Photoautotrophic Production of Docosahexaenoic Acid- and Eicosapentaenoic Acid-Enriched Biomass by Co-Culturing Golden-Brown and Green Microalgae
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
Review report
Title: Photoautotrophic production of DHA and EPA enriched biomass by co-culturing of golden-brown and green microalgae
Comments:
This paper describes the co-culture experiment using golden-brown and green microalgae and the changes in their physiological characteristics. It is basically well written and may be recommended for acceptance, but there is one main concern, which should be addressed.
The authors conclude that wavelength changes biomass production and other physiological properties of the microalgae, as noted in lines L561-562, L589 and other in the text. To confirm this would require an additional experiment in which the light wavelength was varied when incubated alone (single algal culture experiment). The present experiment would certainly change the wavelength of the culture, but I do not know if it really affects them positively. For example, could metabolites released from the cells into the medium or bacteria in culture affect the properties of the algae? If the authors do not carry out additional experiments, it would be better to avoid “definitive conclusions” in the text. Of note, as described in L21 of the abstract, the availability of green light to algae is quite limited, and the authors should support their claims with data or citations to previous studies to see if the organisms used in the experiments can actually use green light.
Minor points:
Captions of Fig. 2,3 and 4:
Please clarify the meaning of the gray circles or squares in the caption.
The caption should also explain why data are presented for each species despite the co-culture experiments (i.e., because readers may not immediately understand why individual data can be produced from co-culture experiments).
Caption of Fig. 5: Specify in which part of the culture vessel the spectrum was measured.
Author Response
#Reviewer 1
The authors conclude that wavelength changes biomass production and other physiological properties of the microalgae, as noted in lines L561-562, L589 and other in the text. To confirm this would require an additional experiment in which the light wavelength was varied when incubated alone (single algal culture experiment). The present experiment would certainly change the wavelength of the culture, but I do not know if it really affects them positively. For example, could metabolites released from the cells into the medium or bacteria in culture affect the properties of the algae? If the authors do not carry out additional experiments, it would be better to avoid “definitive conclusions” in the text.
Response: Done. Unfortunately, due to time constraints, we are unable to conduct any further experiments. However, we tried to avoid definitive conclusions by rephrasing the following sentences:
L595-597: Hence, we suggest the utilization of complementary wavelengths of light in co-cultivation, potentially leading to increased light absorption and improved biomass yield.
L603-607: A high fucoxanthin content of the microalgae I. galbana compared to N. oceanica may explain the decreased absorption intensity of these wavelengths (500 - 580 nm) and, therefore, the enhanced use of the PAR in this range in monoculture.
Of note, as described in L21 of the abstract, the availability of green light to algae is quite limited, and the authors should support their claims with data or citations to previous studies to see if the organisms used in the experiments can actually use green light.
Response: Done. Due to its high content of fucoxanthin, the strain Isochyrsis galbana can use green light. We tried to explain this in the manuscript (see L590-595), including citations from other studies. To clarify, we added another citation that showed the use of green light by Isochyrsis galbana.
L598-603: The golden-brown microalgae T. lutea, a marine microalgae strain with high genetic similarity to the strain I. galbana used in this study, as well as the microalgae strain Isochyrsis aff galbana are known for their high content of the carotenoid fucoxanthin [21, 49-51]. Fucoxanthin, chlorophyll a/c, and proteins form a complex called FCP in the thylakoid membrane [52]. Bound in a complex, the fucoxanthin absorption spectrum is extended from 450 - 540nm in solution to 390 - 580nm [53].
L606-609: The use of green light by the strain Isochyrsis galbana has already been shown in literature: Li and Liu reported an increase in the photosynthetic efficiency by using green light, indicating that green light promoted the photosynthesis of the strain Isochrysis galbana.
Captions of Fig. 2,3 and 4:
Please clarify the meaning of the gray circles or squares in the caption.
The caption should also explain why data are presented for each species despite the co-culture experiments (i.e., because readers may not immediately understand why individual data can be produced from co-culture experiments).
Response: Done.
L264-265: Batch processes with two different nitrogen sources are shown: nitrate (filled grey circles) and urea (filled grey squares).
L322, L440, L534: Flow cytometry allowed the distinction between the two microalgae strains.
Caption of Fig. 5: Specify in which part of the culture vessel the spectrum was measured.
Response: Done.
L573-574: The light measurements were always taken at the same location in the reactor (in the middle of the reactor).
Reviewer 2 Report
Comments and Suggestions for Authors
The authors propose an alternative biotechnological procedure for the production of a biomass with a balanced ratio of DHA and EPA. The co-cultivation is an interesting biological process that requires more studies.
In general the manuscript report some interesting insights but many aspect should be considered:
- The aim section should be rewritten for clarity. Most of all, the authors use future tense verbs, but instead these should be conjugated in the past tense in a scientific paper.
Also, the whole section should be condensed.
- Lines 116-117: the authors stated:"In addition, we will apply a dynamic climate simulation of a repeated summer day in Australia" but why this specific geographical location? A scientific report should consider the reproducibility of the experiment, so please report the details of mean PAR applied during the experiment.
- In the introduction i would mention the potential of new EPA producers such as Pythium sp. which is nowadays phylogenetically classified as Stramenopiles, along with brown algae and diatoms. This could enrich the introduction beyond the common PUFA producers (e. g. Nannochloropsis), check this paper if you want to consider this mention (https://www.mdpi.com/2071-1050/15/2/1147)
- Lines 161-162: the authors stated:"An inoculation ratio of 1:3 (DHA-producing strains versus EPA-producing strains) was chosen for all co-cultures." can you explain why this specific inoculation ratio?
- The figure 1 is not mentioned in the text, and is not introduced. Also, a legend should be introduced since the Figure should be self-explicative.
-In Figure 1A, it appears that the growth trend for M. salina has not been well defined (in particular for urea). In fact, the culture would seem to still be in the exponential phase, but this cannot be observed from the figure provided. This should be fixed or explained. Also in Figure 1, I notice that the samples for urea and NaNO3 were not collected at the same time, but at different times. Why is this?
Moreover, why the error bars are missing? which was the n of the experiment?
- For all the figures, it is not clear to me how the data were treated. In fact, I notice that statistical analysis in this paper is totally absent. This is a serious issue that needs to be resolved before considering publication.
-In table 1, 2, 3 and 4 a standard deviation or standard errors are completely missing, this poses some concerns about the reproducibility. Table 5 also lacks of some standard errors and of any type of descriptive statistics. Without this is impossible to understand the results.
- In the discussions, I see that little attention has been paid to understanding why this co-cultivation increases biomass yield. A more critical thinking about this aspect is required.
Comments on the Quality of English Language
Moderate revisions of English
Author Response
#Reviewer 2:
The authors propose an alternative biotechnological procedure for the production of a biomass with a balanced ratio of DHA and EPA. The co-cultivation is an interesting biological process that requires more studies.
In general the manuscript report some interesting insights but many aspect should be considered:
The aim section should be rewritten for clarity. Most of all, the authors use future tense verbs, but instead these should be conjugated in the past tense in a scientific paper. Also, the whole section should be condensed.
Response: Done, the section has been rephrased.
L113-121: We investigated the phototrophic mono- and co-cultivation of the golden-brown microalgae I. galbana (DHA producer) and the green microalgae M. salina, employing different nitrogen sources (nitrate and urea). Further, we used I. galbana as the DHA-producing strain and co-cultivated it with two other EPA-producing green microalgae strains from the genus Nannochloropsis sp. (N. oceanica and M. gaditana). To explore the potential scalability of these processes to larger outdoor conditions, we applied a dynamic climate simulation of a repeated summer day in Australia (day-night cycles of incident light (PAR) and temperature) for all batch processes performed in flat-plate gas lift photobioreactors with LED illumination.
Lines 116-117: the authors stated:"In addition, we will apply a dynamic climate simulation of a repeated summer day in Australia" but why this specific geographical location? A scientific report should consider the reproducibility of the experiment, so please report the details of mean PAR applied during the experiment.
Response: Done. Large-scale outdoor microalgae production depends on locations that ensure a suitable climate for microalgae growth. A suitable climate for algae growth relies on high solar irritation and a constant temperature of about 30 °C throughout the day. The climate of the western coast of Australia is known for being highly suitable for algae growth; this is why we have chosen this particular geographical location. The mean PAR of the experiment and the following sentence has been added:
L166-171: The chosen target climate was 19 January 2018, in Newcastle, Australia, with a maximal temperature of 30°C and a maximal incident phototrophic photon flux density (PPFD) of 2000 µmol m-2 s-1 (mean PPFD of 879 µmol m-2 s-1). This particular geographical location has been selected, because the eastern coast of Australia offers a suitable climate for algae growth, characterized by high solar irradiation and a constant daytime temperature of around 30°C.
In the introduction i would mention the potential of new EPA producers such as Pythium sp. which is nowadays phylogenetically classified as Stramenopiles, along with brown algae and diatoms. This could enrich the introduction beyond the common PUFA producers (e. g. Nannochloropsis), check this paper if you want to consider this mention (https://www.mdpi.com/2071-1050/15/2/1147)
Response: Done. The introduction has been extended.
L66-67: Other microorganisms, such as Pythium sp. (oomycete) or brown macroalgae, have also lately been shown as promising alternative EPA producers.
Lines 161-162: the authors stated:"An inoculation ratio of 1:3 (DHA-producing strains versus EPA-producing strains) was chosen for all co-cultures." can you explain why this specific inoculation ratio?
Response: Done. In our previous manuscript we investigated the co-cultivation of the two strains T. lutea and M. salina testing different inoculation ratios. The ratio 1:3 (DHA-producing strains versus EPA-producing strains) resulted in the most suitable inoculation ratio regarding biomass growth and omega-3 fatty acid production. In this manuscript, we utilized either the same strain (M. salina) or genetically very similar strains to those previously used. This decision led us to select the same inoculation ratio as before.
The following sentence has been extended:
L162-163: Due to previously obtained results, an inoculation ratio of 1:3 (DHA-producing strains versus EPA-producing strains) was chosen for all co-cultures.
The figure 1 is not mentioned in the text, and is not introduced. Also, a legend should be introduced since the Figure should be self-explicative.
Response: Done, the introduction and the legend of Figure 1 has been added.
L241-244: First, we investigated the influence of two different types of nitrogen sources (nitrate and urea) on biomass growth and omega-3 fatty acid production in phototrophic batch processes of monocultures of I. galbana and M. salina using flat-plate gas-lift photobioreactors (Fig. 1).
L261: Legend has been added.
In Figure 1A, it appears that the growth trend for M. salina has not been well defined (in particular for urea). In fact, the culture would seem to still be in the exponential phase, but this cannot be observed from the figure provided. This should be fixed or explained.
Response: Done. In the batch process of M. salina, data was only collected until day 8 of the process, because the initial supplied urea had already been consumed after 6 days. Omega-3 fatty acids are normally produced during nitrogen-replete conditions; this is why we stopped the process after already 8 days. We tried to clarify this issue by adding the following sentence:
L285-287: Despite further biomass growth in the batch process utilizing urea, the process was stopped after 8 days because the nitrogen source had already been fully consumed.
Also in Figure 1, I notice that the samples for urea and NaNO3 were not collected at the same time, but at different times. Why is this?
Response: Nitrate and urea samples were collected twice a day, in the morning and in the afternoon. Since the algae processes were carried out for 8 to 12 days and couldn't be conducted weekly on a regular schedule (starting on the same day each week), the timing of sample collection varied (especially on the weekend, when either no/ or only one sample was taken).
Moreover, why the error bars are missing? which was the n of the experiment? For all the figures, it is not clear to me how the data were treated. In fact, I notice that statistical analysis in this paper is totally absent. This is a serious issue that needs to be resolved before considering publication. In table 1, 2, 3 and 4 a standard deviation or standard errors are completely missing, this poses some concerns about the reproducibility. Table 5 also lacks of some standard errors and of any type of descriptive statistics. Without this is impossible to understand the results.
Response: We decided to address the following three comments on reproducibility together.
All monoculture experiments were conducted only once. The co-cultivations with I. galbana and M. salina and I. galbana and N. oceanica using nitrate were conducted twice (n=2). This is why in all tables, when compared to the monocultures the standard deviation is missing.
For our purpose, we selected microalgae strains that are commonly used for research and already described in detail in the literature. Growth kinetics and DHA production of I. galbana are consistent with data of other papers (Liu et al., 2013., Zheng et al., 2022; Ge et al., 2023; Sun et al., 2019;). The genetically similar strain Tisochrysis lutea also showed similar growth and DHA production in our previous manuscript (Thurn et al., 2020)
Growth behavior and lipid accumulation of Microchloropsis salina in monoculture using variable incident photon flux densities, which cover the used photon flux densities in our climate simulation, was already characterized in flat-plate gas-lift-photobioreactors previously in our group (Pfaffinger et al., 2016; Pfaffinger et al., 2019) and the presented data are consistent. Further, batch processes with M .salina in flat-plate gas-lift photobioreactors applying a very similar climate simulation (south of Spain) were conducted (data not published) and comparable results regarding growth kinetics and EPA production were obtained. In addition, a comparable EPA content of approx. 50 mg g-1 was published with Nannochloropsis sp. in flat panel reactors (Hulatt et al., 2017). The strains Microchloropsis gaditana and Nannochloropsis oceanica show a strong genetic similarity with M. salina and are also widely described in the literature. Our results are also consistent with the literature (Chen et al., 2018, Meng et al., 2015). As all monocultures are used solely as a reference and the process performances are consistent with published data, we decided to reduce the experimental effort by omitting additional time-consuming reproduction studies. Further, the co-cultivation processes of I. galbana with either M. salina and N. oceanica (and the co-cultivation of T. lutea and M. salina with n=3 from our previous manuscript) showed good reproducibility, we also decided to reduce the experimental effort of the co-cultivations using urea by only conducting it once.
We added the follwing sentences:
L250-251: Since growth kinetics and DHA production of I. galbana were consistent with the literature, we only conducted the monoculture batch processes once [19, 41, 42].
L296-301:Further, batch processes with M .salina in flat-plate gas-lift photobioreactors applying a very similar climate simulation (south of Spain) were conducted (data not published) and comparable results regarding growth kinetics and EPA production were obtained. Regarding this and the consistency of the data with the literature [22], we only conducted the batch processes once.
L265: All performed monoculture batch processes were conducted once.
L324-325: The batch process with nitrate was performed twice (min-max values are shown), and the batch process with urea was performed once.
L442-443: The batch process with nitrate was performed twice (min-max values are shown), and the batch process with urea was performed once.
L534-535: The batch processes using nitrate and urea were performed once.
In the discussions, I see that little attention has been paid to understanding why this co-cultivation increases biomass yield. A more critical thinking about this aspect is required.
Response: Our last section, 3.5 Enhanced light utilization in co-cultures of golden-brown and green microalgae, is fully addressed to try to explain the increased biomass growth in co-cultures compared to monocultures. Light measurements on the light-adverted side of the reactor were conducted and showed clear differences in the light utilization of the two algae strains in monoculture and complementary use of the light in co-culture. We suggest that the increased biomass concentration is due to the complementary and, therefore, enhanced light utilization in co-culture.
See f.e. L595-597: Hence, we suggest the utilization of complementary wavelengths of light in co-cultivation, potentially leading to increased light absorption and improved biomass yield.
Reviewer 3 Report
Comments and Suggestions for Authors
Abstract
Please follow MDPI guidelines and reduce the length of this section to 200 words.
Introduction.
In line 38 the statement “Fish and fish products are the primary dietary sources” is disconnected from the rest of the text
The statement in lines 39-41 is not clear enough. Rewrite it
The explanation of strains on lines 55-65 suits better in the discussion section.
The explanation given by the authors after line 110 is completely unnecessary in the introduction. Please rewrite it and use it in the discussion section.
Materials and methods
Authors must add a table explaining the different co-cultures to be tested (like Table 5, but without data).
Results and discussion
This section is confusing. The linearity of the explanation tends to disappear between figures 1-3. The same continues with the rest of the figures and tables.
Please revise the labels of Figure 2. I think figs 2b, 2c, and 2d have wrong labels.
Why do tables 1 and 2 present data for day 8th, but figures 1 and 2 go as far as 12 days? The authors explain the data for the 10th day in the discussion.
Author Response
#Reviewer 3:
Introduction.
In line 38 the statement “Fish and fish products are the primary dietary sources” is disconnected from the rest of the text
Response: Done, the statement has been removed.
The statement in lines 39-41 is not clear enough. Rewrite it
Response. Done, the statement has been rewritten.
L38-40: The recommended daily human intake of DHA and EPA is approx. 250-500 mg in total. Considering this requirement, 1.4 million tonnes of omega-3 fatty acids need to be supplied by aquaculture, fisheries, and other marine sources.
The explanation of strains on lines 55-65 suits better in the discussion section.
Response: To enhance the clarity and understanding of our research methodology (use of 4 different algae strains and 3 co-cultivation combinations), we believe it's important to provide a brief introduction to the two families of algae strains used in our study — those producing DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid).
The explanation given by the authors after line 110 is completely unnecessary in the introduction. Please rewrite it and use it in the discussion section.
Response: Done, the section has been rewritten.
L113- 121: We studied the phototrophic mono- and co-cultivation of the golden-brown microalgae I. galbana (DHA producer) and the green microalgae M. salina, employing different nitrogen sources (nitrate and urea). Further, we used I. galbana as the DHA-producing strain and co-cultivated it with two other EPA-producing green microalgae strains from the genus Nannochloropsis sp. (N. oceanica and M. gaditana). To explore the potential scalability of these processes to larger outdoor conditions, we applied a dynamic climate simulation of a repeated summer day in Australia (day–night cycles of incident light (PAR) and temperature) for all batch processes performed in flat-plate gas lift photobioreactors with LED illumination.
In this section, we tried to describe the aim of the study. We think this is important in an early stage of the manuscript; this is why we decided to do it directly after the introduction.
To clarify the aim of the discussion part as suggested, the following sentences have been rephrased:
L241-244: First, we investigated the influence of two different types of nitrogen sources (nitrate and urea) on biomass growth and omega-3 fatty acid production in phototrophic batch processes of monocultures of I. galbana and M. salina using flat-plate gas-lift photobioreactors (Fig. 1). A nitrogen concentration of 400 mg L-1 was used.
L304-308: Nitrate use increased the DHA or EPA content of I. galbana and M. salina in phototrophic monocultures compared to urea as a nitrogen source. To obtain DHA and EPA- enriched biomass, a phototrophic co-cultivation process of both microalgae strains was conducted using nitrate as a nitrogen source and applying the same process conditions as in the monocultures.
L425-428: The general concept of a co-cultivation of a DHA and an EPA producing strain should be proven with other microalgae strains. Therefore, a phototrophic batch process was performed with I. galbana as a DHA producer and N. oceanica as an EPA producer with nitrate by applying the same process conditions (n=2)
Materials and methods
Authors must add a table explaining the different co-cultures to be tested (like Table 5, but without data).
Response: Done, table has been added.
See L174
Results and discussion
This section is confusing. The linearity of the explanation tends to disappear between figures 1-3. The same continues with the rest of the figures and tables.
Response: Done, the following sentences have been rephrased for clarification
L 241-244: First, we investigated the influence of two different types of nitrogen sources (nitrate and urea) on biomass growth and omega-3 fatty acid production in phototrophic batch processes of monocultures of I. galbana and M. salina using flat-plate gas-lift photobioreactors (Fig. 1). A nitrogen concentration of 400 mg L-1 was used.
L304-308: Nitrate use increased the DHA or EPA content of I. galbana and M. salina in phototrophic monocultures compared to urea as a nitrogen source. To obtain DHA and EPA- enriched biomass, a phototrophic co-cultivation process of both microalgae strains was conducted using nitrate as a nitrogen source and applying the same process conditions as in the monocultures.
L425-428: The general concept of a co-cultivation of a DHA and an EPA producing strain should be proven with other microalgae strains. Therefore, a phototrophic batch process was performed with I. galbana as a DHA producer and N. oceanica as an EPA producer with nitrate by applying the same process conditions (n=2)
Table 2 has been moved to line 345.
Please revise the labels of Figure 2. I think figs 2b, 2c, and 2d have wrong labels.
Response: Done, labels have been revised in figures 2,3 and 4
Why do tables 1 and 2 present data for day 8th, but figures 1 and 2 go as far as 12 days? The authors explain the data for the 10th day in the discussion.
Response: Done. After 8 days, the total initial supplied nitrogen has been consumed, and no further growth (or only slightly further growth due to lipid production) and constant DHA and EPA contents have been seen in co-culture. This is why we chose this particular process time for the comparison of the mono- and co-cultures. We plotted all data until day 12 to illustrate the whole biomass growth and DHA and EPA production trend (constant trend) after nitrogen consumption.
To clarify we added the following sentence:
L336-337: After 8 days, all the initially supplied nitrate was consumed, leading us to select this process time for comparing mono- and co-culture processes.
The following sentence explaining day 10 in the discussion has been rephrased:
L359-360: After eight days of cultivation, the phototrophic co-cultivation process with urea reached a total cell dry weight concentration of 5.2 g L-1
Round 2
Reviewer 2 Report
Comments and Suggestions for Authors
Dear authors,
The paper has been improved, however i found some errors along the manuscript.
For example in the aim section (Line 133) there is still a verb in future tense.
In Figure 5 there is a repetition in the legend (see the legend in Figure 5C and 5D)
The caption of figure 2 and 4 are very long, they should be condensed.
Most of all, Figure 1-4 are not introduced or cited in the main text. This should be fixed.
Comments on the Quality of English Language
Minor revision
Author Response
Dear authors,
The paper has been improved, however i found some errors along the manuscript.
For example in the aim section (Line 133) there is still a verb in future tense.
Response: Done. Sentence has been resphrased.
L122-125: To test the hypothesis of increased biomass growth in co-culture due to improved light utilization, we measured the remaining light spectra on the light-adverted side of the flat-plate gas lift photobioreactor at different process times and biomass concentrations, both in monoculture and co-culture.
In Figure 5 there is a repetition in the legend (see the legend in Figure 5C and 5D)
Response: Done. Legend has been revised.
The caption of figure 2 and 4 are very long, they should be condensed.
Response: We believe that all the information provided in the captions of the figure (in total we show 6 figures in one) is crucial for comprehension. Shortening the caption may render the figures incomprehensible to the reader.
Most of all, Figure 1-4 are not introduced or cited in the main text. This should be fixed.
Response: Done, all figures have been introduced or/and cited in the main text:
L241-244: First, we investigated the influence of two different types of nitrogen sources (nitrate and urea) on biomass growth and omega-3 fatty acid production in phototrophic batch processes of monocultures of I. galbana and M. salina using flat-plate gas-lift photobioreactors (Fig. 1).
L282-284: After eight days, the phototrophic batch process with M. salina using urea yielded an increased cell dry weight concentration (5.3 g L-1 instead of 3.2 g L-1) compared to the process with nitrate as a nitrogen source (Fig. 1).
L310-311: Figure 2 shows the biomass concentration and the DHA and EPA production of the co-cultivation process as a function of the process time.
L305-308: To obtain DHA and EPA enriched biomass, a phototrophic co-cultivation process of both microalgae strains was conducted using nitrate as a nitrogen source and applying the same process conditions as in the monocultures (Fig. 2).
L429-430: Figure 3 shows the dynamics of the biomass concentration and the DHA and EPA production of the co-cultivation process.
L433-435: In co-culture, total consumption of the initially supplied nitrate was observed after a process time of 6.7 days, resulting in a constant biomass concentration of 4.5 ± 0.2 g L-1 (Fig. 3).
L491-4943 Another co-cultivation process using I. galbana as a DHA producer and M. gaditana as an EPA producer was conducted in flat-plate gas-lift photobioreactors applying the same process conditions as before (Fig. 4).
