Influences of changing inorganic nitrogen concentration on accumulation and degradation of organic components in indigenous microalgae cultivated with secondary effluent

ABSTRACT Climatic changes due to emission of greenhouse gases are a global concern. These emissions occur by combustion of fossil fuels whose drought is near in which case renewable energy is the only alternative. Microalgae are promising sources of sustainable bioenergy production, and utilisation of wastewater as cultures is recommended for economical production cost. In this study, indigenous microalgae, which had adaptability for wastewater samples, were cultivated with a municipal secondary effluent, and influences of changes in inorganic nitrogen (IN) concentration, specifically IN increase, on temporal accumulation and degradation of organic components in indigenous microalgae were investigated. Indigenous microalgae accumulated total lipids and carbohydrates against reduced IN, and increase in superoxide dismutase suggested that the accumulation was possibly induced by generating reactive oxygen species. Continued cultivation of indigenous microalgae under the IN exhausted condition should be avoided because of the resulting total carbohydrate degradation. IN replenishment when IN was decreased but still existed in the culture and that when IN was exhausted in the culture triggered sharp degradation of the total carbohydrate, which possibly utilised to accumulate crude protein and/or chlorophyll a for continuous growth or regrowth. The total carbohydrate was accumulated and recovered after the degradation; meanwhile, two or three days were required for the recovery of the total carbohydrate. In addition, the IN replenishment also resulted in total lipid degradation. Therefore, to produce indigenous microalgae with high and stable total carbohydrate and lipid content, it was critical to prevent IN increase in the culture. GRAPHICAL ABSTRACT


Introduction
Industrialisation and climatic changes have raised global concerns due to greenhouse gas emissions by combustion of fossil fuels whose drought is in sight in which case renewable energy is the only alternative [1]. Microalgae, as the third generation of biofuel feedstock, are promising sources of sustainable bioenergy production due to the faster growth rate and higher light energy utilisation efficiencies than earlier biofuel producer generations and high CO 2 mitigation efficiencies [2]. In the sewerage system, abundant nutrients discharged through human activities are gathered in wastewater treatment plants (WWTPs). Therefore, wastewater utilisation as cultures for microalgae cultivation is highly recommended for economical production costs, although it is necessary to consider the pre-treatment required to use wastewater as the culture [3]. In addition, microalgae cultivation with wastewater enhances nutrient removals in WWTPs, which contributes to potential reduction of eutrophication [4].
Cultivations of a specific microalgae strain, which is commercially available and has very high value, have been extensively investigated. However, because the cultivations of a specific strain with wastewater are susceptible to contamination by other faster growing species, the monoculture has a negative effect on the health of the cultivation system, which may lead to a collapse [5]. In addition, full-scale cultivation combined with pre-treatment (i.e. sterilisation process) of wastewater, which is costly, is not feasible [6]. Meanwhile, cultivations of indigenous strains were conducted with raw municipal wastewater [6], secondary effluent from a sewage WWTP [7], secondary anaerobic effluent of a municipal WWTP [8] and anaerobic effluents from labscale digesters treating various biomass [9], and the adaptation of the cultivation to the wastewater samples were demonstrated. Therefore, indigenous microalgae cultivation is a promising and suitable energy production option in WWTPs.
Biodiesel and bioethanol, which are generated from starch and triacylglycerol, respectively, have attracted attention as alternative energy sources [10]; therefore, organic components in cultivated microalgae is an important indicator for energy generation. Yu et al. [9] reported that nitrogen starvation increased lipid content in indigenous microalgae enriched from anaerobic sludge, based on the comparison of lipid content in cultures before and after nitrogen starvation. The carbohydrate content in Chlorella sp. L06, which were isolated from and continuously cultivated with secondary anaerobic effluent, increased with decrease in the dilution rate, because nutrients in the medium were rapidly assimilated by the biomass and not replenished at the required rate [8]. Therefore, cultivation of indigenous microalgae under nitrogen starved condition was an attractive strategy for lipid enhancement and/or carbohydrate accumulation. Meanwhile, nutrient concentrations in culture, specifically outdoor continuous cultivation, were temporally fluctuated, and the fluctuations were partly attributed to the variation of the water qualities in the secondary effluent [7]. Therefore, production of indigenous microalgae with high and stable organic matter content would be achieved by cultivations based on the response of organic matter accumulation and degradation in indigenous microalgae against nutrient fluctuations. Several studies reported temporal changes of organic matter in indigenous microalgae such as Chlamydomonas incerta, which was isolated from agro-industrial wastewater [11] and algal-bacterial consortium, which were obtained from a high-rate algal pond, with Chlorella sorokiniana [12] against nutrient reduction. However, few studies focused on the response against the nutrient increase.
Differences in the temporal responses of total lipid reduction exist in indigenous microalgae, which naturally grow with treated effluent from a WWTP, to nitrogen increase between the nitrogen-depleted and nitrogen-exhausted conditions. Their detailed mechanism was required to be investigated based on accumulation and reduction of total carbohydrate [13].
In this study, a secondary effluent from a municipal WWTP was used to conduct batch cultivations of indigenous microalgae, which naturally grow without any inoculation of microalgal species. Specifically focusing on increase in inorganic nitrogen (IN), temporal accumulation and degradation of total lipid and total carbohydrate against IN increase and decrease were investigated. In addition, the accumulation and degradation mechanisms against the IN variations were discussed based on the microalgal life strategy.

Secondary effluent collection
The secondary effluent was collected in a municipal WWTP in which the conventional activated sludge treatment process was applied, and the WWTP located in Tottori Prefecture. The secondary effluent was collected from a final sedimentation tank before the chlorination process, and pH and dissolved oxygen (DO) of the secondary effluent were measured with a pH meter (D-71; Horiba, Japan) and a DO meter (AS720; AS ONE, Japan), respectively. The secondary effluent was used for indigenous microalgae cultivation without any pretreatment, as described in 2.2.

Indigenous microalgal cultivation
Indigenous microalgae were cultivated with a polycarbonate columnar reactor whose diameter, height and effective volume were 35 cm, 34 cm and 30 L, respectively. Using a culture pH 8.0 as an indicator, intermittent CO 2 addition to the reactor was conducted to control the culture pH and provide the carbon source, referring to Takabe et al. [14]. The reactor was surrounded by 12 fluorescent lamps, each with a photon flux density of 160 μmol/m 2 /s. Each period of light and darkness was 12 h. The reactor was set under a temperature of 31.1°C ± 1.4°C (average ± standard deviation, n = 43).
Batch cultivation was conducted in the following experiments. Cultivation A (cultivation period: 13 days) was conducted to comprehend changes of organic components against IN reduction; meanwhile, Cultivation B (cultivation period: 9 days) and Cultivation C (cultivation period: 13 days) were conducted to analyse the changes of organic components against IN increase, based on the comparison to Cultivation A. Figure 1 showed a schematic diagram of all experiments. NH + 4 -N and NO − 3 -N mainly consisted of IN in the collected secondary effluent, as described below. Assuming the occurrence of IN increases in the culture due to the variations in the secondary effluent, IN was intentionally increased in the middle of the experiments by adding 137.5 mg of NH 4 Cl (Wako, Japan) and 582.8 mg of NaNO 3 (Wako, Japan) in Cultivation B and C. The weight ratio of the added NH + 4 -N and NO − 3 -N was referred to that of the secondary effluent. When IN that originated from the secondary effluent was decreased but was still found in the culture, IN replenishment was conducted in Cultivation B. The experiment was repeated twice with the secondary effluent (Cultivation B1 and B2). The collection of the secondary effluent for each experiment was conducted on different days to confirm that similar accumulation and degradation of organic components in indigenous microalgae against IN replenishment were observed in the cultivation with the secondary effluent whose water qualities were different due to temporal fluctuations. The IN replenishment was conducted on day 5.0 and 4.0 in Cultivation B1 and B2, respectively. When IN originating from the secondary effluent was exhausted in the culture, IN replenishment was conducted in Cultivation C. The experiment was conducted twice with the secondary effluent whose collection was conducted on different days (Cultivation C1 and C2) for the same reason in Cultivation B, and IN replenishment was conducted on day 9.0. Cultivation A was conducted without IN replenishment. Water samples were continuously collected during each experiment. To better understand temporal changes in microalgal components caused by IN replenishment, more frequent samplings (every 0.25 d) were performed in Cultivation B2 and C2 than in Cultivation B1 and B1 (every 0.5 d).

Analysis of water quality and microalgal components
Suspended solids (SS) measurement was followed by the APHA method (2540-B; [15]) with a GF/B membrane (pore size: 1.0 µm; Whatman, USA). The filtered water samples with the GF/B membrane were used for measuring NH + 4 , NO − 2 , NO − 3 and PO 3− 4 with an ion chromatograph (10A Series; Shimadzu, Japan), and IN was calculated by adding NH + 4 -N, NO − 2 -N and NO − 3 -N. Chlorophyll a (Chl. a) measurement was followed by a standard method [16] with a GF/C membrane (pore size: 1.2 µm; Whatman, USA), and Chl. a content in indigenous microalgae was calculated by dividing the measured Chl. a by SS. DO was measured under light conditions with the DO meter (AS720; AS ONE, Japan).
After water samples were centrifuged at 3000 g for 10 min, sedimented indigenous microalgae were collected. The collected indigenous microalgae samples were frozen and stocked before the lyophilisation. After the indigenous microalgae samples were lyophilised by DC 801 (Yamato Scientific, Japan), microalgal components in the stocked samples were measured. The lipid content was measured, referring to Zhou et al. [17]. After hydrolysis with 72% (w/v) H 2 SO 4 [18], the total carbohydrate content was measured using Total Carbohydrate Assay Kit (Sigma-Aldrich, USA). The nitrogen content was measured by a Vario EL cube (Elementar Analytical, Germany), and crude protein content was calculated by multiplying the nitrogen content by 6.25 [19]. Superoxide dismutase (SOD) was measured by a SOD Assay Kit -WST (Dojindo, Japan).
Identification of all observed microalgae by morphology using a microscope (BH-2; Olympus, Japan) and the cell count of each microalgal species were followed by a standard method [20]. Table 1 shows the water quality of the secondary effluent. NH + 4 -N and NO − 3 -N were the main IN components in the secondary effluent. The mole ratio between IN and PO 3− 4 -P was 3.8: 1. Compared to the molar cell ratio (nitrogen: phosphorus = 16: 1) [21], the secondary effluent contained less IN than PO 3− 4 -P. Figure 2 shows changes in water qualities and microalgal components in Cultivation A. Table 2 showed water qualities before and after the experiment. SS increased until day 8.0 (192 mg-SS/L) with reduction in IN and PO 3− 4 , and the culture in the reactor became green in accordance with the increase in SS. After day 8.0 in which IN was less than the detection limits (NO − 3 : 0.03 mg-N/L, NO − 2 : 0.006 mg-N/L, NH + 4 : 0.04 mg-N/L), increase in SS was slowed. During the whole cultivation, PO 3− 4 and DO were more than 2.44 mg-P/L and 6.67 mg-O 2 /L, respectively. Figure 3 shows cell number ratios of indigenous microalgae which were dominantly observed, and microscopic images of the microalgae were shown in Figure S1. The cell number ratios of all observed indigenous microalgae were shown in Table S2. All Chlorophyceae microalgae, whose order could not be identified because of a quite small size (5-10 μm) without morphological features, are denoted as 'Unidentified Chlorophyceae' in Figure 3 and S1, and it was not evaluated how many species were included in the unidentified Chlorophyceae. During the period from day 4.0 to day 7.0, in which SS increase was observed, and day 10.0, in which the SS increase was slowed, the unidentified Chlorophyceae dominantly existed.

Cultivation A (no IN replenishment)
After Chl. a increased and the maximum value (7.17 μg-Chl. a/mg-SS) was obtained on day 5.5, Chl. a decreased. Crude protein increased from day 4.0 (34.3%) to day 5.5 (41.1%). After that, it decreased slightly and reached 36.4% on day 13.0. The total lipid increased from day 4.0 (7.46%) to day 8.0 (14.5%), and the increase slowed. The total carbohydrate increased from day 4.0 (13.3%) to day 7.0 (19.9%), and the increase slowed. The decrease in the total carbohydrate was observed from day 11.5 (18.5%) to day 13.0 (10.7%). SOD increased from day 4.0 (4.29 U/mg) to day 8.0 (12.1 U/mg); meanwhile, it sharply decreased from day 8.0 to day 9.0 (6.99 U/mg). The unidentified Chlorophyceae dominantly existed except for day 9.0 in which cell number ratio of Scenedesmus sp. was high (see Figure 3).
Chl. a increased during the first half period, which included the time of IN replenishment; meanwhile, Chl. a decreased during the last half period. Crude protein increase was observed after IN replenishment. The total lipid increased before IN replenishment. The increase continued for 0.5 days and 1.0 day after IN replenishment in Cultivation B1 and Cultivation B2,     The unidentified Chlorophyceae dominantly existed throughout the cultivation (see Figure 3).
Chl. a decreased before IN replenishment; meanwhile, Chl. a increased after IN replenishment (e.g. from 1.68 μg-Chl. a/mg-SS on day 9.0 to 3.99 μg-Chl. a/mg-SS on day 10.5 in Cultivation C1). After that, Chl. a decreased again. The total lipid started to decrease just after IN replenishment, and the decrease continued for a while (e.g. from 9.03% on day 9.0 to 5.25% on day 12.0 in Cultivation C1). Total carbohydrate sharply decreased just after IN replenishment (e.g. from 27.7% on day 9.0 to 17.9% on day 9.5 in Cultivation C1); meanwhile, the total carbohydrate increased (e.g. from 16.8% on day 10.0 to 25.2% on day 11.0 in Cultivation C1). Sharp increase in crude protein was observed just after IN replenishment (from 23.7% on day 9.0 to 36.7% on day 9.5 in Cultivation C1). The decrease in crude protein was followed, and crude protein reached 24.9% on day 13.0. SOD increased just after IN replenishment (from 6.79 U/mg on day 9.0 to 12.8 U/mg on day 10.0). The decrease in SOD followed, and SOD reached 8.24 U/mg on day 12.0. The Chl. a and crude protein increase, the total lipid decrease, the decrease and subsequent increase in the total carbohydrate and the increase and subsequent decrease in SOD after IN replenishment in Cultivation C did not occur during the cultivation period, in which IN was under the detection limit, in Cultivation A. In addition, the immediate decrease in the total lipid and total carbohydrate after the IN replenishment was unique, compared to Cultivation B.

Discussion
The increase of SS was observed, accompanied by IN and PO 3− 4 decrease. It was reported that the biomass cultivated with secondary effluent mainly consisted of indigenous microalgae, considering of the biomass amounts of indigenous microalgae, bacteria and zooplankton [7]. In addition, the culture in the reactor became green in accordance with the increase in SS, and microalgae were observed in the culture (see Figure 3). Therefore, it was likely that indigenous microalgae were the main component of SS, and indigenous microalgae grew with IN and PO 3− 4 consumption. Chlorophyceae consistently dominated throughout each cultivation with and without the IN replenishment. A previous study [13] also conducted batch cultivation of indigenous microalgae with 30 L secondary effluent in which the IN (137.5 mg of NH 4 Cl and 582.8 mg of NaNO 3 ) was added when the cultivation started, and the similar composition of indigenous microalgae, in which Nitzschiaceae, Micractiniaceae and Scenedesmaceae were mainly observed, was obtained compared to the cultivation without the IN addition. The cell number ratio of Scenedesmus sp. was uniquely high on day 9.0 in Cultivation B2. Meanwhile, the decrease in the total lipid and the decrease and subsequent increase in the total carbohydrate after IN replenishment occurred by day 7.0 in Cultivation B2. Therefore, the unique domination by Scenedesmus sp. on day 9.0 did not influence the temporal changes. Dominant Chlorophyceae were also reported in cultivations with a mixture of primary effluent and diluted excess sludge [22] and a mixture of secondary effluent and microalgae digestate [23].
The indigenous microalgal growth resulted in IN exhaustion in which IN was under the detection limit, and growth inhibition occurred. Therefore, IN, which was originally less contained than PO 3− 4 -P in the secondary effluent, was the limiting factor of indigenous microalgal growth, and the IN replenishment resulted in continuous growth of indigenous microalgae in Cultivation B and regrowth of indigenous microalgae in Cultivation C, respectively. The lowest PO 3− 4 in the culture (0.17 mg-P/L) was observed in Cultivation C1 due to the low initial PO 3− 4 (2.85 mg-P/L) (see Table 2); meanwhile, based on the half saturation constant of phosphorus (0.002 mg-P/L) [24], the culture in Cultivation C1 still had abundant PO 3− 4 for microalgal growth as is the case with the other experiments. Abundant DO was observed in each experiment due to the DO production through photosynthesis by indigenous microalgae.
Protein was an essential component of biomass production [25]. In addition, microalgae accumulated Chl. a through the uptake of external nitrogen sources in cells in the cultures which provide enough nitrogen sources [26]. Therefore, indigenous microalgae probably accumulated crude protein for the growth and Chl. a as the external nitrogen sources under the cultivation in which the growth of indigenous microalgae and abundant IN was observed in Cultivation A and Cultivation B. Meanwhile, nitrogen-deficient condition made most of the anabolic machinery, including ribosomes and chloroplast, a target for degradation and nutrient recycling [27], and the photosynthetic efficiency might have decreased under nitrogen starvation [2]. In addition, microalgae degraded protein to supply the nitrogen needed for normal metabolic functioning in nitrogen-starved culture [28]. Therefore, the IN exhaustion, which resulted from the growth of indigenous microalgae, probably induced degradation of Chl. a and crude protein. After the IN replenishment in Cultivation C, indigenous microalgae rapidly accumulated Chl. a, which suggested recovery of the photosynthetic efficiency, and crude protein, and the accumulation possibly contributed to the regrowth of indigenous microalgae in Cultivation C. Meanwhile, the replenished IN was rapidly consumed by indigenous microalgae, and IN was exhausted in the culture again, which resulted in the degradation of Chl. a and crude protein in Cultivation C.
Nitrogen starvation induced formation of reactive oxygen species (ROS) such as hydrogen peroxide, superoxide and hydroxyl radical in Chlorella pyrenoidosa [29] and Desmodesmus sp. [30]. SOD provided the first line of defence against toxic effects of ROS by catalysing dismutation of superoxide to form hydrogen peroxide and oxygen [31]. The SOD increase in the first half period of Cultivation A, in which IN existed but decreased, suggested ROS generation in indigenous microalgae. The ROS triggered lipid and carbohydrate synthesis in microalgae [32]. The ROS possibly induced lipid accumulation via gene expression, accumulation of essential molecules and endoplasmic reticulum stress [33]. Gargouri et al. [34] suggested that total lipid accumulation under nitrogen depletion probably has a role for oxidation of NADPH to inhibit ROS production. ROS generation, which was suggested by the SOD increase, possibly induced the total lipid and total carbohydrate accumulation in indigenous microalgae; and the accumulation was terminated, corresponding to the sharp SOD decrease. Continued cultivation of indigenous microalgae under IN exhausted condition resulted in the degradation of the total carbohydrate. The IN exhausted condition also resulted in the degradation of Chl. a in which the photosynthetic efficiency may be decreased [2], and there was a possibility that the accumulated total carbohydrate was consumed as carbon and/or energy source for the survival.
The accumulated starch and lipids under the nutrient depletion condition were utilised as sources of energy and carbon for cell regrowth and division in Parachlorella kessleri [35]. Mulders et al. [36] also reported that metabolites from triacylglycerol and starch served as carbon and energy source for recovery process which was induced by nitrogen resupply in Chromochloris zofingiensis. Meanwhile, Zhu et al. [37] reported that starch served as a source of carbon and energy to support cells recovery and reproduction, while storage lipid preferred to be as a reservoir of fatty acids for membrane lipid construction in Chlorella sp. In this study, indigenous microalgae sharply degraded total carbohydrate after the IN replenishment in Cultivation B and C, and sharp accumulation of crude protein and Chl. a, specifically in Cultivation C, were occurred. Therefore, it was possibly that the accumulated total carbohydrate was utilised for accumulation of crude protein which was an essential component of biomass production, including cellular division [25], and/or Chl. a which assumed the photosynthesis. Total lipid also degraded; meanwhile, the degradation of the total lipid was slower but kept longer than that of the total carbohydrate. The differences of the degradation between total lipid and total carbohydrate likely caused by their different roles in microalgal life strategy against IN increase as suggested by Zhu et al. [37].
The total lipid and total carbohydrate were immediately degraded after the IN replenishment in Cultivation C. Meanwhile, after the IN replenishment, indigenous microalgae kept the accumulation of the total lipid and total carbohydrate for a while, then degraded the total lipid and total carbohydrate in Cultivation B. In both of Cultivation B and C, SOD was increased just after the IN replenishment, and the SOD increase indicated the possibility of the accumulation of total lipid and total carbohydrate, which was induced by generated ROS, just after the IN replenishment. There was a possibility that the total lipid and total carbohydrate accumulation overwhelmed their degradation just after the IN replenishment in Cultivation B, which resulted in the temporal increase of total lipid and total carbohydrate. Meanwhile, regarding Cultivation C in which photosynthetic efficiency may be decreased before the IN replenishment, the degradation of the total lipid and total carbohydrate might overwhelm their accumulation, which resulted in the immediate decrease of total lipid and total carbohydrate.
After the degradation of total carbohydrate induced by the IN replenishment, indigenous microalgae accumulated total carbohydrate in both Cultivation B and C. SOD was decreased during the period in which the total carbohydrate accumulation was observed; therefore, the total carbohydrate accumulation was a different phenome to the accumulation of total carbohydrate and total lipid induced by ROS generation in Cultivation A. A part of assimilated carbon through the photosynthesis was used to produce functional biomass, and the other assimilated carbon was firstly stored in carbohydrate [38]. CO 2 assimilated through the Calvin cycle formed glyceraldehyde-3-phosphate, and starch, which was the main storage form of carbohydrates in the green algae, was finally synthesised through the conversion of glucose-6phosphate, glucose-1-phosphate and ADP-glucose [39]. The Chl. a accumulation, which suggested the recovery of photosynthetic efficiency, was induced by the IN replenishment; therefore, there was a possibility that the total carbohydrate was synthesised by the recovered photosynthetic CO 2 assimilation. Based on the findings described above, it was suggested that the cultivation under the IN exhausted condition was attractive to enhance the content of total lipid and carbohydrate in indigenous microalgae. Meanwhile, the continued cultivation of indigenous microalgae under the IN exhausted condition needed to be avoided due to the decrease of the total carbohydrate content. The IN increase in the culture triggered sharp decrease of total carbohydrate. The total carbohydrate content recovered which followed the decrease; meanwhile, two or three days were required for the recovery of the total carbohydrate content. In addition, the IN increase also resulted in decrease of total lipid. Therefore, to produce indigenous microalgae which contained high and stable total carbohydrate and total lipid, it was required to prevent the IN increase in the culture by appropriate reactor operations such as control of solids retention time and hydraulic retention time.

Conclusions
Chlorophyceae consistently dominated in indigenous microalgae cultivated with municipal secondary effluent. Based on the increase in total lipid from 7.46% to 14.5% for 4.0 days and the increase in total carbohydrate from 13.3% to 19.9% for 3.0 days against IN decrease in Cultivation A, which were possibly induced by ROS generation, the cultivation under the IN exhausted condition was attractive to produce indigenous microalgae with high total lipid and carbohydrate content. Meanwhile, the decrease of total carbohydrate from 18.5% to 10.7% for 1.5 days by prolonged cultivation under IN exhausted condition in Cultivation A suggested that the continued cultivation of indigenous microalgae under the IN exhausted condition needed to be avoided. The IN replenishment resulted in sharp decrease of total carbohydrate in Cultivation B and C (e.g. from 18.5% to 12.2% for 0.25 days in Cultivation B2 and from 27.7% to 17.9% for 0.5 days in Cultivation C1), which possibly utilised to accumulate crude protein and/or Chl. a for continuous growth or regrowth. Recovery of total carbohydrate content was occurred in Cultivation B and C (e.g. from 12.0% to 15.0% for 1.0 days in Cultivation B2 and from 23.7% to 36.7% for 0.5 days in Cultivation C1); meanwhile, two or three days were required for the recovery. The decrease of total lipid was also triggered by the IN replenishment in Cultivation B and C (e.g. from 13.5% to 3.87% for 2.5 days in Cultivation B1 and from 9.03% to 5.25% for 3.0 days in Cultivation C1). Based on the temporal changes in the organic component against IN increase, it was required to prevent IN increase in the culture to produce indigenous microalgae, which contained high and stable total lipid and carbohydrate.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This study was financially supported by Charitable Trust Sewage Works Promotion Fund, Japan Sewage Works Association and JSPS [KAKENHI grant number 19K15123].

Data availability statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.