Partial nitrification of wastewaters with high NaCl concentrations by aerobic granules in continuous-flow reactor

Bioresource Technology 152 (2014) 1–6

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Partial nitrification of wastewaters with high NaCl concentrations by aerobic granules in continuous-flow reactor Chunli Wan a,b, Xue Yang c, Duu-Jong Lee a,b,c,d,⇑, Xiang Liu b, Supu Sun b, Chuan Chen a a

State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China Department of Environmental Science and Engineering, Fudan University, Shanghai 200433, China c Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, Xinjiang 830011, China d Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan b

h i g h l i g h t s  Partial nitrification with aerobic granules was tested under high NaCl stress.  Reactor performance, granular characteristics, microbial community were monitored.  High NaCl led to complete conversion from ammonium to nitrite.  Mechanisms of high-salt tolerance of aerobic granules were discussed.

a r t i c l e

i n f o

Article history: Received 11 September 2013 Received in revised form 27 October 2013 Accepted 30 October 2013 Available online 7 November 2013 Keywords: Aerobic granules NaCl Continuous-flow reactor Osmoprotectants Microbial community

a b s t r a c t Wastewaters with high salinity are yielded that need sufficient treatment. This study applied aerobic granules to conduct partial nitrification reactions for wastewaters with high NaCl concentrations in a continuous-flow reactor. The present granules revealed partial nitrification performances at nitrite accumulation rate >95% and chemical oxygen demand (COD) removal at >85% at salt concentration up to 50 g l1. High salinity led to compact and tough granules. The granules applied electrogenic ion pump and sodium–calcium exchanger to reduce intracellular Na+ concentration; generated amino acids as osmoprotectants to resist the high osmotic pressure; produced excess extracellular polysaccharides and proteins with secretion of c-di-GMP; revised microbial community with halophilic strains. The present continuous-flow aerobic granule reactor (CFAGR) is a promising process to convert ammonium in highly saline wastewaters to nitrite, which can be applied with a subsequent Anammox process for efficient nitrogen removal. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction High saline wastewater is mainly discharged from chemical, pharmaceutical, paper-making, petroleum refinery or dyeing factories, and also as municipal wastewater. Biological treatment of high salinity wastewater, if feasible, would be environmentally friendly, relatively simple and cost-effective compared to physico-chemical clean-up options. However, excess ions such as K+ or Na+ induces high osmotic pressure, and causes desiccation through osmotic movement of water out of cytoplasm (Wood, 1999). Ludzack and Noran (1965) noted that organic removal or nitrification reactions were inhibited when salinity was higher than 20 g l1. Kincannon and Gaudy (1966) found that a noticeable ⇑ Corresponding author at: State Key Laboratory of Urban Water Resource and Environment (SKLUWRE), Harbin Institute of Technology, Harbin 150090, China. Tel.: +86 0451 86286790; fax: +86 0451 86282100. E-mail address: [email protected] (D.-J. Lee). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.112

decrease in BOD removal rate (about 75%) was discovered when sludge developed in non-saline water and then subjected to slug doses of 30 g l1 NaCl. Aerobic granules are self-aggregates coagulated by hydrodynamic shear, which have integrated spatial structure (Adav et al., 2008). Compared with activated sludge flocs, aerobic granules have significantly faster settling rate, good toxicity tolerance and resistance to hydraulic loading rate (Liu and Tay, 2004). Stable granules were noted to exist only in sequencing batch reactor (SBR) rather than in continuous-flow mode (Lee et al., 2010). Liu and Wang (2008) observed that aerobic granules were more slippery and regular in appearance under high salinity. Figueroa et al. (2008) proved that chemical oxygen demand (COD) and ammoniumnitrogen removal were not influenced at up to 10 g l1 of Cl. However, further salinity increase to 2%, nitrification activities of biomass was severely blocked (Dincer and Kargi, 1999). Anaerobic ammonium oxidation (Anammox) is an effective treatment process for wastewaters containing high concentrations

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of nitrogen (Jin et al., 2012). Fernández et al. (2008) and DapenaMora et al. (2010) noted that presence of 2–15 g l1 NaCl could enhance formation of Anammox granules but >13.5 g l1 NaCl would inhibit activities of Anammox bacteria (Dapena-Mora et al., 2007). Karta et al. (2006) successfully adapted Anammox bacteria to 30 g l1 salt (90% NaCl + 10% KCl) without loss of activities. Yang et al. (2011) revealed that Anammox reaction can be achieved by Annamox bacterial community with fed NH4+ + NO2 wastewater at 30 g l1 NaCl. These studies revealed that Anammox reaction can be realized at high efficiency at high levels of NaCl. Restated, the partial nitrification and Annomax can be an effective treatment process for wastewaters containing high concentrations of salts and nitrogen. Pronk et al. (2013) noted that their aerobic granules could stably exist without structural disintegration under up to 32.7 g l1 NaCl in SBR mode. These authors noted that the ammonia oxidation capability of their granule did not deteriorate at high salinity, but nitrite oxidation did. Restated, nitrite accumulation was noted at high salinity for their aerobic granules. Wan et al. (2013) achieved rapid start-up of a continuous-flow aerobic granular reactor (CFAGR) with high partial nitrification efficiency. The partial nitrification by continuous flow reactor had such advantages on facilitating aeration control strategies and maintaining total biomass, comparing with SBR. Besides, the continuous flow reactor was simpler than SBR on operation and automatic control. However, no trial has been made up to date on the use of continuous-flow reactor to achieve partial nitrification under high NaCl concentrations. This study monitored the reactor performance, granular characteristics, microbial community, and the contents of polymeric substances of CFAGR under NaCl up to 50 g1. Mechanisms for high-salinity tolerance of the aerobic granules were discussed.

2. Methods 2.1. Granules and reactor The aerobic granules were cultivated in an SBR and the experimental procedures are referred to a previous study (Wan et al., 2013). The CFAGR has two principal parts: the bottom-structure was 6  105 cm column; the top-structure resembled with triphase separator of anaerobic equipment, and a two-decker stabilizer was set to prevent the loss of aerobic granules. The aerobic granules were cultivated from the SBR and were fed to the CFAGR at volatile suspended solids (VSS) 844.3 ± 50 mg l1. The influent was simulated municipal wastewater at low C/N ratios with compositions as follow: NH4Cl 0.2 g l1; KH2PO4 0.026 g l1; CaCl2 0.01 g l1; MgSO47H2O 0.05 g l1; NaHCO3 0.5 g l1; peptone 0.02 g l1, pH 7.2 ± 0.1. The chemical oxygen demand (COD) was supplied using mixed sodium acetate and propionate (2:1). During 1–23d, 24–54d, and 55–75d, the NaCl concentrations in influent were 15 g l1, 30 g l1 and 50 g l1, respectively. The column temperature was at 28 ± 1 °C. The hydraulic retention time (HRT) of the CFAGR was 0.5d; the applied aeration flow rate was 5 l min1.

2.2. Analysis 2.2.1. Extracellular polymeric substrate and granular strength The EPS of granules was extracted using formaldehyde and NaOH (Liu and Fang, 2002). The contents of extracted proteins and polysaccharides were measured using Folin reagent (Lowry et al., 1951) and phenol-vitriol (Herbert et al., 1971), respectively. The granular strength was evaluated by ultrasound (20–25 kHz, 65 W at 2.5 s (on) 3 s (off) cycles), with the supernatant turbidity

being measured spectrophotometrically at 600 nm (Wan et al., 2013). 2.2.2. Microbial community The microbial community was investigated by polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) technique according to Wan et al. (2013). The genic DNA (100 ll) was extracted by Mo-bio kit (Mobic Inc., USA), and then PCR and DGGE (with 30–60% denaturant) were sequentially conducted as refers. The gene sequences were compared on the GenBank to identify the closest genes using the BLAST alignment tool. The phylogenetic tree was portrayed by MEGA4.1. 2.2.3. Extraction and measurement of intracellular c-di-GMP The extraction of c-di-GMP was amended referred to procedures by Simm et al. (2004). Restated, the granules were firstly freeze dried at 60 °C and 0.2 g of the dried granule and 15 ml ddH2O were loaded in a 50 ml tube. Lysozyme was added with terminal concentration of 1 mg ml1 and some glass beads (0.1 mm) were added and vortex for 15 min. Then the suspension was incubated at 37 °C for 1 h. The mixture was centrifuged at 9000 rpm for 15 min. The supernatant was transferred to a new 50 ml tube with double volumes of ethanol and vortex for 10 s. The tube was incubated at 4 °C for 1 h and shaken every 5–10 min, then was centrifuged at 9000 rpm for 15 min at 4 °C. The precipitate was incubated at 37 °C for 3 h, and then 3 ml of ddH2O was added and vortex for 10 s. The mixture was transferred to a 5 ml tube and centrifuged at 12,000 rpm for 10 min. Finally, 1 ml supernatant was loaded into chromatogram vial for high-performance liquid chromatography (HPLC) analysis to measure concentration of cdi-GMP. The HPLC (Aglient 1260, Aglient Co. Ltd., USA) was performed with a C18 column at 40 °C, detection at 254 nm by diode array detector (DAD). Runs were performed in mixed solvent (95% of solvent A as 0.9% NaCl and 5% solvent B as 100% acetonitrile) at 1 ml min1 (Hyodo et al., 2005). 2.2.4. Extraction and measurement of cation and free amino acids The extraction procedures of cations (Ca2+, Mg2+, Na+, K+) and free amino acids (proline and glutamine) were the same as the extraction of intracellular c-di-GM&P (Section 2.2.4). The supernatant was analyzed by HPLC (Aglient 1260, Aglient Co. Ltd., USA) using pre-column derivatization, equipped with refractive index detector and ZoRBAX eclipse AAA column (4.6  150 mm, 3.5 lm). The mixed solvent (40 mM Na2HPO4 as solvent A and 45:45:10 (v/v/v) of acetonitrile:methanol:H2O as solvent B) at 2 ml min1. All the standards were purchased from Agilent Co., Ltd. 2.2.5. Other analyses The contents of mixed liquor suspended solids (MLSS), COD, NH4+-N, NO2-N, and NO3-N were measured according to the Standard Methods (APHA, 1998). Concentrations of acetate and propionate were determined using a 7890A gas chromatograph (Agilent, USA) fitted with HP-FFAP capillary column (30 m  0.25 mm  0.25 lm) and flame ionization detector. The temperature was programed as follow: 80 °C for 5 min, then increasing to 200 °C at 10 °C min1. The ion concentration was analyzed by inductively coupled plasma mass spectrometry (HITACHI, P-4010). 3. Results and discussion 3.1. Reactor performance Removal efficiencies of COD were all exceeding 85% in continuous-flow aerobic granular reactor with high-salt stress (Fig. 1a). On

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(a)

Inf COD

Eff COD

COD removal rate

240

100

200

NaCl, 50 g l

60

-1

COD (mg l )

160 -1

NaCl, 30 g l

120 -1

NaCl, 15 g l

40

80

Removal rate (%)

80 -1

20

40 0

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Time (d) -

-

Eff NO2 -N

Eff NO3 -N

-

NO2 -N accumulation rate

100

60

80

48 -1

NaCl, 50 g l

-1

NaCl, 30 g l

60

36

-1

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NaCl, 15 g l

-1

+

Eff NH4 -N

-

+

Inf NH4 -N

NH4 -N, NO2 -N, NO3 -N (mgl )

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24

20

12

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-

NO2 -N accumulation rate (%)

(b)

0

0 0

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10

15

20

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30

35

40

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50

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60

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75

Time (d) Fig. 1. Reactor performances for continuous-flow reactor stressed by different concentrations of NaCl. (a) COD (b) nitrogen compounds.

1–23d with 15 g l1 of NaCl, effective COD removal was achieved after a brief acclimation phase, and COD in effluent was less than 25 mg l1. Increase in NaCl to 30 g l1, COD removal was stressed by added salt and was resumed on 30d. Further increase NaCl to 50 g l1 had minimal influence on COD removal, suggesting that the microbial community had adopted to the high salt environment. The GC spectra showed neither acetate nor propionate peaks (spectra not shown), suggesting that the effluent COD was converted to CO2 and biomass. The courses of nitrogen compounds are indicated in Fig. 1b. The ammonium concentration was removed from 50 mg l1 in influent to 5 mg l1 after 10-d acclimation. In this period, NO2-N was accumulated to a level of around 25 mg l1. Increase in NaCl to 30 mgl1 first declined NO2-N concentration to 10 mg l1, then the NO2-N concentration was increased to around 35 mg l1. Further increase NaCl to 50 g l1 the NO2-N conversion rate reached 100% from ammonium. Restated, the AOB in the present granules had adapted to the high-salt environment while the activities of NOB was completely inhibited. The tested CFAGR became a highly efficient reactor for partial nitrification from ammonium wastewaters with high salts.

3.2. Characteristics of aerobic granules Average sizes of aerobic granules were reduced from 3.5– 4.5 mm to 2.5–3.5 mm at increased NaCl concentrations (Fig. 2). At the different NaCl concentration range tested, the structural integrity of most granules remained unchanged. The seed granules

were in smooth surfaces (Fig. 2A). The appearances and granule strength approved that the structure of granules were more compact than the seed granules after cultivation under high salt concentrations. The time evolutions of turbidities of supernatant of ultrasonic treated sample indicates that the adapted, 75-d granules had higher granular strength compared with seed granules.

3.3. Microbial communities of granules The PCR–DGGE profiles for granule samples collected in the reactor on 1 (seed granules), 54 (30 mg l1), and 75d (50 mg l1) (Fig. 4A). The phylogenetic tree of DGGE profile (Fig. 4B) indicates that the incorporated bacteria were belonging to Actinobacteria phylum, Bacteroidetes phylum, TM7 phylum, and fifteen species (account for 79% to all) were belong to Proteobacteria phylum. Bands 5, 9, 11, and 19 disappeared after high salt stress, which were relating to Paracoccus sp., Uncultured Bacteroidetes bacterium, Uncultured Sphaerotilus sp. and Shinella zoogloeoides, respectively. The band 4 (Devosia sp.) was detected at 50 gl1 of NaCl, which was recognized as a halophilic bacterium. The bands 3 (TM7 phylum sp.), 13 (Leucobacter luti strain), 14 (Brevundimonas diminuta strain), and 15 (Rathayibacter festuci) were reduced in intensities but still survived in continuous-flow aerobic granular reactor at all NaCl concentrations. Conversely, the intensities of bands 1 (Arcobacter cloacae), 2 (Rhodobacteraceae bacterium), 8 (Paracoccus alcaliphilus strain), 16 (Alpha proteobacterium), and 18 (Paracoccus sp.) were increased with NaCl addition. The bands 6 (Arcobacter suis), 7 (Acinetobacter sp.), 10 (Rhodobacter sp.), 12

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Fig. 2. Appearance of aerobic granules. A: seed granules, B–D: granules at 10 g l1 (1–23d), 30 g l1 (24–54d), and 50 g l1 (55–75d) of NaCl.

protein

240

polysaccharides

PN/PS 0.80

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0.78 0.25

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0.74

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0.15

80 0.10 0.72

40

0.05

0.70

0 H1d

H23d

H54d

H75d

Sample

0.00 0

10

20

30

40

Time (min)

Fig. 3. Variances of EPS (A) and turbidities of aerobic granules treated by ultrasound tests (B).

(Sinorhizobium sp.), and 17 (Thauera sp.) were maintained in the granules, possibly correlating to the stability of granules and maintenance of reactor performance under salt stress. 3.4. Extracellular and intracellular substances The EPS in seed granules and granules collected at the end of each NaCl dosage is shown in Fig. 3A. At increased NaCl concentrations at 50 mg l1, the contents of polysaccharides were increased

from 190 mg g1 MLSS for seed granules to 210 mg g1 MLSS for adapted granules. The corresponding contents of proteins were increased from 140 mg g1 MLSS to 167 mg g1 MLSS. The concentrations of cations (Ca2+, Mg2+, Na+, K+) and of freed amino acids (proline and glutamine) from granules collected on 1d and 75d were listed in Table 1. With increased NaCl stress, the intracellular Na+ and K+ concentrations were increased from 7.09 and 2.13 mg g1 MLSS to 46.8 and 8.44 mg g1 MLSS, respectively. Meanwhile, the concentration of Ca2+ was decreased from

Fig. 4. The DGGE profile of 16S rDNA gene fragments from sludge at 1d (seed granules), 54d (30 mgl1), and 75d (50 mg l1). A: DGGE patterns; B: Phylogenetic tree.

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C. Wan et al. / Bioresource Technology 152 (2014) 1–6 Table 1 Ion concentrations and compatible solutes contents in seed granules and 75d granules. Sample

Ions concentration (mg g1 MLSS) Ca

Seed 75d

2+

9.97 ± 0.91 4.90 ± 0.71

2+

Amino acids (mg g1 MLSS) +

Mg

Na

0.33 ± 0.05 0.29 ± 0.07

7.09 ± 0.13 46.82 ± 1.44

9.97 mg g1 MLSS to 4.90 mg g1 MLSS. Stressed by high salt, the intracellular free amino acids, glutamine and proline, were increased in concentrations by 5.1–33.3 times. The contents of c-di-GMP in granules were increased slightly with increasing NaCl concentrations. Specifically, the contents of c-di-GMP for seed, 23d, 54d and 75d granules were 3.85, 3.90, 4.01 and 4.13 lg g1 MLSS, respectively. The contents of c-diGMP were mildly increased by 7.3% under 50 g l1 NaCl stress. 3.5. Mechanism In literature, the aerobic granules were shown to effectively treat saline wastewater but with morphology change or even morphology spoiled (Li and Wang, 2008; Taheri et al., 2012). Filament microbes were also noted to dominate microbial community in aerobic granules at higher salinity (Figueroa et al., 2008). The present granules revealed neither severe morphology changes nor integrity deterioration (Fig. 2). The average diameters of granules were mildly reduced, however, the intra-granular strength was enhanced (Fig. 3). Pronk et al. (2013) concluded alike in their SBR studies at high salinity. This study is the first report to have stable granules in continuous-flow reactors for partial nitrification under high salinity. High osmotic pressure was established across the cell membrane under high salinity. The salt tolerance mechanism of aerobic granules in continuous-flow reactor is proposed (Fig. 5). The granules responded to high salinity stress in the following ways. (1) The concentrations of intra-granular cations (Ca2+, Mg2+, Na+, K+) and amino acids (proline and glutamine) were

+

K

Glutamine

Proline

2.13 ± 0.11 8.44 ± 0.27

1.07 ± 0.04 6.63 ± 0.21

0.39 ± 0.03 13.41 ± 0.57

increased (Table 1). Restated, the electrogenic ion pump accommodated the intracellular Na+ and K+ concentration to maintain cell morphology (McLaggan et al., 1994). Meanwhile, the concentration of Ca2+ was decreased, likely owing to the action of antiporter membrane protein called sodium–calcium exchanger (Na+/Ca2+ exchanger) that is triggered when extracellular Na+ concentration was much higher than intracellular Na+ concentration (Philipson, 1999). Influx of K+ could lower deleterious on intracellular enzymes and structural protein; neutralize negative charges of amino acids; and regulate synthesis of compatible solutes (Oren, 1999). Therefore, halophilic prevented desiccation firstly by selective influx of K+ into and discharge Na+ out the cytoplasm (McLaggan et al., 1994). (2) The granules generated amino acids as osmoprotectants to resist the high osmotic pressure (Csonka, 1989). Measures 1975 supported that gram negative bacteria employed glutamate to counteract high saline stress, whereas the gram negative bacteria accumulated proline, both were noted to increase in excess in the granules (Table 1). (3) The granule cells produced excess extracellular polysaccharides and proteins (Table 1). The increase in intracellular c-di-GMP concentrations may be corresponding to the noted increment of polysaccharide contents (Yildiz, 2008; Wan et al. 2013). Excess polysaccharides and proteins in granule could assist enhancing intra-granular strength as noted in Fig. 2. Vyrides and Stuckey (2009) also proposed that anaerobic biomass produce extracellular polysaccharides to help them survive under sodium toxicity. (4) The microbial community was changed to adapt to the high salinity environment. The Arcobacter suis, Acinetobacter sp.,

Fig. 5. High-salt tolerance mechanism of aerobic granules in a continuous-flow reactor.

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Rhodobacter sp., Sinorhizobium sp., and Thauera sp. were predominant microorganisms in aerobic granules, recognizing as halophilic which could survive under 14.63% of NaCl. These bacteria were considered as vital microorganisms maintaining continuous-flow reactor performances and aerobic granules stability under high salinity. High salinity induces high osmotic pressure and desiccation, and the halotolerant organisms employ related osmotic pressure sensing system to counteract high osmotic pressure stress (Bremer, 2000). 3.6. Applications The aerobic granules were effectively used to treat saline wastewater in SBR (Li and Wang, 2008). High saline wastewater induces osmotic pressure, and large numbers of ions freely diffuse into cells and undermine enzyme activity (Ventosa et al., 1998), so the high saline wastewater is more difficult to treat than sewage. In this study, even though the NaCl addition reached 50 g l1, partial nitrification can be achieved with integrated aerobic granules, and the nitrite accumulation rate reached nearly 100%. As Bassin et al. (2011) observed, the activities of NOB were inhibited while those of AOB were not affected by the dosed salinity. This observation makes the present CFAGR a promising process to convert ammonium in highly saline wastewaters to nitrite, which can be used as substrates in the subsequent Anammox process for nitrogen removal. 4. Conclusion This study monitored the reactor performance on partial nitrification, granular characteristics, microbial community, and the contents of intercellular and extracellular substances of CFAGR under NaCl up to 50 g1. At high NaCl concentration, the CFAGR revealed efficient partial nitrification activities and COD removal. The granules adapted to NaCl stress and unfavorable continuousflow environment with various strategies, which were discussed in the present study. The CFAGR is proposed to be used with subsequent Anammox process to achieve efficient nitrogen removal from high-saline wastewaters. Acknowledgements This work was financially supported by Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. HC201324). References Adav, S.S., Lee, D.J., Show, K.Y., Tay, J.H., 2008. Aerobic granular sludge: recent advances. Biotechnol. Adv. 26, 411–423. APHA, 1998. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington DC. Bassin, J.P., Pronk, M., Muyzer, G., Kleerebezem, R., Dezotti, M., van Loosdrecht, M.C.M., 2011. Effect of elevated salt concentrations on the aerobic granular sludge process, linking microbial activity with microbial community structure. Appl. Environ. Microb. 77, 7942–7953. Bremer, E., 2000. Coping with osmotic challenges, osmoregulation through accumulation and release of compatible solutes in bacteria. Bacterial Stress Responses., 79–97.

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