Introduction

Phosphorus is the second most important nutrient in domestic waste-water. It is difficult to remove and hence along with nitrogen is responsible for eutrophication of water bodies, especially where treated sewage is discharged. The most common method of soluble phosphate removal from the wastewater is precipitation [1]. Disposal of the sludge generated by this method is both expensive and difficult to implement, considering low phosphate and nitrate concentrations present in sewage [2]. In contrast, employing algae for the removal of phosphates and nitrates from wastewater has several of several advantages viz.(a) Simultaneous production of O2 and consumption of CO2 in presence of light, (b)Obviating the need for an extraneous supply of organic or inorganic nutrients, (c) Providing a final effluent that is enriched in dissolved oxygen, (d) lesser sludge accumulation (e) absence of generation of secondary pollutants, effective uptake of N and P (g) ecologically safe, (h) generation of a biomass which can have potential use as feedstock for fertilizers, biogas and biofuel [3–6]. However, it is essential to enhance the nutrient uptake kinetics so that the process of sewage treatment will be faster and reduced footprints. The nutrient removal efficiency of the algal cell could be increased by intrinsic and extrinsic factors, which include culture density [7], appropriate algal species, and environmental factors like temperature, pH, CO2 concentration [1] and starvation [8, 9] of algae, etc.

Higher algal inoculums of algae can be achieved with immobilized algae [10]. However, it may also introduce complexity in the operations at larger scale. The previous study reported that immobilized algal cells do not offer an advantage over free cells for removing nutrients from the wastewater [11].

Algal cells accumulate polyphosphate granules under different phosphate condition except the longer phosphate starvation [12]. Though algal cells with phosphate-starved for few days showed faster nutrient uptake rate than the phosphate- saturated algal cells, however how phosphate starvation results in an increase in phosphate uptake rate was not clear [13, 14]. Though some of the recent studies, successfully utilized phosphate-starved, immobilized algal cells for the rapid treatment of the sewage [8, 9, 15], the experimentation was done using either artificial wastewater or filter sterilized domestic wastewater, which makes it difficult for practical implementation.

Here we utilized FTIR spectroscopy to visualize the internal phosphate concentration associated with the various macromolecular pool in phosphate supplemented and starved algal cells and it’s probable relation with the increased phosphate uptake rate under phosphate starvation condition. The changes in the macromolecular pool can be monitored by FTIR on the basis of infrared absorption of the vibrationally active functional group (including O–H, N–H, C=O, =C–H, –C–H, C–O–C and > P=O) in the biological sample. FTIR has been successfully used for algae like Microcystis aeruginosa, Protoceratiun reticulatum, Pandina tetrastromaticato study the physiological changes [16–20].

As algae isolated from the domestic wastewater, can adapt and execute better for treatment of municipal wastewater. Two different algae were isolated from the wastewater and identified as Scenedesmus sp. and Ankistrodesmus sp. Both the isolates were evaluated for phosphate and nitrogen uptake from the sewage. The objective of this study was to develop practically applicable, simple, economical primary treatment of the domestic wastewater using algal cells having accelerated nutrient uptake with minimum footprints. An effort was made to understand the underlying rationale behind this improved cellular mechanism. The depletion of the internal phosphate during starvation and other compositional changes were monitored by FTIR.

Materials and Methods

Algal Cultures and Growth Conditions

The unialgal cultures of Scenedesmus sp. and Ankistrodesmus sp. were isolated from local sewage contaminated water body, from suburban of Pune, India. The isolation was done by agar plating method. Once algae are obtained in pure form, they were cultivated under aseptic conditions and preserved for further experiments. The algae were grown on Bold’s basal medium (BBM) containing following chemicals NaNO3 (0.25 gL−1), K2HPO4 (0.075 gL−1), KH2PO4 (0.175 gL−1), NaCl (0.025 gL−1), MgSO4 (0.075 gL−1), CaCl2 (0.025 gL−1), and trace metals ZnSO4 (5 × 10−6 gL−1), MnSO4. 4H2O (1 × 10−5 gL−1), H3O3 (5 × 10−5 gL−1), Co(NO3)2·6H2O (5 × 10−6 gL−1), Na2MoO4·2H2O (5 × 10−6 gL−1), CuSO4·5H2O (0.025 × 10−6gL−1), FeSO4·7H2O (3.5 × 10−3gL−1), Na·EDTA (4 × 10−3 gL−1) [21]. The cultures were incubated in the average light intensity 7 Wm−2 at 30 °C temperature.

Algal Growth in Raw Sewage Water

The sewage was collected from a wastewater treatment plant located in Pune (India). Untreated 200 mL sewage was taken in 500 mL Erlenmeyer flasks with 10% algal inoculums. The flasks were incubated in the average light intensity 7 Wm−2 at 30 °C temperature. Sewage water then monitored for nutrients (nitrogen and phosphate) concentration, COD and number of algal cells for 3 days. The samples were centrifuged at 3000×g for 5 min before analysis. Total Kjeldahl nitrogen was analyzed by using KjelTron Nitrogen/Protein digestion system (KDIGB 6 M) by the standard procedure mentioned in American Public Health Association (APHA) 4500-NH3 A,B,C [22]. Total phosphate were estimated by using thevanado-molybdo-phosphoric acid calorimetric method as mentioned in APHA 4500 [22]. Chemical oxygen demand (COD) was analyzed by using standard methods of APHA 5220B [22]. The number of algal cells was determined by counting in the hemocytometer. The experiment was done in triplicate.

Phosphate Starvation

The Scenedesmus and Ankistrodesmus growing in the early logarithmic phase were harvested by centrifugation, washed three times with sterile BBM medium without phosphate and nitrate, to remove the media impurity. Part of the washed cells was re-suspended in fresh 200 mL phosphate free BBM and remaining part suspended in phosphate containing BBM (regular medium), such that every flask has approximately 1 × 106 cells mL−1. To determine intracellular phosphate, 10 mL sample was taken from each flask daily. The cells were harvested by centrifugation at the speed of 3000×g for 5 min and re-suspended in phosphate and nitrate free BBM. The cells were digested by boiling in an autoclave for 1 h with 5:1(v:v) alkaline potassium persulfate [23]. Intracellular phosphate was estimated by the phosphomolybdate-blue method as described in APHA 4500 E [22]. The number of cells was monitored by counting in the hemocytometer. All experiments were done in triplicate.

FTIR Analysis of the Phosphate-Starved and Supplemented Cells

The Scenedesmus cells were grown in BBM with and without phosphate for 120 h. The cells were harvested, washed and dried in the vacuumed oven at 100° C. This dried cell mass was utilized for FTIR analysis. For FTIR analysis, sample preparation was carried out as described earlier [18]. Dry algal sample, 2.5 mg was mixed with 150 mg potassium bromide (KBr) using mortar pestle. The mixture was filled in high press 13 mm diameter die to get the pellet. The IR of KBr –algae pellet was recorded at 23 ± 1 °C temperature in the mid-infrared range (4000–450 cm−1) using FTIR (Perkin Elmer, Spectrum One). Thirty scans were single averaged for single spectrum. Each spectrum was displayed in terms of transmission. Analysis, of a peak area estimation was done by Spectrum One software. The carbohydrate-to-protein band ratio was given by the ratio of an area of the carbohydrate region (900–1200 cm−1) and that of amide II band (1300–1500 cm−1).

Utilization of Phosphate-Starved Cells for Sewage Water Treatment

The Scenedesmus culture was grown in 2 L flask containing 800 mL BBM. The culture was harvested by centrifugation in the early logarithmic phase. Cells were washed three times with sterile phosphate and nitrate free BBM medium to remove the media impurity and excess phosphate. Half of the cells were inoculated in phosphate free BBM and incubated in light for 48 h for phosphate starvation. The remaining half (untreated) were inoculated in three, 500 mL conical flasks containing 200 mL raw sewage at three initial inoculums sizes were 1 × 106, 5 × 106 and 10 × 106 cells mL−1. Further, the sewage was monitored for its nutrient (nitrate and phosphate) concentrations and COD.

A similar procedure was adopted for the treated cells. Sewage was monitored further for the nutrients and COD. All experiments were conducted in triplicate.

Results and Discussion

Growth of Algae Isolates in Sewage Water

The algae isolated were checked for their growth rate in raw sewage water after isolation. In the first 24 h, the Scenedesmus was able to remove 72–76% phosphate; 93% phosphate removal occurred in 72 h. Scenedesmus was able to remove 84.5% of nitrogen in 24 h and 98% nitrogen was removal after 72 h. The COD was decreased by 87% during 72 h of incubation with Scenedesmus (Fig. 1a). Ankistrodesmus was able to remove 72% of phosphate in first 24 h, and up to 83 % of phosphate after 72 h. Ankistrodesmus consumed 75% of nitrogen in first 24 h while 100% nitrogen depletion occurred in 72 h. Also, 79% of COD was reduced by Ankistrodesmus culture in 72 h (Fig. 1b). However further incubation of Ankistrodesmus did not reduce the phosphate concentration in the raw waste-water, as nitrogen was completely utilized in 72 h. Values for phosphate, nitrogen, and COD in the control sewage are shown in Fig. 1c. In all these experiments the initial cell density for both of the culture was 1 × 106 cells mL−1. In previous studies, it took 24 days for 97% removal of phosphate and 59% removal of nitrogen, when Chlorella was grown on filtered and autoclaved municipal waste-water [6]. Sing and Thomas [21] used membrane reactor permeated sewage water to grow four different local algal isolates viz. Chlorella, C. vulgaris, Scenedesmus quadricauda, and S. dimorphus and found that 66% PO4 could be removed by the microalgae in first 24 h with an initial cell density of 1.2 × 106 cell mL−1. Scenedesmus obliquus with the initial cell density around 2 × 106 cells mL−1 was able to remove 100% nitrogen and 83% PO4 after 48 h from urban waste-water [11].

Fig. 1
figure 1

Growth of algal isolates in sewage: Growth of algal isolates and concomitant reduction of total phosphate and nitrogen from sewage. a Growth of Scenedesmus, b growth of Ankistrodesmus, c control or un inoculated sewage. X axis shows time in h. Y axis shows filled square Kjealdal nitrogen in sewage (mg L−1), filled circle total phosphate sewage (mg L−1), filled triangle COD of sewage (mg L−1), filled inverted triangle number of algal cells × 106 mL−1

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To make the process more viable for practical application, raw, unsterile sewage was used in the present study, with inoculum of minimum initial cell density. However as it was unsterile, after 24 h, when algae reached their peak growth, zooplanktons (grazers) appeared and reduced the algal cell number. Depletion of nutrient and combating grazers reduced the number of algal cells after 24 h of incubation in the case of Scenedesmus (Fig. 1a). However, cells of Ankistrodesmus were exceptional to this phenomenon of reduction in the cell number, indicating an ability of growth in low concentration of nutrients like phosphate and nitrogen. It may possess some defence mechanism against grazers as the number of algal cells did not reduce like cells of Scenedesmus (Fig. 1b).

Effect of Phosphate Starvation on Algal Cells

When the cells of Scenedesmus were inoculated in phosphate supplemented and phosphate free BBM, the growth of Scenedesmus in phosphate-free BBM is hampered considerable only after 48 h of incubation. In this duration, the cell density was raised to approximately around, 5 × 106 cells mL−1, from the initial 1 × 106cell mL−1 in both types of BBM. The cells growing in phosphate- free medium utilized their internally stored phosphate and able to multiply for first two generations in a similar way as that of normal growing cells. However after 48 h, the growth rate of the phosphate-starved cells was reduced as compared to the algal cells growing in the normal medium. At the end of 96 h incubation, the phosphate supplemented cells were able to multiply for more than four generation while the phosphate-starved cells showed only 2.5 generations. In other words, the phosphate-starved Scenedesmus cells showed the stationary phase of the growth cycle after 48 h (Fig. 2b). In a case of Ankistrodesmus, no reduction in the growth rate was not observed after giving phosphate starvation (data not shown). These algal cells were also observed growing in sewage when nitrogen was almost depleted and with 12 ppm of total phosphate indicating heterotrophic growth (Fig. 1b). Hence, cells of Ankistrodesmus were not selected for the treatment of phosphate starvation and utilizing further.

Fig. 2
figure 2

Phosphate starvation effect on Scenedesmus. a Intracellular phosphate concentration in Scenedesmus. b Growth of Scenedesmus in phosphate free BBM and Phosphate supplemented BBM. X-axis shows time in h for a and b. For a Y axis shows filled triangle intracellular phosphate concentration in the phosphate-starved cells, filled inverted triangle intracellular phosphate concentration in the phosphate supplemented cells (µg mg−1 of fresh weight of the cells). In b Y axis have filled square number of phosphate-starved cells × 106 cells mL−1, filled circle number phosphate supplemented cells × 106 cells mL−1

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The internal phosphate concentration in the phosphate supplemented Scenedesmus cells showed an increasing trend indicating the phosphate accumulation in the daughter cells while growing in the phosphate-rich medium. After 120 h the internal phosphate concentration in the phosphate supplemented Scenedesmus cells was raised up to 0.41 µg mg−1 of fresh weight (Fig. 2a). The cells growing in phosphate free BBM though did not showed significant reduction in the internal total phosphate content for first 96 h of incubation as the stored phosphate might be getting distributed to the daughter cells. However at the end of 120 h of incubation internal phosphate was reduced from 0.12 to 0.078 µg mg−1 of fresh weight in the phosphate-starved cells (Fig. 2a). Cells of Phormidium, Sphaerocystis and Scenedesmus when grown in phosphate supplemented medium showed an increase in the intracellular phosphate content [19]. When grown in phosphate-starved medium algae able to utilize the internal phosphate and could sustain for 3–4 generations under the starvation conditions [14].

FTIR Analysis of Phosphate-Starved and Supplemented Scenedesmus Cell

Effect of the phosphate starvation was further studied with FTIR. To visualize the effect of phosphate starvation stress, the integrated FTIR band of different spectral were studied. The dried cell mass showed transmission peaks over wave number 450–4000 cm−1, are given in Fig. 3. The peaks were tentatively identified on the basis of published FTIR spectra in relation to the specific molecular groups.

Fig. 3
figure 3

FTIR spectrum: FTIR peak of phosphate a starved and b supplemented cells of Scenedesmus. At X axis—wavelength (cm−1), Y axis represents—% Transmission every spectrum is average of thirty scans. The phosphate starved cells showing peaks at 1022 cm−1. This polysaccharide peak is absent in phosphate supplemented cells, however these cells showed broad peak at 1240 and 1085 cm−1

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The FTIR spectra were compared by taking the ratios of areas, of the respective IR bands in the absorbance mode. The region from 900 to 1200 cm−1 are characteristics of C–C, C-O, C–O–C, C–O–P of polysaccharides stretching vibrations of polysaccharides [17, 18]. The cells grown in phosphate free BBM showed the peak at 1022, 1077, 1156 cm−1 (Fig. 3a) These peaks are due to various polysaccharides. Carbohydrate pool containing various polysaccharides increased during the phosphate-starved conditions. The increase in polysaccharides is another indicator of the stationary phase of the algae growth cycle. Cells were grown in phosphate supplemented BBM showed the peak at only at 1085, 1152 cm−1 (Fig. 3b). However, algae growing in phosphate supplemented BBM did not show the peak at 1022 cm−1. Similar findings were reported in Sphaerocystis and Phormidium, where these algae showed strong bonds at 1024, 1080 and 1150 cm−1 under phosphate starvation, however, these bonds were disappeared within 24 h after supplementing cells with phosphate [19].

Phosphodiester bond stretching generates peak at 1245 and 1240 cm−1 in phosphate- starved and supplemented algal cells respectively. We found an increase in intensity and broadening of the aromatic phosphate bond (> P=O) at 1240 cm−1 and symmetric aliphatic phosphate bond (C–O–P) at 1085 cm−1 indicating phosphate storage in the phosphate supplemented Scenedesmus cells. This results of FTIR got confirmed when the intracellular phosphate was quantified with the phosphomolybdate-blue method, the phosphate supplemented cells showed 5.7 times more intracellular phosphate than the phosphate-starved cell (Fig. 2a). Collective effect of phosphate starvation and supplementation on the macromolecular pool is given in (Fig. 4).

Fig. 4
figure 4

Effect of phosphate starvation on macromolecular pool: The bond ratios of the various biomolecules of phosphate-starved (gray bars) and supplemented (white bars) Scenedesmus cells are plotted on X axis. Carbohydrate/phosphor (C/P), carbohydrate/lipid (C/L) and carbohydrate/protein (C/AII) ratio is increased in phosphate starved cells

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The phosphate-starved Scenedesmus cells showed significantly increased in carbohydrate/phosphor (C/P), carbohydrate/lipid (C/L) and carbohydrate/protein (C/AII) ratio. When cells of Microcystis aeruginosa and Phaeodactylum tricornutum were grown in phosphate-limited conditions, they showed decreased growth rate, and increase in (C/AII) ratio [16, 24, 25]. Phaeodactylum tricornutum showed increased in lipid/phosphate after 3 weeks of phosphate starvation [24]. In this study L/P ratio of phosphate supplemented and starved Scenedesmus cell did not differ significantly. This may be due to a short starving period of 120 h. The Lipid/amide II (L/AII) and amide/phosphor (AII/P) did not show a significant difference after phosphate starvation. FTIR study confirms the stationary phase in phosphate-starved Scenedesmus cells as observed in growth curve study (Fig. 2). These starved, stationary algal cells might be able to consume nutrients at faster rate compare to the phosphate supplemented cells which might prove helpful for the faster treatment of raw sewage.

Comparison of Phosphate-Starved and Supplemented Scenedesmus for Sewage Water Treatment

The phosphate-starved and phosphate supplemented Scenedesmus cells were used for the treatment of sewage. The sewage water with initial phosphate concentration 124 mg L−1 when treated with phosphate-starved 1 × 106 cells mL−1, the phosphate was reduced to 17.6 mg L−1(86%) at the end of 24 h (Fig. 5a). Similar cell density of phosphate supplemented cells was able to reduce the phosphate concentration to 27 mg L−1 (76%) (Fig. 5a, b). Previously three days phosphate-starved and immobilized cells of Chlorella sorokiniana able to reduce the 23% phosphate from synthetic waste water in 48 h. This reduction was further enhanced by co- immobilizing C. sorokiniana with bacteria Azospirillum brasilense [8]. Zhang et al. [9] used two days starved cells of Scenedesmus, immobilized 2 × 108 cells mL−1 (cell intensity in the bead) and found 100% removal of PO4 in just 135 min from filtered, sterilized secondary domestic waste water. Similar experimentation was done with 2 days phosphate-starved Chlorella, with immobilized 1.4 ×  108 cells mL−1(cell intensity in the bead), took 4 h for the complete removal of phosphate from filtered, sterilized secondary domestic wastewater [15]. In this study raw sewage water without any prior treatment like filtration and sterilization is used. Hence, it is more viable to the practical application. This unsterilized, uninnoculated sewage has it’s own natural microbial flora which is responsible for reduction in phosphate, nitrogen and COD with time (Figs. 5, 6, 7).

Fig. 5
figure 5

Effect of algal phosphate starvation on phosphate uptake: the phosphate-starved (a) and phosphate supplemented (b) Scenedesmus cells inoculated in sewage. The X-axis represents time in h. Y-axis shows phosphate concentration in mg L−1 utilized by filled square 1 × 106 cells mL−1, filled circle 5 × 106 cells mL−1, filled triangle 10 × 106 cells mL−1, filled inverted triangle un inoculated sewage

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Fig. 6
figure 6

Effect of algal phosphate starvation on nitrogen uptake: The phosphate-starved (a) and phosphate supplemented (b) Scenedesmus cells inoculated in sewage. The X axis represents time in h. Y axis shows nitrogen concentration in mg l−1 utilized by filled square 1 × 106 cells mL−1, filled circle 5 × 106 cells mL−1, filled triangle 10 × 106 cells mL−1, filled inverted triangle un inoculated sewage

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Fig. 7
figure 7

Effect of algal phosphate starvation on COD reduction: The phosphate starved (a) and phosphate supplemented (b) Scenedesmus cells inoculated in sewage. The X axis represents time in h. Y axis shows COD mg L−1 utilized by filled square 1 × 106 cells mL−1, filled circle 5 × 106 cells mL−1, filled triangle 10 × 106 cells mL−1, filled inverted triangle un inoculated sewage

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The phosphate-starved Scenedesmus cells, with initial cell density 1 × 106 cells mL−1, were able to utilize 100% nitrogen within 24 h (Fig. 6a) however the phosphate supplemented cells were able to reduce the nitrogen level up to 24 mg L−1 (52%) within 24 h (Fig. 6b). The initial total nitrogen concentration in the sewage water was 50 mg L−1. Indicating fast uptake of nitrogen by the starved, stationary, Scenedesmus cells. Zhang et al. [9] report 99.1% NH4 removal by two days phosphate-starved Scenedesmus from filtered, sterilized secondary domestic waste water. When 2 × 108 cells mL−1 immobilized (cell intensity in the bead) were used for the treatment. Two days phosphate-starved Chlorella, with immobilized 1.4 × 108 cells mL−1 (cell intensity in the bead), took 4 h for the 98.8% removal of NH4 from filtered, sterilized secondary domestic wastewater [15]. In the present study P-starved Scenedesmus isolate showed no significant increase in phosphate and nitrogen reduction with the increase in a number of free cells (1 × 106 to 10 × 106 cell mL−1) in the sewage water (Figs. 5a, 6a). However, the phosphate supplemented cells at higher cell density showed faster nitrogen reduction with increasing thenumber of free cells in the sewage (Figs. 5b, 6b). Reduction of the COD was similar for both phosphate-starved and supplemented cells (Fig. 7a, b).

Conclusions

The local algal isolate Scenedesmus was entering in the stationary phase of life cycle due to the phosphate starvation. These stationary phase cells of Scenedesmus showed effective phosphate and nitrogen utilization from the untreated sewage water. However, the phosphate starvation did not induce any stationary phase in the cells of Ankistrodesmus. Phosphate starvation of Scenedesmus for 120 h showed a reduction in the internal phosphate and rise in carbohydrate pool. Phosphate starvation of algal cultures appears to be a good technique for an enhanced rate of removal of the total phosphorus and nitrogen contents in sewage. Additional detailed studies need to be conducted under steady state conditions in a chemostat to determine how phosphate starvation triggers an enhanced rate of nitrogen and phosphate.