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.
1
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
2
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 OHwill 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-
mol/L
g/L
mol/L
mol/L
pH
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
3
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.
4
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
Chlorophyta
Character
Fresh
Alkaliphilic
Strain name
Chlorella
sorokiniana
Dunaliella
viridis
Dunaliella
salina
Dunaliella
primolecta
SL 100415A
Fresh Water
Alkaliphilic
Fresh Water
Alkaliphilic
Salt Water
Synechocystis
sp.
Synechococcus
sp.
Cyanothece sp.
Salt Water
Salt Water
Salt Water
Cyanobacteria
Source
UTEX 1602
Medium
Bristol
UTEX LB 200
RCM seawater
medium
RCM seawater
medium
RCM seawater
medium
Bristol
UTEX LB1644
UTEX LB 1000
Isolated from Soap Lake
in Washington State
PCC6803
BG-11
PCC7942
BG-11
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
5
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.
C. sorokiniana
11.0
0.6
0.2
0.05
1.4
0.3
0.1
0.01
0.6
0.2
0.05
1.2
1.0
10.0
OD
pH
10.5
C. sorokiniana
9.5
0.3
0.1
0.01
0.8
0.6
0.4
9.0
0.2
0.0
8.5
0
2
4
Time (day)
6
8
0
2
4
Time (day)
6
8
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).
6
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
LB200
10.5
0.6
0.2
0.05
10
LB200
1.2
0.3
0.1
0.01
0.6
0.2
0.05
1.0
0.3
0.1
0.01
0.8
OD
pH
9.5
9
0.6
0.4
8.5
0.2
8
0.0
0
2
4
Time (day)
6
8
0
2
4
Time (day)
6
8
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.
7
UTEX LB1644
11.0
0.6
0.2
0.05
10.5
UTEX LB1644
1.2
0.3
0.1
0.01
10.0
0.3
0.1
0.01
0.8
9.5
OD
pH
0.6
0.2
0.05
1.0
9.0
0.6
0.4
8.5
0.2
8.0
7.5
0.0
0
2
4
Time (day)
6
8
0
2
4
Time (day)
6
8
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.
UTEX LB1000
UTEX LB1000
11.0
1.4
10.5
1.2
1.0
OD
10.0
pH
0.6
0.2
0.05
9.5
9.0
0.6
0.2
0.05
8.5
0.3
0.1
0.01
0.8
0.6
0.4
0.3
0.1
0.01
0.2
8.0
0.0
0
2
4
Time (day)
6
0
2
4
6
Time (day)
Figure 4. Dunaliella primolecta strains growth with sodium bicarbonate as solely carbon source (a) pH
variation in the culture (b) optical distribution
8
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.
SL100415A
12.0
0.6
0.2
0.05
11.5
0.6
0.2
0.05
1.0
0.3
0.1
0.01
0.8
10.5
OD
pH
11.0
SL100415A
1.2
0.3
0.1
0.01
10.0
0.6
9.5
0.4
9.0
0.2
8.5
0.0
0
2
4
Time (day)
6
8
0
2
4
Time (day)
6
8
Figure 5. Alkaliphilic algae SL100415A growth with sodium bicarbonate as sole carbon source (a) pH
variation in the culture (b) optical distribution
This strain resulted in fast growth in 0.01 M bicarbonate, partly inhibited in 0.05 M and 0.1 M, and
showed severe inhibition in 0.2 M. There was only slight growth in 0.3 M bicarbonate. Although there
was also OD increase in 0.6 mol/L bicarbonate, the observation through microscope found it was actually
caused by dead cells. In 0.01 M bicarbonate, this algae strain resulted in fast growth until the pH
increased to 11.18 at day 5. After that, the growth rate slowed, and resulted in final pH of 11.79 (Figure 5).
In 0.05 M bicarbonate, the pH resulted in 10.37 at day 5, and the OD had further increase until day 7
when the final pH reached 11.97. These results indicated that this strain from Soap Lake can tolerant to
high pH, but not high concentration of bicarbonate. Even 0.05 M bicarbonate culture resulted in much
slower growth than 0.01 M culture. High bicarbonate/carbonate concentration is preferred in the culture,
9
since it provides stronger buffer effect and prevents the pH increasing to too high. Thus, this algae strain
is not an ideal strain, although it had fast growth in high pH environment.
This alkaliphilic algae was isolated from Soap Lake, which usually contains 11.8 g/L (0.51 M) Na + and
1.1 g/L (0.03 M) K+, as well as 2 g/L (0.03 M) bicarbonate and 8.5 g/L (0.14 M) carbonate (Andernson,
19xx). Although it is from this environment, this alkaliphilic algae strain was greatly affected by the
concentration of sodium bicarbonate in the range of 0.01M to 0.2 M. Thus, more strains from soda lakes
are to be isolated and tested as candidate of bicarbonate tolerant strains.
3.1.4 Alkaliphilic cyanobacteria strains
Synechocystis PCC6803 has been reported to have similar growth at pH 7.0 and pH 10.0. Thus, it was
tested as an alkaliphilic cyanobacteria strain in this study. Unlike eukaryotic green algae, this
cyanobacterium had poor growth in 0.01 M bicarbonate. Instead, it had best growth in 0.2 M bicarbonate.
Also, there was compromised growth in 0.3 M, and cells were dead in 0.6 M bicarbonate. The pH
dramatically increased during the first three days culture in 0.1 M bicarbonate and less (Figure 6). At day
3, the pH in 0.01 M, 0.05 M, and 0.1 M are 11.1, 10.9, and 10.6, respectively. This high pH should be the
main reason to cause cell death.
On the contrary, the pH in 0.2 M and 0.3 M was below 10.0 all the culture time, and the OD was
significantly higher than other cultures. This illustrated again that higher concentration of bicarbonate
have a stronger buffer effect than lower concentration. 0.3 M culture resulted in much lower OD than 0.2
mol/L, indicating there was inhibition.
Synechocystis PCC6803
12
0.6
0.2
0.05
Synechocystis PCC6803
1.2
0.3
0.1
0.01
0.6
0.2
0.05
1.0
0.3
0.1
0.01
0.8
pH
OD
11
0.6
0.4
10
0.2
0.0
9
0
2
4
Time (day)
6
8
0
2
4
Time (day)
6
8
Figure 6. Synechocystis PCC 6803 growth with sodium bicarbonate as solely carbon source (a) pH
variation in the culture (b) optical distribution
10
On the whole, this Synechocystis sp. strain is tolerant to high concentration of bicarbonate (up to 0.2M),
and it is an alkaliphilic strain that is tolerant to at least pH 10.0. Thus, it is worth further studying to
determine the optimal culture condition of this strain, and used as one of ideal strains in this study.
Synechococcus PCC 7942 ?
Figure 7. cell growth and pH variation
3.1.5 Halophilic cyanobacteria strains
Figure 8.cell growth and pH variation
3.2 Dunaliella sp. culture with bicarbonate
1. use reduced calcium and magnesium seawater medium with 0.30 M sodium carbonate
2. Use the prepared medium for algae culture in stirred flask (test the effect of agitation to cell
growth and CO2 loss).
3. Use the prepared medium for algae culture in T-flask without agitation (save the energy for
agitation, and may reduce CO2 loss)
4. The culture with same medium, but bubbling with CO2 as the control 1 (same bicarbonate
concentration, with stable pH)
5. The culture without bicarbonate and bubbling with CO2 as the control 2 (no bicarbonate, CO2 as
the carbon source)
6. Measure yield of algae biomass, and bicarbonate consumption rate (TOC analyzer)
7. Test the lipid content in the produced algae
8. Recover the culture medium supernatant
Figure 9. Growth curve of biomass, bicarbonate consumption, pH change, Biomass yield, and lipid
content of Dunaliella for static culture, stirred culture, CO2 sparging culture, and CO2 sparging without
bicarbonate
7/6-7/13
3.3 Carbonation interval:
7/13- 8/13
11
Use pure CO2 to sparge the initial culture medium, measure every 2 hours, to determine carbonation
kinetics
Use the most alkaliphilic Dunaliella strain
Use higher inoculation size 10%, to shorten the lag phase
Harvest every 3 days, then inoculate with same amount of seed cells (20%), run 21 total days
Measure pH before and after sparging CO2
Measure the bicarbonate consumption with each harvesting (every 3 days)
Compensate the evaporated water every day
Use shaker
1. Carbonation continuously (CO2 bubbling all the time)
2. Carbonation every 7 days , Use pure CO2, to the pH 8.0
3. Carbonation every 3 days , Use pure CO2, to the pH 8.0
4. Carbonation every 2 days, Use pure CO2, to the pH 8.0
5. Carbonation every day, Use pure CO2, to the pH 8.0
Figure 10. Growth curve of biomass, pH change, bicarbonate consumption
3.4 recycling the spent medium
7/20-8/20
Recycling 100%, 95%, 90%, and 80% of the spent medium, harvest and carbonation each week, keep
culturing until cell stop growth, or last for 5 weeks
Figure 11. Recycling medium culture results
3.5 Cyanobacteria culture with bicarbonate
Figure 12. Growth curve of biomass, bicarbonate consumption, pH change, Biomass yield, and lipid
content of selected cyanobacteria
3.6 Carbonation interval
Figure 13. Growth curve of biomass, pH change, bicarbonate consumption
12
3.7 recycling the spent medium
Recycling 100%, 95%, 90%, and 80% of the spent medium, harvest and carbonate each week, keep
culturing until cell stop growth, or last for 5 weeks
Figure 14, Recycling medium culture results
Use modeling work explains the CO2 loss in Discussion part. CO2 loss is not a problem for close
system, but it is a severe problem for open system.
13
Discussion
1. This CO2 delivery process will save part of the energy for continuous CO2 sparging,
2.
bicarbonate production process through CA will be used in this process
3. The high pH water should be recycled
4. High pH culture prevents invading species
5. High pH reduce CO2 loss during the culture
6. High bicarbonate concentration utilization faces different problem than saturated CO2 utilization
The effect of high concentration of CO2 to algae growth, and compare this with bicarbonate concentration.
The CO2 concentration balanced with 1 atm CO2 gas is 0.0336 mol/L, and it is less than 0.05 mol/L
bicarbonate we used in this study.
This process will use both of bicarbonate and CO2 transported from power plant. Using only bicarbonate
will produce alkali water, which is potential threat to environment. The CO2 transported from power
plant can be absorbed by the alkali water produced in this process, and it can be recycled into algae
culture process again. However, part of the bicarbonate will be lost in the recycle process, and need to be
supplemented by bicarbonate.
The culture with lower bicarbonate resulted in higher pH. This is because the pH in the solution actually
decided by ratio of carbonate to bicarbonate. Culture with lower bicarbonate concentration consumed the
bicarbonate fast, and produced corresponding concentration of carbonate. This lead to a very high ratio of
carbonate to bicarbonate, and raised the pH.
The mechanism of pH tolerant and high bicarbonate concentration tolerant
Both high pH tolerance and high bicarbonate tolerance are required to screen the optimal strain.
Otherwise, the protocol can be considered that cultures the high bicarbonate tolerant algae strains at first,
and then use the effluent from that process to culture high pH tolerant algae strains.
1. Higher concentration sodium bicarbonate has stronger buffer effect, thus, salt water algae is
preferred
2. The algae strain have to be tolerant to high pH, thus, alkaliphilic algae is preferred
14
Conclusion
Acknowledgements
15
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16