2017 年 23 巻 6 号 p. 819-826
Microalgae are not only potential feed stock of biodiesel, but also an important source of high biological value products. Polysaccharide, as a family member of microalgae, has several important biological activities. In this paper, the culture conditions were optimized by response surface methodology for the production of intracellular water-soluble polysaccharide (IWSP). The highest IWSP yield of 88.1013 mg/L was obtained with the culture condition of NaNO3 362.5 mg/L, NaCl 85.4 mg/L and NaHCO3 2.4925 g/L. Gravimetric analysis revealed a total lipid content of 27.56% in this culture. IWSP was composed of mannose, xylose, galactose and glucose. The fatty acid of lipid was rich in unsaturated fatty acids contributed 55.38%; the main saturated fatty acids were C16:0, which counted for 41.98%. The antimicrobial activity of IWSP showed that antibacterial activity is higher than antifungal activity. It showed the highest antibacterial activity against Pseudomonas aeruginosa and weak antimicrobial activity against Escherichia coli, Staphylococcus albus, Staphylococcus aureus and Saccharomyces cerevisiae.
Conventional biodiesels are mainly produced from soybean and vegetable oils (Bunyakiat et al., 2006), palm oil (Widyan and Shyoukh, 2002), sunflower oil (Antolin et al., 2002), rapeseed oil (Peterson et al., 1996) as well as restaurant waste oil (Bouaid et al., 2007). Producing lipids from microbial fermentation is a new approach of lipids. More and more oleaginous microorganisms are used to produce functional oil and biodiesel. With the advantages of high photosynthetic efficiency, fast growth rate, no restriction of season and climate, and easy to scale-up, microalgae are considered to be raw materials for biodiesel with a lot of potential (Chisti, 2007). Exploiting oil resource with microalgae has attracted more and more attention and biodiesel production with microalgae has become one of the important research areas.
However, high cost is the main problem that limits the production of biodiesel by microalgae (Connemann, 1998). To the best of our knowledge, there has been no breakthrough in microalgae biodiesel industrial production and commercial development. Therefore, decreasing the production cost is very important, only in this way the microalgae biodiesel can truly become an alternative source of energy.
Microalgae are not only raw materials for biodiesel, but also important sources of the additional high-value products, such as carotene (Hejazi et al., 2004), lutein (Sánchez et al., 2008), unsaturated fatty acid (Liang et al., 2005), astaxanthin (Mendes-Pinto et al., 2001), lectin (Alvarez-Hernández et al., 1999), phycobiliproteins (Soni et al., 2008), dinoflagellate toxins, active polysaccharides (Hellio et al., 2004) and other biological active substances. If these valuable products can be exploited and utilized together with the microalgae biodiesel, the production costs will be reduced.
Microalgae can be used to produce a variety of polysaccharides, which have significant biological activities. Although polysaccharide of Spirulina only accounts for about 10% of the dry biomass, it has a unique medicinal value for its unique chemical structure. The results showed that Spirulina polysaccharide could significantly enhance the removal, repair and unscheduled DNA synthesis of DNA damage caused by radiation, which also had certain anti-mutation functions (Qishen et al., 1989). The polysaccharide of Chlorella showed anti-tumor activity on ascites hepatoma AH 44, AH 41c, leukemia L-120, ehrlich ascites tumor and MethA tumors (Yan et al., 2013). Hayashi et al. (1996) isolated and purified a sulfated polysaccharide from Spirulina platensis, which could inhibit the replication of HIV-1 virus, and had inhibition effect on a variety of virus in vitro. Fabregas et al. (1999) reported that the sulfated polysaccharide from water extract of Dunaliella had inhibitory effect on the replication of VHSV and ASFV in vivo.
Since the earliest screening research of algae antibacterial activity by Pratt (1951), many scholars in the world have carried out extensive screening of the algae antibacterial activity, and they have found a lot of seaweed extracts that can inhibit bacteria and fungi. Based on these studies, the further researches find that the antibacterial compounds are mainly lipid and fatty acid, polysaccharide and terpenoid, etc. Padmakumar and Ayyakkannu (1997) reported that the antibacterial activity of the algae extracts was greater than antifungal activity. The experimental results of Richard showed that the microalgae extract had inhibition to Gram positive bacteria rather than gram negative bacteria (Richard et al., 1998).
The polysaccharide productions of microalgae distinguish with their environment, and the dominating factors are light, pH, salt and incubation time. Studies showed that, with the pH of 8.0 in culture medium, the contents of the algal intracellular and extracellular polysaccharide were pH independent and remained at their maximum value. The contents of the intracellular and extracellular polysaccharide were the highest when the concentration of sodium chloride is 0.5 g/L.
At present, the researches on microalgae polysaccharides mainly focusing on marine microalgae, while the researches on freshwater microalgae are less. There has been no study to exploit microalgae polysaccharides together with the microalgae biodiesel. In this research, the high producing oleaginous freshwater microalgae (Chlamydomonas sp. YB-204) was selected as the research strain. We discussed about the effects of different culture conditions on IWSP and lipid production, and the response surface method (RSM) was used to optimize the culture conditions of Chlamydomonas sp. YB-204 for IWSP production.
Materials Chlamydomonas sp. YB-204 was isolated by our laboratory. A SE medium was used in this study, and the microalga grew in a temperature controlled incubator at 25 ± 2°C under a photoperiod of 16:8 h.
Bacillus subtilis (SCTCC 100034), Staphylococcus aureus (SCTCC 100048), Staphylococcus albus (SCTCC 100044), Escherichia coli (SCTCC 100005), Salmonella typhimurium (SCTCC 100403), Pseudomonas aeruginosa (SCTCC 100200), Saccharomyces cerevisiae (SCTCC 300192), Rhizopus chinensis (SCTCC 400512) and Aspergillus niger (SCTCC 400320) are provided by Sichuan Type Culture Collection.
The optimization of culture conditions of IWSP production
Screening of important conditions To determine which variables significantly affected IWSP production, single-factor tests were used. Six variables were screened respectively as follows: NaCl concentration from 25 mg/L to 150 mg/L; NaNO3 concentration from 125 mg/L to 750 mg/L; K2HPO4 concentration from 37.5 mg/L to 300 mg/L; MgSO4 concentration from 50 mg/L to 150 mg/L; Medium initial pH from 6.0 to 9.0; NaHCO3 concentration from 0 to 4.0 g/L.
Optimization of screened conditions The response surface method (RSM) (Masmoudi et al., 2008) was used to optimize the culture conditions of YB-204. According to the results of the single-factor tests, the Plackett-Burman (PB) test was designed to screen the important factors. A central composite design (CCD) was employed in this regard. NaNO3(X1), NaCl(X2) and NaHCO3(X3) were chosen for independent variables. The range and center point values of three independent variables were based on the results of single-factor screening.
The quadratic model for predicting the optimal point was expressed according to:
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where Y is the dependent variables (IWSP production), β0 is the model constant, βi , βij and βjj are the model coefficients. They represent the linear, quadratic and interaction effects of the variables respectively. The variable, Xi is the non-coded independent variables. It should be noted that in the present study three variables are involved (hence, n =3).
Experimental design data analysis and predicted responses calculation were carried out by Design Expert software (trial version 8.0). Further optimization was performed by canonical method (Arteaga et al., 1994). The resulting response surface plots denoted the response function on Z-axis with X- and Y-axes representing the two independent variables while keeping the other constant at their center points. Analysis of variance (ANOVA) was conducted on the actual level of variables to identify the effects of individual variables. Stepwise deletion approach (a process of backward elimination) of individual non-significant terms (p > 0.05) was conducted to simplify the full model. Models that include insignificant variables were termed over-fitted, and often were unrealistically well fitted. Recalculation of the coefficients of regression equation with the exclusion of such terms was done with caution that it should not produce final model with drastically reduced R2 (adjusted) and increased in the estimator of variance, S. The terms would be included back into the model if such things occurred (Kristoa et al., 2003).
The extraction of IWSP The microalgae culture was centrifuged at 5,000 rpm for 10 min and the harvested biomass was dried in vacuum at 60°C until it remains at a constant weight. Ultrasonic-associated hot water extraction was used to extract IWSP: extraction medium of NaOH (4%), solid to liquid ratio of 1:25 (g/mL), ultrasonic extraction 10 min, ultrasonic power 200 W, ultrasonic broken for two times at the same conditions. Then, it was heated in a water bath at 70°C for 180 min, immediately centrifuged for the supernatant. The supernatant was precipitated by ethanol of 3 times volume, kept overnight, centrifuged for precipitation, then adding 3% TCA, fully stirring, until no more precipitate dissolved, repeating the above operation, while the precipitation was the coarse polysaccharide.
In this experiment, anthrone-sulfuric acid colorimetric method was used to determine the polysaccharide concentration (Philippis and Vincenzini, 1998).
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Where Cs is the measured concentration of polysaccharide and Vs is the volume of the polysaccharide solution.
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Where WD is the corresponding dry biomass of microalgae.
Lipid extraction Acid-heating extraction was used to extract the lipid: microalgae collected, dried and weighed, adding 4 mol/L hydrochloric acid, oscillation and mixing, kept at room temperature for 30 min; a boiling water bath for 10 min, then, quickly transferred to a low-temperature refrigerator for 20 min, repeated freezing and thawing three times. About 2 times of the volume of chloroform: methanol (1: 1, v/v) extracting solution was added, oscillation and mixing, centrifugation, drawing the lower chloroform layer into the new container, dried in vacuum.
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Where WL is the lipid production and WD is the corresponding dry biomass of microalgae.
Analysis of fatty acid composition Gas chromatography (MADZU GC-14B) equipped with 30 m DB-5 capillary column was used for qualitative and quantitative determination of fatty acid composition. The oven temperature program started from 150°C, increased by 4°C per min until 250°C, and then maintained for 2 min. Carrier gas, N2, was fed with a constant rate of 15 mL/min. Injector and detector (flame ionization) temperature were kept at 250°C (Chakraborty et al., 2007). The fatty acid methyl ester (FAME) was prepared by KOH-methanol methyl esterification method at room temperature (Christie, 2003). The contents of the fatty acids composition were calculated by area normalization method.
Analysis of monosaccharide composition Gas chromatography–mass spectrometry (GC–MS) (QP 2010, Shimadzu, Japan) was used for identification and quantification of the monosaccharides. IWSP (10 mg) was hydrolyzed with 2 mL of 2 M trifluoroacetic acid (TFA) at 120°C for 6 h in a sealed glass tube. The subsequent steps were carried out according to the method of Xie et al..
The antimicrobial activity of IWSP in vitro The polysaccharide was dissolved to 50 mg/mL with sterile distilled water, filtration sterilization, split charging, and cryopreservation. Antimicrobial activity was determined against the above bacteria and fungi using the paper disk assay method (Salem et al., 2011). Filter paper disk of 6-mm diameter was sterilized by autoclaving for 20 min at 121°C. Agar plates were surface inoculated uniformly from the culture of the tested microorganisms. The sterile disks impregnated with the polysaccharide solution were placed aseptically on the seeded agar plates. Ampicillin (bacteria) and nystatin (fungi) were used as positive controls, and sterile distilled water was used as negative control. The plates of bacteria and fungi were incubated at 37°C for 24 h and 28°C for 48–72 h respectively. Inhibition results were expressed as width of the clear halo surrounding each disc on cultivated agar plats. All the assays were carried out in triplicate.
Single factor experiment
Effects of NaCl concentration on IWSP and lipid production With the change of NaCl concentration, the accumulation of microalgae biomass, IWSP and lipid all had some changes (Fig.1A). The microalgae biomass decreased with the increase of salt concentration, which indicated that the growth of microalgae was inhibited at high concentration of NaCl. In other words, YB-204 was not resistant to high salinity. The content and production of IWSP increased with the rising of NaCl concentration within a certain range. The IWSP achieved maximum production of 26.23 mg/L when NaCl concentration was 100 mg/L. With the continuing increase of NaCl concentration, the IWSP content and production decreased. This might be accounted for that YB-204 would accumulate more polysaccharide to be against the change of external environment in a certain condition of salt stress, but when NaCl concentration exceeded a certain range, the growth of YB-204 would be greatly influenced.
Effects of different factors on the accumulation of microalgae biomass, IWSP and lipid. (A) NaCl, (B) NaNO3, (C) K2HPO4, (D) MgSO4, (E) NaHCO3, (F) pH. (■) Dry biomass, (▴) IWSP production, (▾) IWSP content, (△) Lipid production, (▽) Lipid content. The error bars in the figure indicated the standard deviations from three independent samples.
The lipid production reached a maximum value of 57.2 mg/L when the NaCl concentration was 50 mg/L. Later, the content and production of lipid had a certain degree of reduction with the increase of NaCl concentration. It might be a higher concentration of NaCl inhibits the accumulation of lipid.
Effects of NaNO3 concentration on IWSP and lipid production The accumulation of microalgae biomass, IWSP and lipid had a certain degree of change with the increase of NaNO3 concentration (Fig.1B). The microalgae biomass increased slightly with the rising of NaNO3 concentration. When NaNO3 concentration increased from 125 mg/L to 375 mg/L, the content and production of IWSP increased 1.7 times and 1.6 times respectively. However, IWSP content and production decreased when NaNO3 concentration was further increased. The results indicated that the effect of NaNO3 concentration on the growth of YB-204 was not significant, but it had a significant influence on the synthesis of IWSP. A lower concentration of nitrogen source benefited the synthesis of IWSP.
The content and production of lipid reached to their maximum value when NaNO3 concentration was 375 mg/L. The results showed that high nitrogen source concentration was not conducive to the synthesis of fatty acids, which agree with the results of Yongmanitchai and Ward (1991). Other researches had shown that the lipid content of some species of algae was higher in the case of the high nitrogen source concentration. It results from the different metabolic mechanisms in various microalgae.
Effects of K2HPO4 concentration on IWSP and lipid production Microalgae biomass almost stopped growing when the concentration of K2HPO4 reached 150 mg/L (Fig.1C). Meanwhile, the content and production of IWSP and lipid were the maximum when the K2HPO4 concentration was 75 mg/L, and then they would decrease with the increase of K2HPO4 concentration (Fig.1C).
K+ is not only an indispensable nutrition ion for microalgae growth, but also very important to maintain the osmotic pressure inside and outside cells as well as to promote the transport of other nutriments. What was interesting in our study was that the best K2HPO4 concentration for the synthesis of IWSP was not the optimal condition for the growth of YB-204. Research had shown that K+ nutrient limitation was conducive to the polysaccharide synthesis of Cyanothece and Aphanothece halophytica. In YB-204, K+ nutrient limitation also benefited the synthesis of IWSP and lipid accumulation.
Effects of MgSO4 concentration on IWSP and lipid production The accumulation of microalgae biomass, IWSP and lipid showed the trend of increase first and then decrease with the increase of MgSO4 concentration (Fig.1D). The biomass, IWSP content and production rapidly increased, reaching 289.5 mg/L, 9.84% and 28.49 mg/L, respectively, when the MgSO4 concentration increased from 50 mg/L to 75 mg/L; the biomass had no significant change, while the content and production of IWSP decrease with the further increasing of MgSO4 concentration. The lipid content and production reached to the maximum values when MgSO4 concentration was 100–125 mg/L.
Mg2+ plays a role in algae proliferation, carbon and nitrogen metabolism in the cell, as well as in the synthesis of fat, saccharide and protein. Studies have shown that, the promoting effect of Mg2+ on polysaccharide synthesis is mainly because it has played a role in promoting polysaccharide synthetase activation (Cao et al., 1999); Mg2+ also has an influence on the synthesis of fatty acids, suggesting that Mg2+ plays an activator role of enzymes in fatty acid synthesis. In summary, moderate amounts of Mg2+ could promote the accumulation of IWSP and lipid, but too much would have the opposite effect.
Effects of NaHCO3 concentration on IWSP and lipid production The biomass of microalgae rose sharply with the increasing of NaHCO3 concentration (Fig.1E). The content and production of IWSP reached to the maximum values of 16.24% and 55.59 mg/L, when NaHCO3 concentration was 2.0 g/L; then they would be in rapid decline with the further increase of NaHCO3 concentration. NaHCO3 provided carbon source for the growth of microalgae, so a moderate amount of NaHCO3 could promote the growth of microalgae, which was of benefit to the accumulation of biomass. However, the medium initial pH would be slightly alkaline due to the addition of NaHCO3 and it was not conducive to the accumulation of IWSP.
It was noteworthy that the lipid content reached its maximum when NaHCO3 concentration of 2.0 g/L, and then it would decrease; the lipid production continued increasing. It was because that the biomass increased rapidly while the lipid content decreased.
Effects of medium initial pH on IWSP and lipid production The biomass of YB-204 had no significant change when the initial pH ranged from 6.0 to 8.0 (Fig.1F). The biomass began to decline when the initial pH was higher than 8.0, which indicated a certain degree of inhibition on the growth. The accumulation of IWSP and lipid decreased with the increase of medium initial pH; the content and production of IWSP and lipid were the highest at initial pH 6.0 (Fig.1F). The results indicated that the slightly acidic condition was better for accumulation of IWSP than alkaline condition in YB-204.
Optimization of the screened variables
Statistical analysis and the model fitting According to the results of the single-factor tests, the PB test was designed to screen the important factors from 6 factors, and then the concentration of the important factors closing to the response values were determined by the steepest uphill experiment. Finally, NaNO3 (X1), NaCl (X2) and NaHCO3 (X3) were determined as the three process variables. The results of 17 runs using CCD were presented in Table 1 including the design and the mean observed responses.
The regression equations obtained after the analysis of variance (ANOVA) provided the levels of IWSP production as a function of the values of three process variables. The culture conditions of YB-204 could be predicted by the model:
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The analysis of fit statistics of polysaccharide production(Y) for the selected quadratic predictive model was carried out. The correlation measure for testing the goodness of fit of the regression equation was the adjusted determination coefficient (R2). The value of R2 was 0.9602 that was reasonably close to 1, which indicated a high degree of correlation between the observed and predicted values. A very low value of coefficient of the variation (C.V.) (5.45%) clearly indicated a very high degree of precision and a good deal of reliability of the experimental values. The ‘adequate precision’ measured signal to noise ratio and a value higher than 4 was desirable. The ‘adequate precision’ value of 14.975 for polysaccharide production indicated that the model could be used to navigate the design space.
Statistical testing of the model was performed in the form of analysis of ANOVA, which was required to test the significance and adequacy of the model. F-values for the lack of fit were non-significant (p > 0.05) thereby confirming the validity of the models. The model was found to be adequate for prediction within the range of experimental variables. The significance of each coefficient was determined to use the F-test and p-value in Table 2. The p values were used as a tool to check the significance of each coefficient, which in turn might indicate the pattern of the interactions between the variables. The smaller the p-level, the more significant the corresponding coefficient. It could be seen from the table that the variable with a very significant (p < 0.01) influence on polysaccharide production was linear term of NaNO3 (X1) and NaCl (X2), interaction terms of X1X3, quadratic term of NaNO3 (X12), NaCl (X22) and NaHCO3 (X32). Interaction terms of X1X2 had significant (p < 0.05) effect on polysaccharide production. However, the other terms were found insignificant (p > 0.05). The Model F-value of 18.78 (p < 0.01) implied the model was very significant.
Interpretation of the response surface model The response surface curves were plotted to understand the interaction of the variables and determine the optimum level of each variable for maximum response. In the plots two continuous variables were developed for polysaccharide production responses, while the other variable was held constant at their respective zero level (Fig.2A–C). Fig.2A showed the 3-D response surfaces, the combined effect of NaNO3 (X1) and NaCl (X2) on the IWSP production at constant ratio of NaHCO3 (X3), and it revealed an obvious increase in the response; near the center of X1 and X2 the IWSP production was maximal. When NaCl was fixed at 90 mg/L level, NaNO3 and NaHCO3 displayed an increase in the response at beginning stage, and then slightly decrease at a range of the experiment (Fig.2B). It indicated that the mutual interactions between NaCl and NaHCO3 were insignificant when NaNO3 was fixed at 350 mg/L level (Fig.2C).
(A–C) Three-dimensional plot showing the effect of the three process variables on IWSP production. (A)Y=f(X1, X2), (B) Y=f(X1, X3), (C) Y = f (X2, X3).
Verification of results The optimal values of the selected variables were obtained by solving the regression equation using the Design Expert Software. The suitability of the model equation for predicting optimum response value was investigated under the following optimal conditions: NaNO3 362.5 mg/L, NaCl 85.4 mg/L and NaHCO3 2.4925 g/L. The conditions were determined to be optimum by the RSM optimization process and were also used to predict the values of the response. With the optimal conditions, the experimental IWSP production was 88.1013 mg/L, which agreed with the predicted value of 88.1882 mg/L. Only small deviations were found between the actual values and predicted values. Therefore, the results indicated the suitability of the model employed and the success of RSM in optimizing the cultural conditions.
Fatty acid composition of Chlamydomonas sp. YB-204 In the optimal conditions of the IWSP production, the lipid content of YB-204 could reach to 27.56%. Then, the gas chromatography was used to analyze the fatty acid composition. The fatty acid of the lipid was mainly composed of a mixture of unsaturated fatty acids: C16:3 (3.95%), C16:4 (9.18%), C18:2 (15.38%), C18:3 (23.71%), C18:4 (1.49%), C20:4 (1.67%), and saturated fatty acids: C14:0 (0.29%), C16:0 (41.98%), C18:0 (2.34%). The result showed that the fatty acid contained a variety of polyunsaturated fatty acids, the unsaturated fatty acids accounting for 55.38% of the total fatty acids; the saturated fatty acids were mainly C16:0, which was 41.98% of the total fatty acids.
Monosaccharide composition Monosaccharide composition of IWSP was determined by GC–MS analysis method. Glucose was found as the major monosaccharide in IWSP, and the molar ratio of mannose, xylose, galactose and glucose was 1.86:2.25:2.57:93.32. Ray and Lahaye (1995) isolated three polysaccharides from Ulva rigida, and the monosaccharide composition consisted mainly of xylose, glucose, galactose and rhamnose. Suarez et al. (2008) isolated and purified a polysaccharide from Chlorella pyrenoidosa, consisting essentially of arabinose and galactose. It is now certain that the biological activity of polysaccharides has very important relation with its structure.
The antimicrobial activity of IWSP in vitro IWSP had a different degree of inhibition against the tested bacteria and fungi (Table 3). Among them, IWSP had the highest antibacterial activity against P.aeruginosa and the inhibition halo diameter was 16.43 mm. In addition, it had weak antibacterial activity against E.coli, S.albus and S.aureus. However, there was no antibacterial activity against B.subtilis and S.typhimurium. IWSP only had a weak antifungal activity against S.cerevisiae in the tested fungi, but no activity against R.chinensis and A.niger.
The results showed that, IWSP revealed good antibacterial activity, but weak antifungal activity. It showed a greater antibacterial activity than antifungal activity.
Some studies have shown that the extracts in organic solvent of microalgae had antimicrobial activity. Padmakumar and Ayyakkanna reported that the antibacterial activity of algae extracts is greater than antifungal activity. Rao et al. (1986) found that algae extract had no inhibitory effect on gram-negative bacteria. Richard et al. (1998) reported that microalgae extract had inhibitory activity against gram-positive bacteria but no inhibitory activity against gram-negative bacteria.
The antibacterial and antifungal activity can be different due to the type of extract and different microalgae. The antimicrobial activity of microalgae may be influenced by some factors such as the habitat and the season of algal collection, different growth stages of the algal, as well as experimental methods.
Based on the RSM analysis, the optimal condition was: NaNO3 362.5 mg/L, NaCl 85.4 mg/L and NaHCO3 2.4925 g/L. With the optimal conditions, the IWSP production was 88.1013 mg/L, and the lipid content could reach 27.56%. IWSP was composed of four monosaccharides: mannose, xylose, galactose and glucose in a molar ratio of 1.86:2.25:2.57:93.32. The fatty acid composition was rich in unsaturated fatty acids contributed 55.38%, and the main saturated fatty acids were C16:0 which was 41.98%. The antimicrobial activity experiments of IWSP showed that it had a certain degree of antibacterial activity, but weak antifungal activity. It revealed good to weak antimicrobial activity against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus albus, Staphylococcus aureus and Saccharomyces cerevisiae.
Acknowledgements This work was supported by National Natural Science Foundation of China (31401672), Key Scientific Research Projects of Henan Province (17B180003).
