Utilization of bicarbonate as inorganic carbon source for microalgae culture
Abstract
1) Test the algae or cyanobacteria that can tolerant high pH and high concentration (to buffer the pH increase)
2) Test screened algae strain in lab scale culture system to provide primary information on the culture
3) Determine algal biomass productivity, lipid yield, and yield of algal biomass from consumed bicarbonate
4) The effect of light intensity, agitation rate, temperature, salinity etc, with pH controlled CO2 bubbling culture as control
Keywords: alkaline, microalgae, cyanobacteria, bicarbonate, carbon dioxide.
Introduction
Among all carbon capture technologies, some of them convert CO2 into bicarbonate. For example, UOP’s Benfield TM process use potassium carbonate to absorb CO2, and convert it into potassium bicarbonate (1). Also, there are some researchers working on using ammonium to absorb CO2 and convert it into ammonium bicarbonate (1). There are also technologies using carbonic anhydrase to convert CO2 in the flue gas into bicarbonate (2). Besides, traditional method for sodium bicarbonate production, such as Solvay process and Hou's process, which pumps carbon dioxide and ammonia into saturated sodium chloride to produce sodium bicarbonate and ammonium chloride, may be used as a potential method for CO2 capture if cheap ammonium can be produced with renewable energy.
Benemann (2009) have pointed out several key challenges to capture CO2 produced from thermal power plant with algae culture. Firstly, piping of flue gas any distance is a major cost problem, and transfer flue gas CO2 into algae ponds cost very much and may not be economical viable. For example, among the $40.5/ton’s cost in Kadam’s estimate (3), $28.72/ton is for MEA extraction, $8.48/ton for compression and drying, and $3.30 /ton for transportation to an place 100 km away from the power plant. Thus, the major cost for transportation cost is actually from compression of the captured CO2, rather than transport by pipeline itself. If the flue gas was pumped directly into the algae pond, and combined with the cost of compression and drying, the cost was $46.64/ton, with a $10.58/ton of flue gas transportation cost. The total annualized cost was $57.21/ton. Thus, it is actually not economically viable to transport flue gas directly. Secondly, CO2 in flue gas cannot be stored during night time or winter, and supply to algae pond when it is needed in day time or summer. Thirdly, there is a significant loss of CO2 from outgasing in algae pond. These problems result in a maximum ratio of only 25% for carbon capture with algae culture. However, if the CO2 in flue gas is converted into bicarbonate, it can be stored and transported as solids or water solution. This will significantly reduce the cost of compression of CO2. Also, the energy cost for bubbling CO2 into algae pond can be reduced. In addition, converting CO2 into bicarbonate and be used in high pH environment would lead to much less CO2 loss during the algae culture process.
There have been tremendous literatures on the research of carbon concentration mechanisms , and it was found that most of the examined algae and cyanobacteria can take up both of HCO3- and CO2 (4, 5). However, there was no research on how high concentration of bicarbonate the algae species can survive and growth in. As a matter of fact, algae can be found in lagoon in volcano area that is hyper alkaline (pH 11) and contains concentrations of salt five times higher than those of sea water (http://www.nature.com/news/2010/100402/full/news.2010.161.html?s=news_rss). If the algae species that can survive in high concentration of bicarbonate screened, it can be used in the algae culture process for biofuel and bioproducts production. With consumption of bicarbonate, the pH in the water will be increased since HCO3- is actually converted into CO2 and OH-. The reaction is:
Reaction 1: HCO3- →CO2 + OH-
The CO2 is consumed and the leftover OH- will significantly increase the pH. Fortunately, this can be buffered by the rest HCO3- , since it reacts with OH- to form CO32-.
Reaction 2: OH- + HCO3- → CO32- + H2O
The pKa for dissolution of HCO3- is 10.3, via the reaction:
Reaction 3: HCO3- → CO32- + H+
The mixture of HCO3- / CO32- actually works as a strong pH buffer around pH 10.3, and the leftover OH- will not be able to increase the pH to a very high level.
Theoretically, if the initial bicarbonate concentration is 0.3 mol/L, the pH in the solution will be 9.92 (Table 1).
Table 1. Theoretical pH of difference concentration of sodium bicarbonate
[HCO3-] / NaHCO3 / [CO32- ] / OH- / pHmol/L / g/L / mol/L / mol/L
0.6 / 50.4 / 0.000119 / 0.000119 / 10.07
0.3 / 25.2 / 8.38E-05 / 8.38E-05 / 9.92
0.2 / 16.8 / 6.85E-05 / 6.85E-05 / 9.84
0.1 / 8.4 / 4.84E-05 / 4.84E-05 / 9.68
0.01 / 0.84 / 1.53E-05 / 1.53E-05 / 9.18
* Pka = 10.3, and Ka = 2.34×10-8,
If 0.1 mol/L of HCO3- was consumed in the algae culture process, then 0.1 mol/L of OH- will be produced. This 0.1 mol/L of OH- will react with another 0.1 mol/L of HCO3- (among the rest 0.2 mol/L of HCO3- ), and produce 0.1 mol/L of CO32- . Thus, the theoretical pH in the solution can be calculated as:
pH = pKa + log([CO32- ]/[HCO3-] = 10.3 +log(0.1/0.1) = 10.3+ 0 =10.3. Thus, if the initial concentration is 0.3 mol/L, the consumption of 0.1 mol/L of bicarbonate will lead to pH increase from 9.92 to 10.3, which is about 0.4. Once the culture finished, the high pH water should be recycled to absorb more CO2, and used again in the culture.
The algae strains that can tolerant high concentration of bicarbonate were screened at first. Then, they were tested in the culture system. The productivity of algae biomass dry weight, lipid yield was determined. Also, the yield of biomass from bicarbonate (g/g) was investigated. Also, the water produced in this culture was re-charged with CO2, and then used again in the culture with supplementation of nutrients.
Using saturated bicarbonate solution will significantly reduce the evaporation rate, and this will save a lot of cost for supplying fresh water to make up the evaporated water.
Bicarbonate as carbon source for algae culture:
Recycling the alkaline water to absorb more CO2, this is a close loop process. To save energy, higher concentration is preferred in CO2 absorption process. Thus, we need to screen high bicarbonate concentration tolerant algae strains.
Most algae strains cannot tolerant to too high concentration of bicarbonate. So, the actual optimal concentration has to be determined based on the compromise between carbon absorption and tolerance ability of algae strains to bicarbonate.
Using high concentration bicarbonate will cause more CO2 loss due to outgassing. Thus, higher pH should be used in this process.
Too high concentration of CO2 causes problem in algae culture, but if the high concentration bicarbonate can cause the same problem has not been investigated.
The CO2 loss rate is determined in this process.
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Materials and methods
1. Strains and medium
Strains have fast growth, and could be tolerant to high pH or high salinity was compared.
Table 1.Strains were tested to utilize bicarbonate as the carbon source in high concentration
Character / Strain name / Source / MediumChlorophyta / Fresh / Chlorella sorokiniana / UTEX 1602 / Bristol
Salt Water / Dunaliella viridis / UTEX LB 200 / RCM seawater medium
Salt Water / Dunaliella salina / UTEX LB1644 / RCM seawater medium
Salt Water / Dunaliella primolecta / UTEX LB 1000 / RCM seawater medium
Alkaliphilic / SL 100415A / Isolated from Soap Lake in Washington State / Bristol
Cyanobacteria / Fresh Water Alkaliphilic / Synechocystis sp. / PCC6803 / BG-11
Fresh Water Alkaliphilic / Synechococcus sp. / PCC7942 / BG-11
Salt Water / Cyanothece sp. / ATCC 51142 / BG-11
2. Culture conditions
The cells were cultured in the 24-well plate, 2 mL for each well. The culture room temperature was controlled at 20 °C. Different concentrations of sodium bicarbonate were used as the inorganic carbon source, and no CO2 gas is delivered into the culture. The optical distribution was tested with 750nm wavelength light for each sample. The light intensity was controlled at approximately ?????.
Results
3.1 Screening of microalgae and cyanobacteria strains for culture with bicarbonate
The purpose of this experiment is to test different strains of alga and cyanobacteria on their capability of growth in different concentration of bicarbonate, and screen the ideal strains that have fast growth in high concentration bicarbonate and high pH culture systems.
3.1.1 Fresh water green algae strains: C. sorokiniana
C. sorokiniana was tested as the candidate strain is because of its fast growth. When bicarbonate is used as inorganic carbon source, C. sorokiniana grow well in the concentration of 0.01 M, but partly inhibited by 0.05 M, and severe inhibited in 0.1 M (Figure 1). Also, there was no growth in bicarbonate concentration higher than 0.1M. In the low bicarbonate concentration (0.01 M), it had fast growth when pH is low (9.95 at day 3). When pH increased to 10.21 at day 5 and 10.77 at day 7, it resulted in much slower growth. These results indicated that C. sorokiniana can successfully take bicarbonate as the major carbon source, and resulted in fast growth. However, its growth in high concentration bicarbonate and high pH was much slower. Thus, it is not an ideal strain for the high pH and high bicarbonate concentration culture system.
Figure 1. C. sorokiniana growth with sodium bicarbonate as carbon source (a) pH variation; (b) optical distribution
3.1.3 Dunaliella strains
When high concentration of bicarbonate is presented, it leads to not only high pH, but also high ion strength. It is known that fresh water algae are usually tolerant to 0.2 M Na+ concentration (reference), and higher concentration than this may inhibit its growth. Dunaliella is well-known halophilic green algae, and it was reported some Dunaliella strains can even grow in saturated NaCl, which is about 6.1 M (reference). If some strains from this halophilic species are tolerant to high pH, they can be used as ideal strains for this study. Thus, three Dunaliella strains were tested (Table 1).
As an algae strain that have optimal growth at two times seawater salinity, it is not surprise that Duanliella viridis is tolerant to 0.3 mol/L of sodium bicarbonate. However, this algae strain had minimal growth in 0.6 M bicarbonate, indicating that the inhibition was not caused by the high pH, rather than the high sodium concentration.
When bicarbonate concentration is lower than 0.3M, the cell growths were at the same level (Figure 2). It is notable that 0.01 M bicarbonate resulted in a pH of 9.13, which is at same level as other culture’s initial pH, and should have the slightest inhibition by pH. Other concentrations led to a little less OD than 0.01 M, and this indicated that D. viridis only has slight inhibition when pH is lower than 10.0. However, D. viridis had a slower growth than C. sorokiniana in 0.01 M bicarbonate.
Prepare the seed culture 2/9, inoculate 2/16, harvest 2/23
Figure 2. Dunaliella viridis strains growth with sodium bicarbonate as solely carbon source (a) pH variation in the culture (b) optical distribution
Similar to D. viridis, D. salina culture with 0.3 M bicarbonate had a growth at the same level as that with lower bicarbonate concentration. Also, the D. salina with 0.6 M resulted in an OD greater than 0.4, which is higher than that of D. viridis. These results indicated D. salina is tolerant to higher concentration of bicarbonate. At day 3, the culture from 0.05 M to 0.3 M resulted in pH from 9.73 to 9.87. After that, all cultures had less growth rate during day 3 to day 5 than that during day 1 to day 3. This indicated that pH higher than 9.7 inhibit the cell growth, and it is not an ideal strain.
Figure 3. Dunaliella salina strains growth with sodium bicarbonate as solely carbon source (a) pH variation in the culture (b) optical distribution
Dunaliella primolecta grows faster than D. viridis and D. salina. Most cultures reached their maximum growth at day 3, rather than day 5. At day 3, the pH in cultures with 0.05 M to 0.3 M are from 10.21 to 10.38, and this increased to 10.46 to 10.63 at day 5. However, OD in all these cultures decreased, indicating too high pH killed the algae. Also, the 0.01 M culture resulted in pH 9.79 at day 3, and the growth rate from day 3 to day 5 was much less than that from day 1 to day 3. This indicated that pH higher than 9.79 also inhibited the cell growth. However, this algae strain has the fastest growth in high concentration of bicarbonate (0.3 M), and selected as one of ideal stains in this study.
Figure 4. Dunaliella primolecta strains growth with sodium bicarbonate as solely carbon source (a) pH variation in the culture (b) optical distribution
With the results of three Dualiella strains, it can be concluded Dunaliella can grow at pH greater than 10.0, and it all had good growth at high concentration of 0.3 M bicarbonate. D. salina also had some growth in 0.6 M bicarbonate. It is notable that the pH in 0.6 M bicarbonate is not higher than 0.3 M culture, indicating that the key limiting factor is not the high pH, but the high bicarbonate concentration.
3.1.3 Alkaliphilic algae strains from soda lakes
This algae strain was isolated from Soap Lake in Washington State, USA, and it was identified as a strain of xxxxx with 18S RNA test. Usually, soda lakes have high pH. For example, Soap Lake has a pH of 9.68 (data not shown). Thus, alkaliphilic algae strain may be isolated, and can be tested as candidate strains for this study.