The concept of bioremediation is quickly becoming the norm in the resolution of environmental issues. The steady increase in carbon dioxide (CO2) levels, as documented by NASA [1], inspired scientists to engineer plants to absorb excess CO2 from the atmosphere. Here, we have explored the consequences of the uptake of excess CO2 by select plants. Carbon dioxide dissolves in H2O to produce H2CO3, which dissociates to yield H+ ions. We hypothesized that increased CO2 absorption results in decrease in pH of plant sap. Three plants (Byophyllum pinnatum, Romaine Lettuce and Nevada Lettuce), exposed to increased CO2 concentrations (15%), demonstrated a consistent increase in pH towards alkalinity compared to control plants. Based on the outcome being opposite of what we have hypothesized, our results suggest Byophyllum pinnatum, Romaine lettuce and Nevada lettuce, all have a unique homeostatic system to prevent over-absorption of CO2 in a CO2-rich environment.

This work is inspired by the concept of bioremediation; the use of naturally occurring or genetically engineered organisms to consume or break down pollutants in the environment. Carbon dioxide levels have steadily increased over the years, raising concerns for individuals who want to solve this problem. In Scientist’s quests to address this problem, the idea of engineering plants to increase the capacity of CO2 absorption from the environment is still in the early stages. Others have pursued the use of a semi-synthetic rubisco, which has the ability to increase the rate of photosynthesis [2]. Apart from the positive side of bioremediation, we contemplate if there may be any negative consequences of increased CO2 in plants; so, we ask the question, what will be the effect of excess CO2 on the pH of plant sap? To our knowledge, there is currently no comprehensive study which specifically addresses the effect of increased CO2 concentration on the pH of plants and how plants potentially regulate the increased levels of CO2.

By principle, when carbon dioxide dissolves in water, carbonic acid is produced according to equation [1].

CO2 (aq) + H2O ⇓∇ H2CO3 (aq)                         [1]

H2CO3 (aq) ⇓∇ H+ (aq) + HCO3- (aq)               [2]

According to equation [2], dissolved CO2 in the form of H2CO3 may lose up to two protons through the acid equilibrium. The relatively small amounts of H+ produced, when built up, is anticipated to decrease the pH of plant cytosol. We hypothesize that the exposure of plants to increased CO2 levels results in a decrease in the pH of plant cytosol. 

• Monitor pH of plant sap at increased CO2 concentrations 
• Determine whether bioremediation is a safe solution to combat CO2 pollution based on pH data
• Further understand the relationship between CO2 and pH of plant sap

• pH meter
• Garlic press
• CO2 incubator
• Lamp for light source
• Plant soil heater

Plants (e.g. Byophyllum pinnatum, Romaine lettuce, Nevada lettuce) were incubated in the presence of increased CO2 with a light source or under normal atmospheric conditions with a light source. The lighting schedule, temperature, and watering of the two species of plants were kept the same, with the only variable being the difference in CO2 concentration. The control plant was maintained in atmospheric CO2 concentrations and the test plant was maintained in 15% CO2 concentration, using a CO2 incubator. The light sources were on for approximately ten hours per day and the plants were watered once per week. The same set up was observed for 6 stalks each of Romaine lettuce and Nevada lettuce. Every 2 to 3 days, plant sap from leaves were squeezed using garlic press and pH of the sap were measured using the Horiba LAQUAtwin compact pH meter. The pH was measured for triplicate samples each of controls and test plants, and averaged. The results are as follows: 

Byophyllum pinnatum

Table 1 shows days of pH measurement of test and control samples on days 3, 5, 7, 11 and 12 of experiment set-up. The pH of the control samples were consistently lower compared to that of the test samples (plants exposed to 15% CO2). The result also shows that sap pH generally increased as the number of days of CO2 exposure increases (Figure 1). 

Table 1: Mean pH readings from triplicate samples vs exposure time in Byophyllum pinnatum

Figure 1: The results were not what we expected. The plant exposed to excess CO2 became more alkaline instead of more acidic, while the control plant remained well under pH=5. This suggests Bryophyllum pinnatum has a homeostatic system to prevent over-absorption of CO2 in a CO2-rich environment. 

Romaine lettuce (Lactuca sativa)

Table 2 shows days of pH measurement of test and control samples on days 4, 7, 11 and 14 of experiment set-up. The pH of the control samples were consistently lower compared to that of the test samples (plants exposed to 15% CO2). The result also shows that sap pH increases as the number of days of CO2 exposure increases (Figure 2).

Table 2: Mean pH readings from triplicate samples vs exposure time (Days) in Romaine lettuce

Figure 2: The results were not what we expected. The plant exposed to excess CO2 became more alkaline instead of more acidic, while the control plant remained mostly under pH=6.3. This suggests Romaine lettuce also has a homeostatic system to prevent over-absorption of CO2 in a CO2-rich environment. 

Nevada lettuce

Table 3 shows days of pH measurement of test and control samples on days 4, 7, 11 and 14 of experiment set-up. The pH of the control samples were consistently lower compared to that of the test samples (plants exposed to 15% CO2). The result also shows that sap pH increases as the number of days of CO2 exposure increases (Figure 3).

Table 3: Mean pH readings from triplicate samples vs exposure time (Days) in Nevada lettuce

Figure 3: The results were not what we expected. The plant exposed to excess CO2 became more alkaline instead of more acidic, while the control plant remained well under pH=6.3. This suggests Nevada lettuce also has a homeostatic system to prevent over-absorption of CO2 in a CO2-rich environment. 

Due to the consistent increase in sap pH of all plants exposed to increased CO2 levels, our result demonstrates that edible plants, such as lettuce, share a common mechanism of excess CO2 level regulation with crassulacean acid metabolism plants, such as Bryophyllum pinnatum. Both species of plants have shown a similar response in pH when they were exposed to excess CO2 levels in a controlled environment.

Although increased CO2 concentration in the atmosphere has been a main concern of the general public, there are some merits of excess CO2 to plants. Increased CO2 levels increases the yields of crops such as soybean, wheat and rice by up to 14% [3, 4]. The amount of carbohydrate production measured in unit areas of plant leaf was found to have increased by up to 40%, accompanied by a 13

We have used the chambered method of plant exposure to increased CO2 levels because it is affordable and within our budget. We understand that the chambered method of plant exposure to increased CO2 levels may not mimic the exposure of plants to increased CO2 in a more natural setting. Nevertheless, our result is significant and we welcome fellow scientists with the capability to use Free-Air Carbon dioxide Enrichment (FACE) technique [7] of plant exposure to increase CO2 to monitor plant pH to replicate our data.

We further hypothesize that the exposure of plants to increased CO2 accelerates the rate of plant carbohydrate breakdown.

We thank the Friends University VPAA office, and the division Chair, Dr. Nora Strasser, for the provision of starter funds to support research involving undergraduates. We thank Ms. Amy Morgan for the procurement of equipment and materials needed to execute all projects. We remain indebted to Dr. J.C. Moore for his timely intervention on ideas with experimental set up.

P.N.A. conceived the project. P.N.A, J.S., T.P. and P.H. designed the experiments. J.S., T.P. and P.H. performed all experiments. J.S. and N.S. analyzed the data. J.S. and P.N.A. wrote the manuscript. P.N.A. and G.B. edited and proofread the manuscript. P.N.A. procured funding. 

Graphical Abstract

Suppl. Figure 1: Graphical abstract of plant exposure to increased CO2 levels and the consequent increase in the pH of the sap of the plant towards alkalinity.

Suppl Figure 2: Mean pH Data of triplicate samples. Plants exposed to increased CO2 levels show consistent increase in pH compared to control plants.

Suppl. Figure 3: 2-way Anova test results