in recent decades , the use of pharmaceutical and personal care products ( ppcps ) has increased dramatically [ 1 , 2 ] . they are members of a group of chemicals newly classified as organic microcontaminants in water after pesticide and endocrine disrupting compounds , which stably exist in nature , have properties of being hard - biodegraded , bioaccumulation , and long - range hazardous , posing far - reaching and unrecoverable hazard on ecosystem [ 35 ] . antibiotic medicine sulfamethoxazole features classic ppcps , with very low removal ratio in water treatment and high frequency to be detected . in recent decades , although the consume of sulfamethoxazole has been reduced , it is the most popular germifuga in animal food production . it is reported that smx applied in veterinary directly discharges into the aquatic environment , which has high toxicity . however , most attention has been focused on identification , fate , and distribution of ppcps in municipal wastewater treatment plants [ 9 , 10 ] . the commonly used treatment methods include advanced oxidation process , adsorption , and membrane technology [ 1113 ] . bioflocculation absorption method has several advantages over other methods , such as going green , being environmentally protective , no second pollution , and being biodegradable . what is more , bioflocculation has been proved to be highly effective and wildly applied , and yet there is no published research on bioflocculation removal of ppcps . bioflocculant mfx is a metabolized production with good flocculant activity , generated and secreted by klebsiella sp . into the extracellular environment , composed of macromolecular polysaccharide and protein . in this study , based on its physical and chemical property , the effective ingredient of mfx is extracted by water abstraction and alcohol precipitation , transformed into dry powder . the study aims at developing an effective treatment method of sulfamethoxazole and expanding the applied range of bioflocculation . the strain was screened by our laboratory from activated sludge in municipal wastewater treatment plants and preserved in china general microbiological culture collection center ( cgmcc number 6243 ) . peptone 10 , nacl 5 , beef extract 3 , agar 15~18 , water 1000  ml , ph 7.0~7.2 ; flocuclant fermentation medium ( g / l ) : glucose 10 , yeast extract 0.5 , urea 0.5 , mgso47h2o 0.2 , nacl 0.1 , k2hpo4 5 , kh2po4 2 , h2o 1000  ml , ph 7.2~7.5 . flocculating rate : 5.0  g chemically pure kaolin clay , 1000  ml tap water , and 1.5  ml 10% cacl2 liquid are added into a beaker , ph is adjusted to 7.2 by adding naoh , then 10  ml flocculant is added , compared with control without flocculant addition . flocculator is applied during the experiment , after 40  s fast mixing , and changed into slow mixing for 4 minutes , after 20  min settling , and the absorbance of the supernatant is measured under 550  nm by 721 uv spectrometer . the flocculation efficiency is calculated as follows : 
 ( 1)flocculation  efficiency=(ab)a100% , 
 where a is turbidity of the supernatant in control ( light transmittance ) ; b is turbidity of the supernatant in sample . the removal efficiency of sulfamethoxazole is calculated by the following equation : 
 ( 2)remove  efficiency=(cd)c100% , 
 where c is the concentration in control and d is the sulfamethoxazole concentration after treatment :  polysaccharide measurement : phenol - sulphuric acid method . add 2x volume absolute alcohol ( precooled under 4c ) to fermentation liquid , and filter and collect the white flocs after mixing . add 1x volume absolute alcohol to filtered liquid , and then collect the white flocs again . add small amount of di water to collected flocs , after uniformly dissolving , freeze the flocculants in the ultralow temperature freezer for 24  h , and then put them into freeze drying to change the flocculants into dry powder . chromatographic column : c18 ( 2504.6  mm , 5  um ) ; mobile phase is formic acid water : acetonitrlle ( 60  :  40  v / v ) ; flow rate 1.0  ml / min ; sample size 10  l ; column temperature 30c ; wave length 265  nm . add flocculants into 1  mg / l sulfamethoxazothe liquid with dosage 0  ml , 1  ml , 3  ml , 5  ml , 7  ml , and 9  ml ; set the coagulant aids dosage as 0  ml , 0.5  ml , 1  ml , 1.5  ml , and 2  ml ; adjust ph value to 4 , 5 , 6 , 7 , and 8 under 5c , 15c , 25c , 35c , 45c , and 55c ; change the reaction time as 0  h , 0.25  h , 0.5  h , 0.75  h , 1  h , 2  h , 4  h , 6  h , 8  h , 10  h , and 12  h ; calculate the removal rate . based on the preliminary obtained optimum condition , flocculant dosage , coagulant - aid dosage , ph , reaction time , and temperature are considered as influencing parameters . mix the flocculant mfx and sulfamethoxazole with initial concentration as 0.8  mg / l , 1  mg / l , 1.2  mg / l , 1.4  mg / l , 1.6  mg / l , and 1.8  mg / l separately , under different temperature condition as 15c , 35c , put on 140  rpm shaking table with constant temperature , conduct adsorption isotherm experiment , and adsorption time is 1  h. the strain j1 was short rod - shaped , cream - colored , viscous , smooth , and gram - positive . the j1 showed a high yield of flocculant and good flocculation activity toward kaolin suspension . the active ingredients of bioflocculant mfx produced by j1 distributed mainly in the supernatant after the first centrifugation of fermentation broth , that is to say , the flocculation active extracellular secretions remain freely in fermentation broth . flocculation ratio after first centrifugation appeared negatively , which proved that the j1 itself was not responsible for the flocculation . after ultrasonic crushing and centrifugation , bacteria cells were broken and the intercellular content went into the supernatant ; the negativity of flocculation ratio showed the fact that the intercellular content may not have flocculation effect . what is more , fermentation broth without inoculation has relatively high flocculation ratio , and it may be caused by the flocculation effect and coagulation aid effect of the phosphate or other inorganic salts . thus , we may reach the conclusion that the flocculant active ingredient is the metabolized production ; in the meantime , the growth medium also contributes to the flocculation . by the isolation and purification of flocculation active ingredients , removing disturbance of growth medium , a further conclusion may be reached ; that , is the active ingredient is the secondary metabolites of bacteria fermentation ( extracellular polymeric substances ( eps ) ) . table 2 presents mfx that has apparent results in the saccharides and protein chromogenic reactions . according to table 2 , we can conclude qualitatively that the major ingredients of the flocculant produced by j1 are polysaccharides and protein , the polysaccharides content is 0.0656  mg / ml , and the protein content is 0.1021  mg / ml . after the enzymatic digestion of eps , polysaccharides were removed , while proteins remained . the proteins accounted for 15.05% ( cellulase ) , 61.9% ( -amylase ) , and 11.4% ( -amylase ) of the flocculation activity . obvious decrease in flocculation activity was observed after the flocculant mfx was exposed at 70c for 20  min , indicating that it was low thermostable . this implies that the active constituents in mfx were proteins and polysaccharides , and proteins dominant accounted for the flocculation activity . five parameters : ph value , flocculant dosage , coagulation aid ratio , flocculation time , and temperature are measured to see the effects of these factors on sulfamethoxazole removal efficiency . along with the changes of ph value , the removal efficiency increases firstly then decrease , and changes sharply , from where we could know that ph does affect removal ratio a lot . it is shown in figure 2(a ) that , between ph 4 and 5 , the removal efficiency increases along with ph increment . when ph is 5 , mfx possesses the strongest flocculation capacity and the highest flocculation efficiency , which is 67.2% . between ph 6 and 8 , the removal efficiency falls steeply when ph is 8 , and the removal efficiency is only 1.61% . the result demonstrated that the bioflocculant has higher removal efficiency on sulfamethoxazole in the acidic condition , while the alkali condition results in relatively poor removal efficiency . figure 2(b ) shows that along , with the increase of flocculant dosage , the removal efficiency increases firstly , then decreases , and changes acutely . when flocculant dosage varies within the range from 1  ml to 5  ml , the removal efficiency improved with the increasing dosage . when flocculant dosage is 5  ml , the optimum removal efficiency is obtained , which is 57.89% . as seen in figure 2(c ) , the dosage of coagulant aid also has impact on the removal efficiency . when coagulant aid dosage is 0.1 times of flocculation dosage , which is 0.5  ml , more than 50% removal efficiency is reached . afterwards , the increment of coagulant aid dosage decreases flocculation capability and removal efficiency . in the meantime , the removal efficiency is around 20% without coagulant aid addition , which proves that bioflocculant could remove sulfamethoxazole without coagulant aid . figure 2(d ) shows that the removal efficiency increases firstly , then decreases with the change of temperature , but within narrow fluctuation range . when temperature is between 5c and 25c , the removal efficiency increases slowly . when temperature is 35c , the strongest flocculation capability is obtained and the highest removal efficiency is reached , which is 67.20% . the removal efficiency decreases on the temperature of 45c and 55c . according to figure 2(e ) , the removal efficiency increases exponentially within the first 30 minutes , after 30 minutes the increment slows down and remains the same after 1 hour , reaching equilibrium . this is because of that a large amount of adsorbate exists in the liquid in the beginning of the reaction and is adsorbed quickly by adsorbent . with the occupation of the adsorption sites , the optimum flocculation condition is ph 5 , flocculant dosage 5  ml , coagulant aid dosage 0.5  ml , flocculant reaction time 1  h , and temperature 35c . under this condition , it is reported that the removal efficiency using conventional water treatment process including preoxidation , coagulation , and sand filtration is 36% , and the removal efficiency by microelectrolysis fentonis is 65.5% . it can be seen that bioflocculant is obviously more efficient than normal treatment . in order to evaluate the removal effect of bioflocculant mfx on sulfamethoxazole in actual wastewater , domestic wastewater 9 sets of experiments are conducted according to the orthogonal design table l9 ( 3 ) , and results are shown in table 4 . as is shown in table 4 , according to average value analysis , optimum flocculation condition is the combination of a1b3c2d2 , which is ph 5 , flocculant dosage 8  ml , coagulant aid dosage 0.2  ml , and reaction time 1  h. it has been proved that , under this condition , the removal efficiency of sulfamethoxazole is 53.27% . by comparing the extremums , we may reach a conclusion that the effect degrees of factors obey the following order : ra > rb > rc > rd . that is to say , ph value affects mostly , followed by flocculant dosage , coagulant aid dosage , and reaction time . proper ph value could increase the dissociation degree , lead to a higher charge density of flocculant , benefits the spreading of the flocculant molecules , and facilitates the bridging action of the bioflocculant . when the flocculant dosage is relatively low , early adsorption saturation may be reached , the removal efficiency of contaminants decreases . when flocculant dosage is high , extraflocculant weakens the bridging effect due to adsorption sites overlapping and finally affects the removal efficiency of specific contaminant . sulfamethoxazole flocs in the water are negatively charged , and when approaching positively charged flocculant hydrolyzates and calcium ions in coagulant aid , charge neutralization occurs on the surface of sulfamethoxazole and makes the colloidal particles to sediment , exacerbating the collision of colloids and collision between colloids and flocculant , integrating a whole group under van der waals ' force , finally precipitating from water by gravity . in the research of removal mechanism of sulfamethoxazole aqueous solution , the highest removal efficiency is more than 60% , but , in the removal of sulfamethoxazole in wastewater , the highest removal efficiency under optimum condition is only 53.27% . this may be caused by the relatively low concentration of sulfamethoxazole in wastewater , which is 23.26  ug / l . the limitation of measurement methods may lead to potential systematic errors . on the other hand , domestic wastewater has complex component , and there exists reversible and irreversible competitions among substrates , liming the combination of flocculant and sulfamethoxazole . what is more , some unknown ions and organic compounds may also decrease the removal efficiency . the dry power of mfx is white , sparses and reticulate , while the aqueous solution is milky white , ropy , and turbid . the material of mfx adsorbent is glycoprotein which has hydrophobicity and displays a wide range of sorption behavior for hydrophobic organic compounds in aqueous solutions . the adsorption isotherms of sulfamethoxazole on mfx adsorbents are presented in figure 3 and table 5 . adsorption data are fitted to freundlich isotherm model which is the most widely used models for describing adsorption phenomena in aqueous solutions . the freundlich isotherm model is an empirical equation accurate for describing adsorption in aqueous solutions at low solute concentrations . expressions and interpretations are as follows . the maximum adsorption capacity of the adsorbent is represented by kf ( mg / g ) , while n is constant which is indicative of the adsorption energy and intensity : 
 ( 3)cs = kfcw1/nlgcs=1nlgcw+lgkf , 
 where cs is mass concentration in solid phase after adsorption equilibrium ( mg / g ) ; cw is concentration in liquid phase after adsorption equilibrium ( mg / l ) . the results show that mfx exhibited high - adsorption capacities for sulfamethoxazole in water . a maximum adsorption capacity ( kf ) of 176.6445  mg / g was obtained with mfx in 35c . compared figure 3(a ) with figure 3(b ) , it can be perceived that linear correlation is marked in 35c . according to n value , it is known that , under this temperature , the adsorptive property of mfx to sulfamethoxazole is high . however , mfx has poorer adsorption efficiency in 15c . this is due to the effect of temperature on chemical reaction and molecular movement , which finally promote or restrain flocculent effect . on the one hand , providing appropriate temperature , colloidal particle is bombarded intensively by molecules of flocculation to form a whole . if temperature is excessively low , molecular movement slows down , and reaction rate lessens , leading to bad adsorptive property . on the other hand , some active groups between bioflocculant and sulfamethoxazole start the chemical reaction to separate out of aqueous solution system . what is more , when hydrophobic chain polymer - bioflocculation mfx comes into sulfamethoxazole aqueous solution system , with the help of appropriate ph and ca , sulfamethoxazole becomes unstable rapidly , passing into flocculation phase . driven by such mechanism , the adsorption on mfx does not rely on a high porosity and a resultant high specific surface area to reach a high adsorption capacity , as for most activated carbon and polymeric adsorbents . this result implies that hydrophobic partitioning is an important factor in determining the adsorption capacity of mfx for sulfamethoxazole solutes in water ; meanwhile , some chemical reaction probably occurs . this work demonstrates the efficient removal of sulfamethoxazole from water using bioflocculant mfx as adsorbents . the findings are summarized as follows.the active flocculent constituents of mfx are eps which is composed by polysaccharides and proteins fermented by j1 . the optimum condition is 5  mg / l bioflocculant , 0.5  mg / l coagulant aid , initial ph 5 , and 1  h reaction time , and the removal efficiency could reach 67.82% . using mfx , the removal rate of sulfamethoxazole in domestic wastewater can reach 53.27% . the effect size of ecological factor is as follow : ph > flocculant dosage > coagulant aid > reaction time.it is efficient for mfx to remove sulfamethoxazole in aqueous environment in 35c . it is inferred that hydrophobic partitioning is an important factor in determining the adsorption capacity of mfx for sulfamethoxazole solutes in water . the active flocculent constituents of mfx are eps which is composed by polysaccharides and proteins fermented by j1 . proteins mainly accounted for the flocculation activity .   the mfx displays great sorption behavior for sulfamethoxazole in aqueous solutions . the optimum condition is 5  mg / l bioflocculant , 0.5  mg / l coagulant aid , initial ph 5 , and 1  h reaction time , and the removal efficiency could reach 67.82% .   using mfx , the removal rate of sulfamethoxazole in domestic wastewater can reach 53.27% . the effect size of ecological factor is as follow : ph > flocculant dosage > coagulant aid > reaction time . it is inferred that hydrophobic partitioning is an important factor in determining the adsorption capacity of mfx for sulfamethoxazole solutes in water . the study shows that bioflocculation mfx can be used as an efficient alternative adsorbent for the removal of sulfamethoxazole in water , with high - adsorption capacities observed in actual wastewater . further studies are underway to make mathematical models of the relationship between flocculant and contaminant . when many ppcps coexist , the research on removal efficiency with bioflocculant is more significant .
although the treatment technology of sulfamethoxazole has been investigated widely , there are various issues such as the high cost , inefficiency , and secondary pollution which restricted its application . 
 bioflocculant , as a novel method , is proposed to improve the removal efficiency of ppcps , which has an advantage over other methods . 
 bioflocculant mfx , composed by high polymer polysaccharide and protein , is the metabolism product generated and secreted by klebsiella sp . in this paper , 
 mfx is added to 1  mg / l sulfanilamide aqueous solution substrate , and the removal ratio is evaluated . according to literatures review , for mfx absorption of sulfanilamide , 
 flocculant dosage , coagulant - aid dosage , ph , reaction time , and temperature are considered as influence parameters . 
 the result shows that the optimum condition is 5  mg / l bioflocculant mfx , 0.5  mg / l coagulant aid , initial ph 5 , and 1  h reaction time , and the removal efficiency could reach 67.82% . in this condition , 
 mfx could remove 53.27% sulfamethoxazole in domestic wastewater , and the process obeys freundlich equation . 
 r2 value equals 0.9641 . 
 it is inferred that hydrophobic partitioning is an important factor in determining the adsorption capacity of mfx for sulfamethoxazole solutes in water ; meanwhile , some chemical reaction probably occurs .