Summer microalgae report, Sept 2014

Growing C. vulgaris in mixotrophic conditions and high light intensity; UPRA

Observed Growth Rate of Chlorella Vulgaris under Mixotrophic Conditions –

Observed Growth Rate of C.v. under Hight Light Intensity in Autotrophic Conditions

Undergraduate Investigation QUIM 4999

Commenced: July 8, 2014 Ended: August 22, 2014

Student: Joseph A. Barnes (440-13-0709)

Laboratory Supervisor: Dr. Hirohito Torres

University of Puerto Rico at Arecibo

Abstract

Green microalgae can serve as a source for biodiesel production, providing a means of generating renewable energy, which is eco-friendly and comparatively simple to manage. This experimental study has been partitioned into two phases. The first phase of this study was to evaluate the effects of subjecting Chlorella vulgaris, a species of green microalgae, to mixotrophic conditions. Three groups, each in duplicate, were observed over a period of eight days in order to analyze the effects of feeding C. vulgaris simple and complex sugars (heterotrophic conditions). The autotrophic effects were satisfied by a steady light-source regulated to a 12:12 hour light-dark cycle. The experimental study was evaluated on the basis of observed changes in cell-concentration and light absorbance. Initial findings demonstrated a failure of the algae to successfully thrive under heterotrophic effects (sugar supplementation plus aeration). The second phase of this study was to evaluate the outcomes of subjecting the remaining batches of C. vulgaris to a highly intensified light source, with wavelengths of blue and red, optimal for photosynthetic activity. Initial findings revealed a positive correlation between cell concentration and light intensity, although without a substantial gain in cell biomass.

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Introduction

The topic of microalgae has been gaining considerable attention in recent years, due to its capacity of serving as a source of renewable energy and as a means of finding economical independence from costly oil importation. Green microalgae, including the species of Chlorella vulgaris, the principal specimen of this experimental study, is capable of autotrophic growth, absorbing sunlight and carbon dioxide in order to produce sugars for the purpose of sustaining and reproducing itself. Once collected and dried, fatty acids and triglycerides can be extracted from the algae, subjected to the process of transesterification, and converted into biodiesel.

This method of producing biodiesel possesses clear advantages over other implemented techniques. First, green microalgae can be grown relatively quickly in shallow bodies of water or in photobioreactors, without the use of the excessive square-miles of surface terrain as in the case of other oil-producing crops (e.g. coconut trees, corn, soybeans, etc.) (Hirohito, 2013; Scarsella et al., 2010). In other words, a good crop of green microalgae can generate a much higher quantity of biodiesel per square-mile of land than any other oil crop. Second, green microalgae absorb vast quantities of carbon-dioxide. Producing large masses of microalgae means sequestering even larger amounts of carbon-dioxide from the atmosphere, which signifies that growing microalgae can be a carbon-neutral process, that is, the carbon dioxide emitted by the burning of biodiesel is reabsorbed by the microalgae. This cycle may inhibit any long-term accumulation of carbon-

dioxide in the upper atmosphere from the combustion of biodiesel. In fact, so potential is the algae’s property of sequestering carbon-dioxide, that it is the subject of research as a potential means of reducing overall carbon-dioxide emissions in the environment (Sahoo et al., 2012). A third advantage of microalgae is its ability to utilize heterotrophic metabolism in order to absorb sugars and other organic-carbon substances (Debjani et al., 2012; Xiaoyu et al., 2014; Leesing and Kookkhuntod, 2011; Scarsella et al., 2010). This makes a two-fold benefit. For one, by being able to grow and reproduce under heterotrophic conditions, the farming of microalgae will not be inextricably linked to the environment. Some measures can be taken to maintain high microalgae production in the absence of sufficient sunlight and environmental carbon dioxide. And for the other, microalgae can prove a viable means for removing industrial waste and unwanted byproducts, which substances would serve as the principal sources of organic carbon for the algae (Debjani et al., 2012; Xiaoyu et al., 2014).

The study was divided into two phases, wherein the first was focused on observing the effects of growing Chlorella vulgaris under mixotrophic conditions as opposed to just autotrophic. In order to satisfy these mixotrophic conditions, the microalgae were fed a simple sugar (dextrose) and a mixture of simple and complex sugars (molasses). The autotrophic metabolism was to be maintained by exposure to a steady source of light regulated to a 12:12 hour light-dark cycle. The effects were evaluated on the basis of cell-concentration and light absorbance. The results were observed over a period of eight days.

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Following the sugar supplementation in the first phase, the study was carried to a second phase aimed at analyzing the effects of a stark increase in the light intensity sustaining the autotrophic conditions. The parameters observed were cell concentration and light absorbance.

Materials and Method

Phase 1

The microalgae Chlorella vulgaris seed culture was purchased from the UTEX culture collection in Texas. The growth medium used in this study consisted of a solution of distilled water with a 0.75 g/L concentration of a standard brand-X 20-20-20 all-purpose fertilizer, which provided 0.15 g/L concentrations of nitrogen, phosphoric acid and potassium, essential nutrients necessary for promoting and sustaining the growth of autotrophs (table 2). Six 1-liter flasks were prepared with 630 mL of growth medium in each. Next, each flask was inoculated using 70 mL of seed culture, establishing a 9:1 ratio of growth medium to cell volume. Two flasks were set aside for the control group (Control Group 1 & 2); two other flasks were supplemented with dextrose at a concentration of 10 g/L (Dextrose Group 1 & 2); the final two flasks were supplemented with molasses at a concentration of 10.0 g/L (Molasses Group 1 & 2) (photo a). All six flasks were placed upon an industrial mixer (Environ Shaker), which was set to shake the solutions at a speed of 125 rpm. The light source consisted of four regular household fluorescent light bulbs, providing an average light intensity of 1,400 lux. The bulbs were set on a timer which

regulated the exposure of light to a 12:12 hour light/dark cycle. All six flasks were aerated via two pumps with the air hoses arranged in series, in order to supplement the media with carbon-dioxide drawn from the surrounding air. The average rate of aeration was over 660 ml/minute.

Phase 2 One of the four standard fluorescent light-bulbs was replaced with a highly efficient LED light device that emits high intensity light at blue and red wavelengths. Measured light intensity climbed to an average of 8,770 lux. The remaining flasks from phase 1 (CG 1 & 2) were subjected to the new light intensity.

Analysis Procedures

Changes in cell concentration were measured directly by cell-counting using a hemacytometer and a light-microscope. Light absorbance was measured with a spectrophotometer at red wavelengths (690 nm). Dry biomass was measured in triplicate using 15 mL samples from each unit and applying centrifugation to form pellets. The samples were diluted and subjected to a second centrifugation to remove salts; samples were placed in an incubator at 80°C for 24 hours to remove humidity.

Results and Discussion

Phase 1 As the graph provided will demonstrate (figure 1), the dextrose and molasses groups experienced a stagnation coupled with a steady decline in algae cell concentrations. After the

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eighth day, both sugar groups were discarded and phase 1 was terminated. The sugars and aeration had promoted the growth of bacteria and protozoa, which presence was easily detectable by the microscope as well as by macroscopic growths (photo b). In a short while, large concentrations of bacteria had developed which had stifled and significantly reduced the cell concentration of C. vulgaris. The absorbance tests with regards to the sugar supplemented groups do not correlate with the cell-concentration of algae. The pattern evinced is irregular due to the nature of contamination by bacteria and other microbes (figure 2). We can gather from our studies that in the presence of initial high sugar concentrations and aeration, the C. vulgaris is quickly supplanted by bacteria and protozoa. Other measures need to be taken (e.g. sterilization) in order to sustain algae growth under these particular mixotrophic conditions.

Phase 2 The LED light was installed on the 13th day of the experiment, and the effects observed until the 28th day, a duration of 15 days. As the graphs will demonstrate, the initial spike in light intensity (from 1,400 lux to 8,770 lux) is correlated to a steep climb in cell concentration and absorbance (figure 3 and figure 4). By the end of the phase, the cell concentrations and absorbance were still climbing, albeit at a lesser rate. Increase in oxygen output was also visibly noticeable by foaming at the surface. The main reactor containing the seed culture experienced certain changes in color and other properties, for reasons not conclusively established (photo c and d). The change in biomass as calculated by day 28 (table 1), although

not significant, is still perceptible and warrants consideration. For this section, a very rough estimate for the specific growth rate (u) was given, using biomass concentration calculated at day 8, as the biomass concentration at the beginning of the exponential growth phase (Xo). The specific growth rates for control groups 1 and 2 were very low, falling far short of given expectations (less than 0.07 d^-1) (table 1). It must be noted though, that these specific growth rates were measured in an interval of time which included a brief period before the LED light was installed. We can gather from these results that an improvement in the autotrophic conditions (higher light intensity in red and blue light spectra) has positive effects on the cell concentration and absorbance of the C. vulgaris. However, it remains to be seen by how much the algae can be affected by these new conditions (i.e. maximum output). As shown here, there is potential in amplifying autotrophic effects (e.g. supplying higher light intensity and carbon dioxide supplementation) in order to improve microalgae biomass.

Conclusion and Recommendations

Unless we undertake measures to sterilize the samples, such as by autoclaving, high initial concentrations of sugars combined with aeration will favor bacterial and protozoan growth, which is debilitating to the microalgae. Steps to be considered in order to avoid sterilization, may include using reduced concentrations of sugar, avoiding aeration, and fortifying the conditions which are favorable for photosynthesis (e.g. more light and more carbon-dioxide). Here listed are a few propositions to consider:

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1. Growth of algae solely by autotrophic means, with a given high light intensity and graduated boosts of carbon-dioxide supplementation.

2. Growth of algae initially under only autotrophic conditions, but then after reaching the maximum yield of cell-concentration, follow with a supplementation of sugar at a low concentration (e.g. 2 g/L).

It is certainly recommended that further research be done to study more accurately the effects of light intensity on C. vulgaris. According to earlier investigations (Cheirsilp and Salwa, 2012), high yields of biomass were attained for Chlorella sp. at ideal light intensities of 3000 to 5000 lux. The intensity from sunlight on a clear day at noontime (12:00 pm) measured up to 104,000 lux in the Arecibo area of Puerto Rico in mid-August (19/8/2014). However, it should be noted that very high light intensities do not necessarily correlate positively with biomass yields, but in fact could actually limit cell growth (Cheirsilp and Salwa, 2012).


Figures, Tables, and Images Figure 1

Cell Concentration versus Time (Phase 1)

050

100150200250300350400450

1 3 5 7 9

Time (days)

Ce

ll c

on

ce

ntr

ati

on

[1

0^

4]

(ce

lls/m

L)

Control Group 1 Control Group 2 Dextrose Group 1

Dextrose Group 2 Molasses Group 1 Molasses Group 2

Sugar groups were discarded on day 8 due to contamination by microbes other than algae. Figure 2

Absorbance versus Time (Phase 1)

0

0.2

0.4

0.6

0.8

1

1.2

1 3 5 7 9

Time (days)

Ab

sorb

ance

(A

) [6

90 n

m]

Control Group 1 Control Group 2 Dextrose Group 1

Dextrose Group 2 Molasses Group 1 Molasses Group 2

Absorbance tests were made at 100% dilution (cell volume was diluted with an equal volume of distilled water; 0.75mL cell volume + 0.75mL distilled water).

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Figure 3

Cell Concentration versus Time (Phase 2)

0

500

1000

1500

2000

2500

3000

3500

4000

13 15 17 19 21 23

Time (days)

Ce

ll c

on

ce

ntr

ati

on

[1

0^

4]

(ce

lls/m

L)

Control Group 1 Control Group 2

CG 2 received a higher intensity of light due to uneven distribution of LED light.

Figure 4

Absorbance versus Time (Phase 2)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

13 18 23 28

Time (days)

Ab

so

rba

nc

e (

A)

[69

0 n

m]

Control Group 1 Control Group 2

Absorbance tests were made at 100% dilution (cell volume was diluted with an equal volume of distilled water; 0.75mL cell volume + 0.75mL distilled water).

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Table 1 Dry biomass per liter of media

Sample Source Day 1 Day 8 Day 28 u (days^-1) Main Reactor 0.28 g/L - 0.20 g/L -

Control Group 1 - 0.033 g/L 0.33 g/L 0.0607 d^-1 Control Group 2 - 0.053 g/L 0.39 g/L 0.0544 d^-1

Note: specific growth rate (u) was calculated according to the Monod model, wherein, u = ln(Xt – Xo)/t, where Xt is biomass concentration at time t and Xo is biomass concentration at the beginning of the exponential growth phase. Biomass at day 8 will be regarded as the value at the beginning of exponential growth phase, thus t = 20.

Table 2 Contents of 20-20-20 All Purpose Fertilizer Percent of mass Nitrate nitrogen (NO2) Ammoniacal nitrogen (NH3) Water soluble organic nitrogen Total available nitrogen

6.09% 3.85% 10.06%

20%

Available phosphoric acid (P2O5)

20%

Water soluble potassium (K2O)

20%

Note: a 0.75 g/L concentration of 20-20-20 all-purpose fertilizer would yield a concentration of nitrogen of 0.15 g/L; phosphoric acid concentration would be at 0.15 g/L and potassium concentration would be at 0.15 g/L.

Data Table for the Figures 1, 2, 3 and 4 Time Cell concentration [10^4] (cells/mL) – Sugar Groups Days DG 1 DG 2 MG 1 MG 2

1 3 6 8

52.7 32.0

0 0.67

47.3 64.7 5.33 4.67

21.0 39.3 42.6 34.0

28.3 39.0 21.3 14.7

Time Cell concentration [10^4] (cell/mL) – Control Groups Days CG 1 CG 2

1 3 6 8 10 13 16 22 24

41.3 100

250.3 297.4 330.6 513.9 2119 2606 3078

48.7 125 213 356

406.6 513.9 2244 3374 3766

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Time Absorbance (A) at 690 nm – Sugar Groups Days DG 1 DG 2 MG 1 MG 2

1 3 6 8

0.053 0.502 0.493 1.132

0.053 0.505 0.415 1.004

0.141 0.612 0.253 0.643

0.179 0.935 0.254 0.252

Time Absorbance (A) at 690 nm – Control Groups Days CG 1 CG 2

1 3 6 8 10 13 16 22 24 28

0.037 0.052 0.084 0.079 0.111 0.148 0.308 0.518 0.564 0.627

0.044 0.065 0.084 0.104 0.127 0.170 0.337 0.616 0.674 0.714

Photos a, b, c, and d

(a) (b)

(c) (d)

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References

Cheirsilp, B., Salwa, T., 2012. Enhanced growth and lipid production of microalgae

under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresource Technology 110, 510-516

Debjani, M., van Leeuwen, J.H., Lamsal, B., 2012 Heterotrophic/mixotrophic cultivation

of oleaginous Chlorella vulgaris on industrial co-products. Algal Research 1, 40-48.

Leesing, R., Kookkhunthod, S., 2011. Heterotrophic growth of Chlorella sp. kku-s2 for

lipid production using molasses as a carbon substrate. Internat. Conf. on Food Engin. and Biotech. IPCBEE vol. 9

Sahoo, D., Elangbam, G., Devi, S.S., 2012. Using algae for carbon dioxide capture and

bio-fuel production to combat climate change. Phykos 42 (1), 32-38. Scarsella, M., Belotti, G., De Filippis, P., Bravi, M., 2010. Study on the optimal growing

conditions of Chlorella vulgaris in bubble column photobioreactors. Paper prepared by the Dept. of Chem. Engin. Mater. Environ., Sapienza Uni. of Roma.

Torres, H., 2013. On the growth of Chlorella vulgaris for lipid production. Poster

presentation at the University of Puerto Rico. Xiaoyu F., Walker, T.H., Bridges W.C., Thornton, C., Gopalakrishnan, K., 2014. Biomass and lipid production of Chlorella protothecoides under heterotrophic

cultivation on a mixed waste substrate of brewer fermentation and crude glycerol. Bioresource Technology 166, 17-23.

Documents

Summer microalgae report, Sept 2014