Evaluation of the Growth Performance of Microalgae Based on Fine pH Changes

    

    

    Figure 2: Representation of the C. vulgaris photobioreactor.
    

The bioreactor was equipped with a pH (EasyFerm Bio Arc 325, Hamilton, Swiss) probe and a dissolved oxygen sensor (VisiFerm DO Arc 325, Hamilton, Swiss). The pH probe was calibrated before autoclaving at pH 4.01 and 7.00 or 7.00 and 9.03 depending on the pH range of the experiment, and then recalibrated at a single point each week against a calibrated bench-top pH meter (PL-700 PC, GOnDO benchtop pH/mV/Temperature Meter).

The dissolved oxygen sensor was calibrated at two points: at 0% immediately after autoclaving and at 100% after reaching an equilibrium with air, following continuous air injection under operating conditions. During the experiments, the pH of the culture was adjusted automatically using injection of CO₂ into the constant air flow (200 ml/min) at a maximum concentration 2 % (v/v) CO₂. The supply of the carbon dioxide was regulated by a mass flow meter (Bronkhorst). For the lowest pH value, the adjustment of the pH was achieved by adding 1 mol/L H₃PO₄. benchtop pH/mV/Temperature Meter). The dissolved oxygen sensor was calibrated at two points: at 0% immediately after autoclaving and at 100% after reaching an equilibrium with air, following continuous air injection under operating conditions. During the experiments, the pH of the culture was adjusted automatically using injection of the tested pH values are 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0 and 10.0. The inoculation of the bioreactor with C. vulgaris was carried out with 1% of pre-culture giving 1-2 million cells.ml-1 at the beginning of the culture.

During growth, several parameters such as cellular concentration (Multisizer 4, Beckman), dry weight, absorbance at 800 nm (UV-1800, SHIMADZU), total CO₂ supply, chlorophyll content (Porra method, [39]), internal carbon content, ionics analysis, pH and dissolved oxygen concentration were measured. The experiments were always started on a Monday and two sample were collected and analyzed every day. Samples were not taken in duplicate but several of the analyses were performed in replicated: Cell counts and size distribution by Coulter counter were performed in triplicate and the results presented as mean averages, CHNS analyses were performed in duplicate on single samples and the other off-line analyses were performed without repeats.

Analytical methods

Biomass quantification: The biomass concentration was estimated by three different methods: i) a spectrophotometric measurement of Optical Density (OD) at 800 nm; ii) the cell count and the distribution of the cell size were obtained using the Multisizer 4e (Beckman Coulter); Measurements were made in triplicate on single samples. iii) The dry weight of the biomass, DW in g.L^-1, was determined by centrifugation (3200 g, 10 min) of 15 ml of microalgal sample (centrifuge 5840R). The pellet was re-suspended in the same volume of deionised water and was then subjected to a second centrifugation. The final pellet was frozen (-18°C) for at least 24 h, then the frozen pellet was dried for 24 h by lyophilisation (Alpha 1-2 LD Entry Freeze Dryer, MARTIN CHRIST).

Chlorophyll concentration: The concentration of chlorophyll a and b was obtained based on a modified extraction method of Porra [39]. Chlorophyll a and b were obtained according to the following equations after extracting the chlorophyll:

Chl a (mg/L) = 16.41A₆₆₄ - 8.09A₆₅₀ (1)

Chl b (mg/L) = 30.82A₆₅₀- 12.57A₆₆₄ (2)

Determination of mineral ions concentration: The sample supernatants obtained through centrifugation (3214 g, 10 min) were diluted appropriately with deionized (MilliQ) water, filtered through C18 and 0.2 μm filters in order to retain all proteins and macromolecules that could interfere with the analysis. The ions analysis was performed with ionic chromatography (DIONEX ICS 5000⁺ Thermo Scientific equipped with anionic column DIONEX IONPACTM AS11-HC 2x250 mm and cationic column DIONEX IONPAC^TM CS16 3x250 mm). This method allowed to determinate the concentrations of the major anions and cations, such as sodium, ammonium, potassium, calcium, magnesium, sulphate, orthophosphate and nitrate.

Elemental content analysis: The elemental intracellular content of microalgal cells was obtained by the CHNS-O Elemental Analysis (ThermoFisherScientific). This technic allows quantification of the percentage by weight of carbon, hydrogen, nitrogen, sulfur and oxygen contained in homogeneous organic materials.

For the elemental determination, 2 mg of the dried microalgal sample, was transferred into an oven at 930°C filled with oxygen for very fast and complete dynamic combustion “flash”, using an elemental analyzer Flash 2000 (ThermoFisherScientific). The detection of carbon, hydrogen and sulphur is based on the thermal conductivity of combustion gases.

For the determination of oxygen, a complementary module allowed the analysis of this element by a pyrolysis process in an oven at a temperature of 1000°C.

Results

C. vulgaris growth

C. vulgaris grew at all the pH values (5.0-10.0) tested, however, at the extremes of the tested pH-values, the growth of this organism was severely retarded (lower μ). The lag phase duration was also influenced by the culture-pH with considerably longer lag phases encountered at the extremes of pH.

Growth of C. vulgaris under autotrophic conditions can be divided into three phases: a lag phase, a short exponential phase and a long linear growth phase [40]. C. vulgaris grew best (Figure 3) at neutral pH (μ=0.047 h^-1 and μ=0.049 h^-1 for pH 6.5 and 7, respectively). On either side of pH 7, the specific growth rate of C. vulgaris decreased to a value of approximately 0.03 h^-1. The impact of pH variation around the neutral value on the biological growth of C. vulgaris could be, at least partially, caused by the energetic demand of cells in order to adapt their cell physiology to maintain intracellular stasis. Moreover, the photosynthetic activity of C. vulgaris is also affected by the external pH variations; specifically, at low pH, the total carbon accumulation and the nutrient uptakes rate are reduced. The lower external pH may cause undesirable effects on cell metabolism [41,42]. In alkaline environments the carbonate species are dominant inducing carbon limitation in its CO₂ form.

Figure 3: Growth rate of C.vulgaris culture at different pH values. The μ values were determined at the beginning of the exponential phase based on 6 time points.

    

    

    Figure 3: Growth rate of C.vulgaris culture at different pH values. The μ
values were determined at the beginning of the exponential phase based on
6 time points.
    

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The biomass produced by the end of the exponential phase decreased on either side of neutral pH (Figure 4) reaching its lowest value at pH 10.0 (0.235 g.L^-1). The highest value of final algal biomass at the end of exponential phase was obtained at pH 7.0, (0.824 g.L¹). This reflects that varying growth rate with pH. The variation of the external pH may have caused a modification of cell metabolism in order to counteract the unfavourable environmental pH by changing the internal cellular pH [25-26] or in order to maintain internal stasis. The data obtained leads to the conclusion that the optimum pH value of C. vulgaris growth is 7.0 (Table 2). At neutral pH, the distribution of the carbon species is dominated by CO₂ and HCO₃^-, with the majority of the distribution tending towards the latter species. Carbon assimilation can be directly from free CO₂ or by active transport of HCO₃^-. The modification of the cell metabolism is less drastic compared with acid or basic environments, and therefore, the cell requires less energy to adapt to a supposedly aggressive environment. The growth performance obtained at pH 6.5, is slightly lower than for pH 7.0. There may be an optimal carbon species (HCO₃^-/CO₂) ratio for carbon assimilation by C. vulgaris.

Figure 4: C. vulgaris biomass concentration at the end of the exponential growth phase. (n=3, Sd generally less than 5% of the mean).

    

    

    Figure 4: C. vulgaris biomass concentration at the end of the exponential
growth phase. (n=3, Sd generally less than 5% of the mean).
    

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Table 2: Biological parameters of C. vulgaris culture at different pH values.

  

    
Parameter
    
Units
    
pH
  
  

    

    
5.0
    
6.0
    
6.5
    
7.0
    
8.0
    
9.0
    
10.0
  
  

    
Lag Phase
    
h 
    
65
    
51
    
8 
    
6 
    
25
    
23
    
27
  
  

    
Growth Rate
    
h-1
    
0.02
    
0.036
    
0.047
    
0.049
    
0.029
    
0.033
    
0.031
  
  

    
Doubling Time
    
h 
    
34
    
20
    
15
    
14
    
24
    
21
    
22
  
  

    
Biomass Productivity
    
g.L-1.day-1 
    
0.034
    
0.058
    
0.1
    
0.12
    
0.11
    
0.095
    
0.035
  
  

    
Final Biomass Produced
    
g.L-1 
    
0.229
    
0.407
    
0.619
    
0.824
    
0.767
    
0.636
    
0.235
  

Table 2: Biological parameters of C. vulgaris culture at different pH values.

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The average C. vulgaris cell size varied from 3.177 μm to 2.972 μm for the pH values between 6.5 and 7.5 (Figure 5). The variation observed in cell size suggests that at pH values between 6.5 and 7.5 the cells were “younger” as on average they would not have time to grow in size due to the faster replication rate. Under acidic conditions, the cell size of C. vulgaris was larger (increase up to 19.6%) than at neutral pH values. The higher pH induces a larger cell size (increase up to 8 %), indicating a possible cell physical modification possibly due to an accumulation of storage lipids increasing fatty acid content (reserves). The modification of the cell size is perhaps related to the growth rate of the microalga.

Figure 5: Average cell size of C .vulgaris at different pH values at the end of exponential growth phase. (n=3, Sd generally less than 5% of the mean).

    

    

    Figure 5: Average cell size of C .vulgaris at different pH values at the end
of exponential growth phase. (n=3, Sd generally less than 5% of the mean).
    

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The acclimatization of C. vulgaris toward pH variation could be observed from the duration of the lag phase of the cultures (Figure 6). The shortest lag phases were observed for near neutral pH values, with a minimum value (6 h) at an optimal pH 7.0. The variation of the external pH led to a prolongation of the lag phase, reaching 65 h under acidic environments and 27 h under alkaline environments. The variation of the external pH may induce metabolic adaptation to counteract the changes in conditions and overcome the high pH gradient [17]. This biological modification would have prolonged the lag phase, while a new equilibrium between the inside and the outside of the cell was established.

Figure 6: Duration of lag phase of C.vulgaris at different culture pH values.

    

    

    Figure 6: Duration of lag phase of C.vulgaris at different culture pH values.
    

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Nitrogen consumption

Besides the distribution of the carbon species, another culture parameter that can affect the growth of C. vulgaris and that is affected by the pH is the availability of nitrogen species. Based on the previously obtained growth results (data not shown), the authors decided to no longer focus on the strongly alkaline pH (pH 9.0 and pH 10.0).

The specific nitrogen consumption rates of C. vulgaris at different pH values were determined (Figure 7). The best nitrogen utilization was observed at pH 7.0, while the lowest utilizations were observed in acidic environments. To explain these findings, it would be reasonable to suppose that the acidic and basic extreme pH environments significantly affect the microalgal enzymatic activity and thus reduce the assimilation capacity of nutrients in the medium, such as nitrogen source, therefore, inducing a starvation phenomenon of the nitrogen source.

Figure 7: Specific nitrogen consumption rate of C. vulgaris at different pH values measured at the end of the exponential growth phase (n=1).

    

    

    Figure 7: Specific nitrogen consumption rate of C. vulgaris at different pH
values measured at the end of the exponential growth phase (n=1).
    

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Quantification of C:N ratio

The effect of the variation of the external pH on the carbon and nitrogen uptake can be estimated by the quantification of the cellular elemental content of carbon and nitrogen. The maximal intracellular contents of carbon and nitrogen were observed at a pH of 7.0. For acidic pH values, there was a decrease in the intracellular nitrogen content (26% approx. compared with pH 7.0) suggesting that the pH, under these conditions, alters the function of certain enzyme and thus, the assimilation capacity of nutrients such as nitrogen. Thus, the cellular carbon metabolism of C. vulgaris seems to change at different culture pH values. The major proportion of carbon in a cell is diverted towards lipid accumulation inside the cell. Acidic pH can inhibit the activity of nitrate reductase, involved in nitrate consumption, and therefore, for nitrogen uptake [32].

In alkaline environments, the intracellular carbon content is higher than in the acidic environments (45% and 48% for pH 5 and 8, respectively). As the culture pH was increased to a more neutral pH, it seems that C. vulgaris showed a better affinity to assimilate the HCO₃^-than the CO₂. These results suggest that in an alkaline environment, when the primary metabolic pathway of MCC’s uses active transport of HCO₃^-, the assimilation of carbon is higher than that at lower pH. In the latter case, the carbon source is diverted towards protein synthesis.

One of the most important parameters that indicates the effect of the carbon and nitrogen limitation is the C:N ratio of the medium. When this ratio is low, carbon limitation occurs and the carbon source is privileged for protein synthesis. On the other hand, a low C:N ratio, tends to induce a lipid accumulation. For microalgae, the optimum C:N ratio is around 10 [43]. Another study [44] indicates that for fast growth, this ratio is approximately 6. The profile of C:N ratio of C. vulgaris at different external pH values (Figure 5) showed that the rise of the pH decreases the C:N ratio with a lower value for pH 7.0 (C:N ratio equal to 5.51). The increase in the C:N ratio and the decrease of the intracellular nitrogen content are possible consequences of nitrate starvation in the culture medium. These results are in accordance with other studies [45,46]. It could be that the C/N ratio should be taken into consideration in combination with μ. With fast μ, the effective carbon flux can be considered to be reduced. Industrially, this suggests that lipid accumulation could be more extensive at lower culture pH values.

Chlorophyll content

For cultures at the optimum pH of 7.0 (Figure 10), the chlorophyll content of the microalgae cells peaks at 72 hours of growth corresponding to the end of the exponential phase (3.1 ng/g biomass). Lower contents were observed at the other pH values used (0.60-0.42 ng/g biomass). C. vulgaris exhibited the best performance in terms of photosynthetic activity at pH 7.0. The effect of pH on chlorophyll production in C. vulgaris is illustrated by the assimilation of nitrogen and carbon. The maximum specific yield coefficient (mg/g dry biomass) of chlorophyll correlated with the carbon and hydrogen content of the cells, at pH 7.0. When the pH was lower than 7.0, the chlorophyll accumulation was less. This might suggest that, at acidic culture conditions, denaturation of enzymatic activity and/or the decreasing of total accumulated carbon limit the photosynthesis activity of C. vulgaris. In fact, lower pH affects the uptake of nitrogen and the cellular metabolism changes by using the fixed carbon for lipid accumulation [28]. Nitrogen deficiency can decrease chlorophyll production [47]. A peak in chlorophyll production was observed after 24 hours of culture, under all operating conditions. This may suggest an “optimal” cell concentration that can be considered to have the maximal photosynthetic activity with respect to the culture conditions applied. This could be the case particularly in relation to the phenomenon of cellular auto-shading. The optimum photosynthetic activity coincided with the point when the cell concentration was approximately 20 million cells/ml. This of course is dependent on the geometry of the photobioreactor. The primary objective of this study was to identify the optimal growth pH for C. vulgaris growth in pHcontrolled cultures where the pH was continuously adjusted by CO₂ addition. The results showed the best growth at pH 7.0. Under these operating conditions of light and temperature, C. vulgaris showed the maximal growth rate, productivity, specific nitrogen consumption and chlorophyll content over a pH range of 7.0 to 10.0. In non-pH adjusted batch cultures, the final pH of the culture can approach 10.0.

Figure 8: Elemental cellular content of C. vulgaris at different culture pH values. (n=2, Sd generally less than 5% of the mean).

    

    

    Figure 8: Elemental cellular content of C. vulgaris at different culture pH
values. (n=2, Sd generally less than 5% of the mean).
    

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Figure 9: C:N ratio of C. vulgaris biomass at different pH values. (n=2, Sd generally less than 5% of the mean).

    

    

    Figure 9: C:N ratio of C. vulgaris biomass at different pH values. (n=2, Sd
generally less than 5% of the mean).
    

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Figure 10: Changes in chlorophyll content of C. vulgaris culture in different pH values (n=1).

    

    

    Figure 10: Changes in chlorophyll content of C. vulgaris culture in different
pH values (n=1).
    

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In terms of intracellular elemental content, the microalgae cells had the highest carbon and nitrogen content and the lowest C:N ratio when grown at pH 7.0. The effect of pH variation can be explained by the modification of C. vulgaris metabolism to counteract the unfavourable pH gradient between the inside and the outside cell. This postulated metabolic modification could cause enzyme denaturation, interfere with the uptake of assimilable nitrogen and diminish photosynthetic activity. Nitrogen source limitation can induce the microalgae to switch their metabolism from protein synthesis to lipid accumulation.

The culture pH has a directly impact on the distribution of carbonate species in the medium. During this study, the affinity of C. vulgaris for carbon dioxide and bicarbonate species was considered. It seems that C. vulgaris has a better affinity to assimilate the HCO₃^- than the CO₂, not surprising in the context of MCC’s biological phenomena.

Microalgae culture pH-control can be optimized by regulating the mineral acids or base content of the medium in combination with automated CO₂ supply. Further work will involve confronting the various hypotheses developed during this study; the effect of pH on fatty acid accumulation. And photosynthetic activity Moreover, the affinity of C. vulgaris toward CO₂ and HCO₃^- may be investigated by using different media with different carbon sources (CO₂ gas or bicarbonate salts) under the same operating conditions. Another interesting perspective concerns the identification of the optimal C:N ratio of balanced photoautotrophic C. vulgaris culture in order to identify the transition point between carbon and nitrogen starvation.

Acknowledgement

We are grateful to Pr. PERRE Patrick, director of the Chair of Biotechnology of CentraleSupélec for founding all the work presented in this article.

Declaration of Authors Contributions

Rayen FILALI and Behnam TAIDI conceived and designed the study, performed the analysis and the interpretation of data, drafting the article and critical revising. Rayen FILALI and Hao TIAN have principally preformed the experiments with a contribution from Emilie MICHIELS. Rayen FILALI and Behnam TAIDI contributed to the manuscript preparation and revisions and all authors approved the final version.

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Citation: Filali R,Tian H, Micheils E and Taidi B. Evaluation of the Growth Performance of Microalgae Based on Fine pH Changes. Austin J Biotechnol Bioeng. 2021; 8(1): 1109.

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