3 L, as shown in Figure1. The polyvinylidene fluoride (PVDF) hollow-fiber ultrafiltration membrane (Motian Co. Ltd., Tianjin, China) was installed in the aeration zone. The pore size and surface area of the membrane was 0.03 µm and 0.02 m2, respectively. Local aeration and central baffle were used to circulate microalgae liquid in the reactor and slow down the precipitation of microalgae. The air flow rate was 0.8 L/min during the experiment. Meanwhile, the surface of membrane was scoured via the shear stress of bubble. The liquid level of the reactor was maintained by the control system. The effluent was filtered out from the membrane module by peristaltic pump. By adjusting the speed of the peristaltic pump, the flow rate of effluent was controlled. The membrane module was operated at a constant flux with a filtration cycle of 8 min on and 2 min off via the time relay to alleviate membrane fouling. The hydraulic retention times (HRT) was kept at 1 day with the room temperature and 12 h light–12 h dark cycle. Transmembrane pressure (TMP) was recorded through vacuum gauge. The TMP data presented were based on the measurements conducted after the MMBR reached steady-state. The steady-state, herein, referred to the experimental period approximately after 20 cycle periods. Only the steady-state results were reported in the paper. Once the TMP reached 30 kPa in the MMBR, the membrane modules were taken out and cleaned. The membrane foulants were carefully scraped off from the membrane surface. The membrane modules were reloaded into the bioreactors to run after cleaning. Membranes 2020, 10, 353 3 of 10 Membranes 2020, 10, x FOR PEER REVIEW 3 of 10

Influent pump vacuum gauge Peristaltic pump

Liquid level control system

Time relay

Membrane module

Saline wastewater

Lamp

Air compressor

FigureFigure 1. The 1. The schematic schematic representation representation ofof the microalgae microalgae membrane membrane bioreactor bioreactor (MMBR) (MMBR) with withinternal internal circulatingcirculating ﬂuidized fluidized bed bed (ICFB). (ICFB).

2.2.The Microalgae fluorescent and lampCultivation was placed around the reactor with a total illumination intensity of about 4000 lx. TheIn this influent experiment pH remained, Platymonas at about helgolandica 7 by adding tsingtaoensis 1 mol from/L HCl the andInstitute 1 mol of/L Oceanography, NaOH. Chinese Academy of Sciences was used as biogenic source. The microalgae cells were cultured in 2.2. Microalgae and Cultivation light incubator with f/2 medium. The flasks were shaken 3 times a day to prevent precipitation. In this experiment, Platymonas helgolandica tsingtaoensis from the Institute of Oceanography, Chinese2.3. Marine Academy Aquaculture of Sciences Wastewater was used as biogenic source. The microalgae cells were cultured in light incubatorSeawater with f/2 collected medium. from The Huang flasks Hai were (Shidao, shaken Weihai, 3 times China) a day was to preventused to synthetize precipitation. the saline wastewater. The main pollutants in wastewater were as follows: 30 mg/L NaNO3, 55 mg/L NH4Cl, 17 2.3. Marinemg/L KH Aquaculture2PO4. No buffering Wastewater agent such as NaHCO3 was added. In addition, a proper amount of trace elements was added according to the composition of f/2 medium. Seawater collected from Huang Hai (Shidao, Weihai, China) was used to synthetize the saline wastewater.2.4. Treatment The P mainerformance pollutants and Membrane in wastewater Fouling were as follows: 30 mg/L NaNO3, 55 mg/L NH4Cl, 17 mg/L KH2PO4. No buffering agent such as NaHCO3 was added. In addition, a proper amount of The influent and effluent samples of the reactor were collected every two days. After filtration trace elements was added according to the composition of f/2 medium. with 0.45 μm filter membrane, the ammonia (NH4+–N), nitrate (NO3−–N), nitrite (NO2−–N), phosphate (PO43−–P), total nitrogen (TN) and pH value of the samples were determined. Meanwhile, the 2.4. Treatment Performance and Membrane Fouling microalgae biomass and membrane fouling of the reactor were also investigated. TMP was determinedThe influent directly and e ffl viauent precise samples vacuum of the pressure. reactor The were extracellular collected every polymeric two days. substance After (EPS) filtration + withextracted 0.45 µm from filter the mixed membrane, liquor samples the ammonia was also (NHanalyzed.4 –N), nitrate (NO3−–N), nitrite (NO2−–N), 3 phosphate (PO4 −–P), total nitrogen (TN) and pH value of the samples were determined. Meanwhile, 2.5. Analytical Methods the microalgae biomass and membrane fouling of the reactor were also investigated. TMP was determinedMeasurement directly viaof ammonia precise nitrogen vacuum (NH pressure.4+–N), nitrate The nitrogen extracellular (NO3−–N), polymeric nitrite nitrogen substance (NO2−– (EPS) extractedN) and from phosphorus the mixed (PO liquor43−–P) were samples carried was out also using analyzed. an ultraviolet-visible spectrophotometer (UV- 1800, Shimadzu, Kyoto, Japan) according to the standard methods [17]. The biomass of microalgae 2.5. Analyticalwas determined Methods via optical density (OD) method [18]. The EPS consists of soluble EPS (S-EPS) and bound EPS (B-EPS). The composition of EPS was analyzed in terms of protein and carbohydrate. + ProteinsMeasurement were analyzed of ammonia by the nitrogen Lowry method (NH4 [–N),19] and nitrate carbohydrates nitrogen were (NO 3 assessed−–N), nitrite using nitrogenthe 3 (NO2 −–N) and phosphorus (PO4 −–P) were carried out using an ultraviolet-visible spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) according to the standard methods [17]. The biomass of microalgae was determined via optical density (OD) method [18]. The EPS consists of soluble EPS (S-EPS) and Membranes 2020, 10, 353 4 of 10 bound EPS (B-EPS). The composition of EPS was analyzed in terms of protein and carbohydrate. Proteins were analyzed by the Lowry method [19] and carbohydrates were assessed using the phenol-sulfuric method [20], respectively. The pH value was measured by a pH meter (FE20K, Mettler Toledo, Switzerland). Nutrient removal efficiency and removal amount were calculated by the following Membranes 2020, 10, x FOR PEER REVIEW 4 of 10 two formulas: phenol-sulfuric method [20], respectively. The pH value was measured by a pH meter (FE20K, NutrientMettler Toledo, removal Switzerland). efficiency Nutrient= (1—e removalffluent concentrationefficiency and removal/influent amount concentration) were calculated100% by × the following two formulas: Nutrient removal amount = (influent concentration effluent concentration)/HRT Nutrient removal efficiency = (1—effluent concentration/influent− concentration) × 100% Nutrient removal amount = (influent concentration − effluent concentration)/HRT 3. Results and Discussion 3. Results and Discussion 3.1. Microalgae Growth in the ICFB-MMBR 3.1. Microalgae Growth in the ICFB-MMBR During the 40 days’ operation of the reactor, the growth of microalgae in the reactor was continuouslyDuring monitored, the 40 days’ and operation the results of arethe reactor, shown the in Figuregrowth 2 of. Themicroalgae initial in biomass the reactor was was 0.12 g /L. continuously monitored, and the results are shown in Figure 2. The initial biomass was 0.12 g/L. The The biomass concentration of microalgae reached 1.32 g/L on the 20th day, and the average growth biomass concentration of microalgae reached 1.32 g/L on the 20th day, and the average growth rate rate was 60.0 mg/(L d). In order to realize the resource utilization of microalgae and not affect was 60.0 mg/(L·d· ). In order to realize the resource utilization of microalgae and not affect the the pollutantpollutant treatment treatment levellevel of of the the reactor reactor excessively, excessively, the algal the liquid algal was liquid collected was in collected the reactor. in From the reactor. From thethe 20th20th day, day, the the biomass biomass concentration concentration of microalgae of microalgae increased from increased 0.86 g/L from to 1.85 0.86 g/L g(t/heL 40th to 1.85 g/L (the 40thday), day), and and the theaverage average growth growth rate was rate 49.5 was mg/( 49.5L·d). mg/(L d). · 2.00

1.75 microalgae harvesting

1.50

1.25

1.00

0.75

0.50

Biomass concentration (g/L) concentration Biomass

0.25

0.00 0 5 10 15 20 25 30 35 40 Time (day)

Figure 2.FigureMicroalgae 2. Microalgae growth growth in the microalgae in the microalgae membrane membrane bioreactor bioreactor (MMBR) (MMBR) with with internal internal circulating ﬂuidizedcirculating bed (ICFB). fluidized bed (ICFB).

3.2. Pollutant3.2. Pollutant Removal Removal in the in ICFB-MMBRthe ICFB-MMBR The removalThe removal efficiency efficiency of pollutants of pollutants was monitoredwas monitored with with mariculture mariculture wastewater wastewater asas influentinfluent during + 40 days’during operation 40 days’ of the operation reactor. of The the removal reactor. The effi removalciency of efficiency NH +–N of wasNH4 about–N was 25%, about the 25%, concentration the + 4 +concentration of NH4 –N in effluent was 11.7 mg/L, and the quality of effluent is poor, as shown in of NH4 –N in effluent was 11.7 mg/L, and the quality of effluent is poor, as shown in Figure3a. Figure 3a. With the growth of microalgae biomass, the effluent NH4+-N concentration decreased to 7 + With themg/L growth (on the of 10th microalgae day). The removal biomass, efficiency the effl NHuent4+–N NH was4 stable-N concentration at 65% and microalgae decreased biomass to 7 mg/L + (on the 10threached day 1.32). The g/L removal (on the 20th efficiency day). Some NH4 studies–N was have stable repor atted 65% that and longer microalgae retention biomass time of reached 1.32 g/Lmicroalgae (on the 20th will day). lead to Some a decrease studies in TN have removal reported efficiency that longerand reduce retention the productivity time of microalgae of microalgae will lead to a decreasebiomass in TN[21] removal. In order e ffi to ciency achieve and better reduce utilization the productivity of microalgae of andmicroalgae not affect biomass the efficiency [21]. In of order to nitrogen and phosphorus removal efficiency due to the low biomass in the reactor, 1 L microalgae achieve better utilization of microalgae and not affect the efficiency of nitrogen and phosphorus removal liquid in the reactor was harvested on the 20th day. After microalgae harvesting, the biomass of efficiency due to the low biomass in the reactor, 1 L microalgae liquid in the reactor was harvested microalgae in the reactor decreased to about 0.86 g/L, and the NH4+–N concentration in effluent on the 20thincreased day. slightly. After microalgae However, the harvesting, effluent NH the4+–Nbiomass concentration of microalgae decreased to in 5.4 the mg/L reactor on the decreased 26th to + about 0.86 g/L, and the NH4 –N concentration in effluent increased slightly. However, the effluent

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+ NH4 –N concentration decreased to 5.4 mg/L on the 26th day, and the growth rate of microalgae biomass was consistent with that of the previous stage. In the following period, with the increase + of biomass, the concentration of NH4 –N in effluent was stable at about 2.5 mg/L, and the removal + efficiency of NH4 –N reached about 84%. On the 36th day, the growth of biomass tended to be slow, and the maximum biomass 1.85 g/L was reached on the 40th day. In addition, the flow pattern of microalgae in the reactor was more uniform during the whole operation period. Except for a small amount of microalgae on the inner wall of the reactor, there was no obvious precipitation, whichMembranes indicated 2020, that 10, x FOR MMBR PEER couldREVIEW provide a good environment for the growth of microalgae.6 of 10

(a) (b) 6 16 80 5 60 12 4 Influent 60 Effluent 40 8 3 Removal efficiency 40 2 4 Influent 20 Effluent

-N Concentration (mg/L) -N Concentration

-N concentration (mg/L) -N concentration

- 1

-N removal efficiency (%) -N removal efficiency

3 Removal efficiency -

20 4

NH 0 NO 0 0

NO 0 5 10 15 20 25 30 35 40 NH 0 5 10 15 20 25 30 35 40 Time (day) Time (days)

(d) (c) 5 3.0 Influent Effluent Removal efficiency 80 2.5 4 Effluent 2.0 3 60 1.5 2 40 1.0 1

-P concentration (mg/L) -P concentration

-N Concentration (mg/L) -N Concentration

- 0.5 3- 20

-P removal efficiency (%) efficiency -P removal

3- 0 4

NO 0.0 PO 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 PO Time (day) Time (day)

++ − − 3− 3 FigureFigure 3. The 3. The removal removal of of NH NH4 4–N–N ((aa),), NONO33−––NN ( (bb),), NO NO22–−N–N (c) ( cand) and PO PO4 4–P− –P(d) (ind )the in internal the internal circulatingcirculating ﬂuidized fluidized bed bed (ICFB)—microalgae (ICFB)—microalgae membranemembrane bioreactor bioreactor (MMBR). (MMBR).

- During3.3. Variations the 40 of days’ pH in operationthe ICFB-MMBR of MMBR, the removal efficiency of NO3 –N was 26–55%, as shown - + in Figure3Duringb. By comparing the cultivation the removalof microalgae, e fficiency there are of NO many3 –N factors and NHaffecting4 –N, the it isgrowth known of thatmicroalgae, the removal + + - efficiencyamong of which NH4 pH–N is isone better. of the most It had impor beentant reported environmental that when factors NH in microalgae4 –N and culture, NO3 –N because coexisted, + + microalgaepH determines preferentially the solubility utilized and NH mass4 –N,transfer and efficiency the presence of carbon of NH dioxide4 –N and inhibited matrix, theand absorption has a of NOsignificant3−–N by impact algae cellson the [22 metabolism]. Similar of experimental microalgae [26 phenomena]. When the pH have of wastewater been reported, is lower which than may 8, be it is more conducive to the formation of CO2, which was easy to be immobilized by microalgae [27]. the main reason for the low removal efficiency of NO3−–N in this experiment. NitriteIn this isexperiment, the intermediate the influent product pH of was many stabilized biochemical at about reactions 7 by adding of microorganisms, acid and alkali, but and excessive the effluent pH was between 7.2 and 8, as shown in Figure 4. Within this pH range, chemical processes accumulation of NO –N will cause toxic effects on microorganisms and inhibit biochemical such as volatilization2− and precipitation have little effect on ammonia and phosphate removal. Thus, reactions, which is not conducive to the growth of microalgae and the stable operation of the reactor. it can be inferred that nitrogen and phosphorus removal mainly depends on the growth of microalgae Thus,biomass. the concentration of NO2−–N was also monitored during the operation of the reactor. The results showed thatMany low studies concentration showed that of that NO CO2−2 –Nfixation was would detected lead into a the gradual effluent pH increase, in a few but days, excessive as shown in FigurepH would3c. Overall, affect the there growth was no of algaeobvious cells. accumulation Furthermore, of ammonia NO 2−-N nitrogen during is40 more days’ toxic operation. to Mostmicroalgae studies have under shown the condition that nitrate of high reductase pH [28 and]. During nitrite the reductase microalgae successively wastewater transformed treatment, many NO3 −–N + + and NOresearchers2−–N to adjust NH4 the–N pH in algaevalue cells,by adding and finallyCO2 to assimilatedthe system. However, to amino overuse acids in of the carbon form dioxide of NH4 –N. will increase operating costs. In this experiment, aeration (air) equipment was used without Therefore, NO2−–N in effluent may be due to the transformation of NO3−–N. Praveen and Loh (2016) additional CO2 supply, and the effluent pH could be controlled below 8 without a buffer. Meanwhile, constructed a microalgae photobioreactor to treat tertiary effluent, and no NO2−-N was accumulated the effluent pH tended to increase with the improvement of nitrogen and phosphorus removal under HRT of 4 days [23]. Therefore, the longer HRT may help to alleviate the accumulation of efficiency of the reactor, which showed that ICFB-MMBR could create a good environment for the NO –N. 2−growth of microalgae under the operating parameters.

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Compared with other microorganisms, microalgae are more likely to absorb phosphorus 3- in wastewater to increase biomass. The PO4 –P concentration in effluent was about 2.3 mg/L 3 during the first 6 days of operation, as shown in Figure3d. From the 16th to the 20th day, the PO 4 −–P concentration in effluent could be maintained at 1.2 mg/L. After harvesting microalgae on the 20th day, 3 + the PO4 −–P concentration in the effluent increased slightly, which was similar to that of NH4 –N. 3 Thereafter, with the increase of microalgae biomass, the concentration of PO4 −–P in effluent was stable below 0.9 mg/L with a high removal efficiency. Praveen and Loh (2016) reported that in the continuous microalgae-membrane bioreactor to treat tertiary effluent, the removal amount of TN and TP were 3.6 mg/(L d) and 0.8 mg/(L d), respectively [23]. · · Gao et al. (2016) showed that in the continuous streamer-membrane bioreactor, the growth rate of microalgae was 42.6 mg/(L d), and the removal amount of TN and TP was 5.85 mg/(L d) and · · 0.42 mg/(L d), respectively [24]. In this experiment, through continuous monitoring of water quality, · the TN and TP removal amount of the reactor was investigated. With the increase of microalgae biomass, the removal amount of TN and TP could reach 13–16.2 mg/(L d) and 2.3–3.2 mg/(L d), · · respectively. The average growth rate of microalgae was 55 mg/(L d). The removal efficiency of · pollutants and the growth rate of microalgae are higher than those reported above. This may be due to the uniform distribution of microalgae in MMBR, which can significantly improve the efficiency of mass transfer and biochemical reaction of microalgae. During the operation of the reactor, the maximum biomass of microalgae was 1.85 g/L, and the average growth rates of microalgae before and after harvesting were 60 mg/(L d) and 49.5 mg/(L d), respectively. The reason for this difference may be that · · with the increase of biomass concentration, limited light intensity will become a limiting factor for the growth of microalgae [25]. During the later stage of operation, more algal adhesion was observed in the inner wall of the reactor, which might reduce the light intensity to some extent. As a result, the growth rate of microalgae slowed down in the last few days. Overall, the growth rate of microalgae was at a higher level in the current study.

3.3. Variations of pH in the ICFB-MMBR During the cultivation of microalgae, there are many factors affecting the growth of microalgae, among which pH is one of the most important environmental factors in microalgae culture, because pH determines the solubility and mass transfer efficiency of carbon dioxide and matrix, and has a significant impact on the metabolism of microalgae [26]. When the pH of wastewater is lower than 8, it is more conducive to the formation of CO2, which was easy to be immobilized by microalgae [27]. In this experiment, the influent pH was stabilized at about 7 by adding acid and alkali, and the effluent pH was between 7.2 and 8, as shown in Figure4. Within this pH range, chemical processes such as volatilization and precipitation have little effect on ammonia and phosphate removal. Thus, it can be inferred that nitrogen and phosphorus removal mainly depends on the growth of microalgae biomass. Many studies showed that that CO2 fixation would lead to a gradual pH increase, but excessive pH would affect the growth of algae cells. Furthermore, ammonia nitrogen is more toxic to microalgae under the condition of high pH [28]. During the microalgae wastewater treatment, many researchers adjust the pH value by adding CO2 to the system. However, overuse of carbon dioxide will increase operating costs. In this experiment, aeration (air) equipment was used without additional CO2 supply, and the effluent pH could be controlled below 8 without a buffer. Meanwhile, the effluent pH tended to increase with the improvement of nitrogen and phosphorus removal efficiency of the reactor, which showed that ICFB-MMBR could create a good environment for the growth of microalgae under the operating parameters. Membranes 2020, 10, x FOR PEER REVIEW 7 of 10

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9.0

9.0 8.5 Influent Effluent 8.5 Influent 8.0 Effluent 8.0

pH 7.5

pH 7.5 7.0 7.0 6.5 6.5 6.0 0 5 10 15 20 25 30 35 40 6.0 0 5 10 15 Time20 (days)25 30 35 40

Time (days) Figure 4. Variations of pH in the influent (blue) and effluent (red) of the internal circulating fluidized FigureFigurebed (ICFB) 4. 4.Variations Variations—microalgae of of pH pH membrane in in the the influent influent bioreactor (blue) (blue) and(MMBR). and e ffleffluentuent (red) (red) of of the the internal internal circulating circulating fluidized fluidized bedbed (ICFB)—microalgae (ICFB)—microalgae membrane membrane bioreactor bioreactor (MMBR). (MMBR). 3.4. Variations of TMP in the ICFB-MMBR 3.4. Variations of TMP in the ICFB-MMBR 3.4. VariationsTMP can ofdirectly TMP in reflect the ICFB membrane-MMBR fouling of the reactor. Therefore, TMP was continuously TMP can directly reflect membrane fouling of the reactor. Therefore, TMP was continuously monitored within 40 days of reactor operation, during which no physical cleaning of membrane monitoredTMP withincan directly 40 days reflect of reactor membrane operation, fouling during of the whichreactor. no Therefore, physical cleaningTMP was of continuously membrane module was carried out. It can be seen that the variation of TMP almost increased at the constant rate, modulemonitored was withincarried 40out. days It can of be reactor seen that operation, the variation during of whichTMP almost no physical increased cleaning at the constant of membrane rate, as shown in Figure 5. In the first 7 days, TMP rose rapidly from 0.5 kPa to 2.3 kPa. However, the TMP asmodule shown was in Figure carried5. Inout. the It firstcan be 7 days, seen TMPthat the rose variation rapidly of from TMP 0.5 almost kPa to i 2.3ncreased kPa. However, at the constant the TMP rate, rising speed slowed down on the 8th day. At the same time, microalgae attachment was observed on risingas shown speed in slowedFigure down5. In the on first the 7 8th days, day. TMP At the rose same rapidly time, from microalgae 0.5 kPa to attachment 2.3 kPa. However, was observed the TMP on the surface membrane module. Subsequently, the TMP rising speed began to increase on the 36th therising surface speed membrane slowed down module. on the Subsequently, 8th day. At thethe TMPsame rising time, speedmicroalgae began attachment to increase was on the observed 36th day. on day. Meanwhile, it was observed that there were much more microalgae attached to the membrane Meanwhile,the surface itmembrane was observed modul thate. there Subsequently, were much the more TMP microalgae rising speed attached began to to the increase membrane on surface,the 36th surface, which indicated that the membrane pore blockage was more serious. Finally, the TMP whichday. Meanwhile, indicated that it was the observed membrane that pore there blockage were much was more microalgae serious. Finally, attached the to TMP the membrane increased increased to 24 kPa on the 40th day. Overall, the membrane fouling was not severe in the reactor tosurface, 24 kPa which on the indicated 40th day. that Overall, the themembrane membrane pore fouling blockage was was not moresevere serious. in the reactor Finally, operation. the TMP operation. This had also been reported, that the membrane fouling of microalgae system was lighter Thisincreased had also to been24 kPa reported, on the that40th the day. membrane Overall, foulingthe membrane of microalgae fouling system was not was sever lightere in than the thatreactor of than that of activated sludge MBR [29]. activatedoperation. sludge This had MBR also [29 ].been reported, that the membrane fouling of microalgae system was lighter than that of activated sludge MBR [29].

30 25 25 20 20 15

TMP (kPa) 15 10

TMP (kPa) 10 5 5 0 0 5 10 15 20 25 30 35 40 0 0 5 10 15 Time20 (days)25 30 35 40

Figure 5. The variations in TMP throughoutTime (days) the experimental period. Figure 5. The variations in TMP throughout the experimental period. 3.5. Variations of EPSsFigure in the 5. ICFB-MMBR The variations in TMP throughout the experimental period. 3.5. Variations of EPSs in the ICFB-MMBR EPS is closely related to the physiological state of microorganisms and plays an important role in3.5. the Variations formationEPS is closely of of EPSs membrane related in the to ICFB fouling. the- physiologicalMMBR Carbohydrate state and of proteinmicroorganisms are known and to be plays the mainan impor componentstant role ofin EPSs. theEPS formation Therefore, is closely ofrelated the membrane EPSs to contentthe physiological fouling. was monitored Carbohydrate state of during microorganisms and the protein operation areand of knownplays the reactor, an to impor be as thetant shown main role inincomponents Figure the formation6. As of for EPSs. S-EPS, of Therefore, membrane carbohydrate the fouling. EPSs accounted content Carbohydrate for was 70–75% monitored and of S-EPS protein during content are the known comparedoperation to withof be the the protein reactor, main components of EPSs. Therefore, the EPSs content was monitored during the operation of the reactor,

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as shown in Figure 6. As for S-EPS, carbohydrate accounted for 70–75% of S-EPS content compared with protein (25–30%). Because carbohydrate is one of the main parts of the algae cells wall, it would be released into the mixed liquor when the microalgae multiply [30]. In contrast, protein was the Membranesmain component2020, 10, 353 of B-EPS, as shown in Figure 6b. Since N is an essential constituting element8 of 10of proteins, higher N removal efficiency observed in this experiment might result in production of large amounts of protein in B-EPS. Additionally, EPS and cells were co-deposited during membrane (25–30%).filtration, Because forming carbohydrate the cake layer is one formation, of the main which parts could of the alleviate algae cells membrane wall, it fouling would be to released a certain intoextent the [ mixed31]. liquor when the microalgae multiply [30]. In contrast, protein was the main component of B-EPS,In general as shown, the in Figure contents6b. of Since S-EPS N is and an essential B-EPS increased constituting significantly element of with proteins, the higher growth N of removalmicroalgae eﬃciency biomass. observed However, in this the experiment increase of might EPS will result lead in to production serious membrane of large amounts fouling. ofTherefore, protein inthe B-EPS. proper Additionally, harvesting EPSof microalgae and cells were is of co-depositedgreat significance during to membranealleviate membrane ﬁltration, fouling. forming the cake layer formation, which could alleviate membrane fouling to a certain extent [31].

(a) 40 Protein Carbohydrate 30

S-EPS Concentration (mg/L)

0 10 20 30 40 Time (days)

(b) 70 Protein 60 Carbohydrate

B-EPS Concentration (mg/L) 0 10 20 30 40 Time (days)

FigureFigure 6. 6. ConcentrationsConcentrations of of S-EPS S-EPS (a) (anda) and B-EPS B-EPS (b) in (b the) in internal the internal circulating circulating fluidized ﬂuidized bed (ICFB) bed— (ICFB)—microalgaemicroalgae membrane membrane bioreactor bioreactor (MMBR). (MMBR).

4. ConclusionsIn general, the contents of S-EPS and B-EPS increased significantly with the growth of microalgae biomass. However, the increase of EPS will lead to serious membrane fouling. Therefore,In this the study, proper a microalgae harvesting membrane of microalgae bioreactor is of great (MMBR) significance with internal to alleviate circulating membrane fluidized fouling. bed (ICFB) was constructed to treat high salinity wastewater. The operation results showed that ICFB- 4.MMBR Conclusions could achieve high removal efficiency of nitrogen and phosphorus, especially for ammonia nitrogen and phosphate in wastewater. Moreover, microalgae could be enriched quickly in the In this study, a microalgae membrane bioreactor (MMBR) with internal circulating fluidized reactor. Meanwhile, the removal capacity of the reactor was not significantly affected by the bed (ICFB) was constructed to treat high salinity wastewater. The operation results showed that ICFB-MMBR could achieve high removal efficiency of nitrogen and phosphorus, especially for ammonia nitrogen and phosphate in wastewater. Moreover, microalgae could be enriched quickly in the reactor. Meanwhile, the removal capacity of the reactor was not significantly affected by the harvesting of Membranes 2020, 10, 353 9 of 10 microalgae on the 20th day. The results of TMP variations showed that the membrane fouling of ICFB-MMBR system was relatively light.

Author Contributions: Data curation, Z.G.; formal analysis, J.M.; investigation, Y.D. and Z.L. (Zhenlin Liang); writing—original draft, Z.L. (Zhipeng Li); writing—review and editing, X.H. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (No. 51408158), and the Natural Science Foundation of Shandong Province of China (No. ZR2019QEE012). Conﬂicts of Interest: The authors declare no conﬂict of interest.

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