Evaluation of Aurantiochytrium mangrovei Biomass Grown on Digestate as a Sustainable Feed Ingredient of Sea Bass, Dicentrarchus labrax, Juveniles and Larvae

Round 1

Reviewer 1 Report

See review attached.

Comments for author File: Comments.pdf

Author Response

Reviewer 1

 

General comments

The manuscript presents new results for feeding trials for sea bass using a thraustochytrid strain – A.

mangrovei grown in a novel (using a waste digestate) manner.

One major issue, which is also covered below, is the overall low survival in the feeding trial using

both diets tested. This aspect of the results is of concern and could be expanded on to sufficiently

justify that the study as it stands can be published.

The results are generally well presented and written and are suitable for publication in Sustainability.

Should all matters raised in this review be adequately addressed, publication would be

recommended.

 

Response: We agree with reviewer 1 that low larvae survival was an issue and make our conclusion a bit difficult to establish. As mentioned in the discussion, artificial micro-diets did not significantly affect larvae survival as compared to live diet and we may have introduce the artificial diet too early. But, we were constrained by zootechnical facilities availability and could not repeat the experiment in the imposed project framework. Thus, such dietary stressful conditions may have pile up with low larvae quality provided by the commercial hatchery.

Nevertheless, we wished to look at the bright side. It is the first time that hydrolyzed Thraustochytrid biomass was included in a larvae artificial microalgae-diet. The hydrolysis process is fully mastered even though we currently keep working on its optimization. Then, we managed manufacturing the micro-diets on which larvae were able to feed on. We believe we should pursue such line of nutrition research.

Indeed, the fish hatchery stakeholders could directly apply it using commercial thraustochytrid biomass such as DHAgold, perform the hydrolysis and include the hydrolysate into larvae feed.

 

Specific minor comments

Introduction

L21. Three FA are listed. Add ‘and’ between the second and third FA.

 

Done

 

L42. Delete ‘tons’ here. This is a monetary unit here, and not a weight unit.

 

The euro symbol “€” was misleading. We certainly overlooked this sentence when editing the MS. It was indeed tons of fish oil. The sentence has been reworded in order to cite a more recent reference (Tacon and Metian 2015).

 

L53-54. Add reference.

Reviewers 1 and 2 brought our attention on missing references for introductive part between lines 47 and 54. Accordingly we did revise this part of the introduction that has not been enough verified for ref and edition.

“Furthermore, as fishmeal and fish oil were becoming more expensive and the awareness of aquaculture effect on small pelagic stocks was rising, substitution of these ingredients by plant-based and animal-based ingredients increased during the last 30 years, potentially affecting the nutritional composition and value of the farmed fish (Kok et al, 2020). Heterotrophic production of n-3 LC-PUFA by marine microorganisms as an alternative to fish oils from small pelagic fish (e.g., anchovy, sardine) have received growing interest in the past few years. The dinoflagellate Crythecodinium cohnii and the Thraustochytrids are the most commonly grown marine protists for n-3 LC-PUFA production. Although the production cost under controlled heterotrophic fermentation of these microorganisms remained high, the resulting biomass is generally free of contaminants (Sprague et al, 2017).”

 

L57-58. The wording here is needing assistance. ‘…of the Thraustochytrid phylum for having …’

 

We combined this sentence with the previous one, as they were partially repetitive. They were reworded as follow:

“The Thraustochytrids, most notably the species of Auranthiochytrium and Schizochytrium genus, are  particularly relevant for this type of application, mainly due to their high content in n-3 LC-PUFA [6,7, 8].”

 

L59. ‘genus’.

 

Corrected

 

L83. Replace – ‘limit’ with ‘reduce’.

 

Done

 

 

L101. The 15% target is actually higher than trialled/used in previous studies. The authors could

make more of this aspect, including in the Discussion.

 

We agree. To have a significant impact on fishmeal and fish oil substitution, we targeted 15% as a first step. This statement and justification is included in the introduction and discussion as follow:

(Introduction) “The applicative objective is to partially replace fishmeal and fish oil ingredients generally obtained from the small pelagic fish industry in fish feed with 15% of microalgae biomass. It meant to be a first step towards higher replacement percentage.”

(Discussion)“The Sea bass juvenile experimental feed included 15% of non-hydrolyzed biomass. Inclusion level of 15% was targeted to have a significant impact on fishmeal and fish oil replacement in fish feed. The Seabass larvae experimental feed included the same level (15%) of A. mangrovei biomass 15% but in a hydrolyzed form as protein hydrolysates were previously shown enhancing larval growth and/or survival performance of European sea bass Dicentrarchus labrax larvae.”

 

L105. It would be useful to state the origin and source of A. mangrovei.

 

It is now mentioned in the material and methods as RCC893

 

Methods

L185. Define DH (?) here. Or is it something else?

 

It is degree of hydrolysis. Text has been changed accordingly. The abbreviation is after all unnecessary.

 

L191-L246. The juvenile feed contained biomass, with the larval feed containing hydrolysed

biomass. Why was this the case? It would have been useful to use biomass for the larvae also. Was

the hydrolysis step that has been used perhaps a contributing issue regarding the low survival that

has been reported?

 

We agree that it would have been better to use a dietary condition with non-hydrolysed biomass but the zootechnical facilities allowed only six tanks (3 per each condition). As mentioned above, , we compared three dietary conditions, control diet, 15% hydrolysed biomass and 15% non-hydrolysed biomass, on small sea bass juvenile (2.5 g) in order to investigate the pretreatment step on the fish health and survival.

The hydrolysis was unlikely responsible for the low survival and high variability, which were similar with larvae fed control diet.

 

L324. Change ‘…and fed one day…’ to ‘…and then fed one day…’

 

Done

 

L390-392. Several of the solvents are not defined. It would be helpful for readers to see the common

names of the solvents rather than formulas; there may be multiple options for the formulas used.

 

Text has been changed using solvent names as follow:

“First the plate was immersed in a solution of methyl acetate:isopropanol:chloroform:methanol:KCl 0.25% (10:10:10:4:3.6; v/v), allowing polar lipids (PL) separation. Subsequently the plates were immersed first in a solution of hexane:diethyl ether:acetic acid (20:5:0.5; v/v) and then in a solution of hexane:diethyl ether (97:3; v/v), for neutral lipids (NL) separation.”

 

L438. The terms no1 and no3 are used, with the o present as a superscript. What are these terms

here?

 

Our mistakes. According to supplier (Supelco) website, the proper writing is PUFA No.1 and PUFA No.3. Text was changed accordingly.

 

Results

L469. Change ‘thr’ to ‘the’.

 

Changed

 

L515. Use NL and PL here.

 

Our mistakes. It was written the French way.  Text has been changed as requested.

 

L516. Delete the ‘of’.

 

Done

 

L517. Use ‘…fractions at about…’. Add the ‘at’.

 

Done

 

L545. 16 PUFA, should read C16 PUFA. And at L735.

 

Done

 

L593. ‘over total fatty acids’ would read better as ‘of total fatty acids’.

 

Changed accordingly

 

L595 and following. Several significant differences are noted. A number of these such as liver 16:0,

muscle 18:1, muscle 20:1, muscle EPA appear rather minor.

 

We agree. We mentioned all significant differences based on p<0.05 threshold but p value was far below 0.05 for the FA we discussed later on, most notably DHA, DPA and ARA in liver.

 

L640-643. The survival data (only 0.9 to 26%, Table 4) presented (for both the control diet and microalgae diet) are very low indeed. What is normally encountered by the CNRS/IFREMER aquaculture team performing such feeding trials for Sea bass at this age? And what suggestions can be made to improve overall survival? Where such low survival is encountered, are other parameters such as the many reported here (FA etc), compromised? Discussion is provided on the survival topic by the authors at L849-863, although further discussion on these overall low survival data would be pertinent to include. It appears that a presently unknown system issue(s) of some kind, perhaps related to overall water quality based on our experiences, has occurred in the study (as raised at L863).

 

It is difficult to discuss  the low survival rate as it was below our expectations. It will be too speculative to try to provide more hypothesis than those already proposed in the discussion.

The survival percentage we obtained in this experiment is below those generally obtained in Ifremer hatchery facilities (15-35%). We further detailed this point in the discussion as follow:

“Survival percentages between 15-35% were usually reported in the present experimental rearing facility with sea bass larvae, fed exclusively artificial micro-diet as compared to >50% for larvae fed live prey…”

Regarding FA, the composition of polar (membrane) lipids appeared to be  confirmed. PUFA was around 51% with no difference for all compared effect of the chosen diets. Thus, it seems that larvae were able to well regulate the composition of their membrane whatever dietary conditions were provided .

As mentioned above, we believe the obtained information could be useful to the hatchery industry aiming to reduce their dependency on fishmeal and fish oil as well as improving performance by including microalgae peptides. Process/manufacturing steps for developed micro-algal diets could be applied using commercial Thraustochytrid biomass by commercial hatchery.

Regarding the overall water quality, we do not think it could be the issue as seawater underwent multiple treatments to ensure its high quality. We added the following information in the material and methods:

“Sea water prior the experiment use was passed through two sand filters (~500 μm), heated (tungsten, Plate Heat Exchanger), degassed using a column, filtered using a 2 μm membrane and finally UV sterilized assuring high water quality.”

 

Discussion

It would be useful to add text at the start of the Discussion on the use of pig fecal matter derived digestate for nutrient ingredients in feed (algal biomass) preparation for animals (fish) and in due course human (as consumers of fish) consideration. Are there health and other regulations that need to be considered?

 

We fully agree that using digestate derived from pig manure has some safety and regulatory issues check prior to be used in the feed and food application. This point is indeed discussed in a companion paper (de la Broise et al, 2022) and the following sentence is now included at the last part of the discussion.

“However, European regulation stated that animal-based digestates could not be used for feed production including aquafeed (de la Broise et al, 2022). Up to day, only crop-based digestates can be used in the developed process. Alternatively, other by-products from the food industry could be investigated as potential sources of carbon and/or nitrogen to produce Thraustochytrid biomass.”

De la Broise et al (2022) provides additional information on the ALG-AD project including a report on the safety issue at the following supplementary files link.

https://www.mdpi.com/article/10.3390/md20080499/s1 It includes three reports named: Interreg NWE ALG-AD project Seasonal variation of algal biomass cultivated using nutrient rich digestate; Interreg NWE ALG-AD project Safety analysis digestate and algal biomass produced by the three ALG-AD pilots; Interreg NWE ALG-AD project A Regulatory Review on the Use of Digestate to Cultivate Algal Biomass for Animal Feed.

 

L694, L716 (and L724 – spelling is different, and L842 yet another term). Different terms are used for

the main NL. One term is preferable. Use and standardize on one term only.

 

We used the term “triacylglerides” in the whole manuscript.

 

L721 (and following). The term LC n-3 PUFA is used and in following text at p19. The term n-3 LCPUFA

has been used previously and is the preferred term. Standard here and elsewhere in the

manuscript on n-3 LC-PUFA.

 

We agree. We followed reviewer recommendation and homogenized the text accordingly

 

L731 and L780. 22C should read C22.

 

Agree. We used a bad French way. Changes were made.

 

L801. Use ‘arachidonic acid’.

 

Done

 

L879. ‘…pigments,…’. Change to ‘…pigments.’

 

Done

Reviewer 2 Report

This work study on the production of Auranthiochytrium mangrovei biomass and evaluate the potential to use as fish oil/meal replacement for seabass production (both juveniles and larvae).

Overall, the topic is interesting, and the findings may useful for the fish industry. However, some presented data, statistical analysis or discussion can be more improved. My comments are as following:

 

ABSTRACT

- The structure of abstract is well-written. I suggested author state new novel or new finding of your work (likes first time study on seabass juveniles? first time study by using this algae), this will gain the comprehensive of this part.

- Moreover, some data should be added / appeared as numeric, for example, the survival percentage of each group / proportion of PUFA, etc.

 

INTRODUCTION

- Line42: Please add the ref.

- Line49,54: No need to start the new paragraph.

- Line96: Author should state earlier that Schizochytrium sp.? is the former name of Auranthiochytrium (maybe on line68). Then, I suggested author to use the up-to-date name (same with the topic) throughout the text.

- Line96: Other use the heterotrophic fermentation in this experiment but did not state in the review why? Please add the reason in this part.

- Why author use 15% of them to instead the fish oil? Please add the reason in the introduction part as well.

 

RESULTS
- Fig.1: I suggested to use the line graph or bar graph for this figure, this will help reader know the exactly amount of the data. Also, how’s about the replication and its statistical value? 

- Same comment for Fig.3 Please add the statistical value to clarify the significance of the treatment.

- Fig2,3: What’s difference between Total and free MDA? How’s they represent to lipid oxidation?

- Table4: Why the survival rate is too low? Is it normal? For control, why the survival rate of each tank is too much difference? Please check the data and clarify this point.

 

DISCUSSION

- Line676-677: Why author use microalgae for the juveniles but using the hydrolyzed form for larvae? Please describe and state on the text as well.

- I suggest author describe why author measure both total and free MDA? Please add/discuss more how the level of lipid oxidation occur in this fish affect to other parameters like survival rate? Growth performance?

- Why the fish feed with control diet still have the low survival rate? Please clarify this point.

Comments for author File: Comments.pdf

Author Response

Reviewer 2

 

Comments and Suggestions for Authors

This work study on the production of Auranthiochytrium mangrovei biomass and evaluate the potential to use as fish oil/meal replacement for seabass production (both juveniles and larvae).

Overall, the topic is interesting, and the findings may useful for the fish industry. However, some presented data, statistical analysis or discussion can be more improved. My comments are as following:

 ABSTRACT

- The structure of abstract is well-written. I suggested author state new novel or new finding of your work (likes first time study on seabass juveniles? first time study by using this algae), this will gain the comprehensive of this part.

Thanks to reviewer 2 to bring our attention on the “first time study” issue. Thus, I re-run my literature search and found a recent work using Schizochytrium (now Aurantiochytrium) limacinum at 5-10% of sea bass juvenile diet (ref below). Although, in our study, our strain was grown on digestate (different waste than glycerol) and its inclusion percentage was slightly higher (15% vs 5% and 10%), we cannot claim a true novelty as A. limacinum is closely to A. mangrovei. We rather stay modest on this point.

Terova, G., Moroni, F., Antonini, M., Bertacchi, S., Pesciaroli, C., Branduardi, P., ... & Rimoldi, S. (2021). Using Glycerol to Produce European Sea Bass Feed With Oleaginous Microbial Biomass: Effects on Growth Performance, Filet Fatty Acid Profile, and FADS2 Gene Expression. Frontiers in Marine Science, 1115

We missed the above reference. We now included it in the introduction and discussion. Furthermore, I included in the introduction other recent references, which are pertinent to this MS as they were using a whole biomass of Thraustochytrids into Salmon diets. It encouraged us further continuing this line of research by increasing replacement of fishmeal and fish oil by Thraustochytrid biomass produced using agri-agro waste or by-product.

- Moreover, some data should be added / appeared as numeric, for example, the survival percentage of each group / proportion of PUFA, etc.

Abstract was further extended as follow:

“…Two 800 L bioreactors were used to produce Auranthiochytrium biomass in non-axenic conditions. Biomass was then filtered through a crossflow filtration system (300 Kda ceramic membrane). The harvested biomass was freeze dried and included at 15% in experimental diet.  Sea bass juveniles (32.7±4.2 g) were fed both a control diet and a diet containing 15% of freeze-dried A. mangrovei biomass for 38 days. Juvenile survival percentage was 90% on average in both dietary conditions. Similar growth was observed between fish fed both diets, demonstrating the feasibility to replace 15% of a standard fish feed by Aurantiochytrium biomass. The liver of sea bass juveniles fed the A. mangrovei diet contained significantly higher proportions of 22:6n-3, 22:5n-6, and 20:4n-6 than those fed control diet, while the proportions of 16:0, 16:1n-7, and 18:1n-9 were significantly lower. The secondary oxidation, as measured by malonylaldehyde (MDA) content, in the liver and muscle of juveniles fed the microalgae diet tended to be higher than in fish fed the control diet but the differences were not statistically significant….””

INTRODUCTION

- Line42: Please add the ref.

 

As mentioned, both reviewers brought our attention on missing references for introductive part between lines 47 and 54. Accordingly we did revise this part of the introduction that has not been enough verified for ref and edition.

“Furthermore, as fishmeal and fish oil were becoming more expensive and the awareness of aquaculture effect on small pelagic stocks was rising, substitution of these ingredients by plant-based and animal-based ingredients increased during the last 30 years, potentially affecting the nutritional composition and value of the farmed fish (Kok et al, 2020). Heterotrophic production of n-3 LC-PUFA by marine microorganisms as an alternative to fish oils from small pelagic fish (e.g., anchovy, sardine) have received growing interest in the past few years. The dinoflagellate Crythecodinium cohnii and the Thraustochytrids are the most commonly grown marine protists for n-3 LC-PUFA production. Although the production cost under controlled heterotrophic fermentation of these microorganisms remained high, the resulting biomass is generally free of contaminants (Sprague et al, 2017).”

- Line49,54: No need to start the new paragraph.

Done

- Line96: Author should state earlier that Schizochytrium sp.? is the former name of Auranthiochytrium (maybe on line68). Then, I suggested author to use the up-to-date name (same with the topic) throughout the text.

 

According to reviewer 1, we revised Lines 57-58 (now lines 66-68) by merging two sentences partially repeating each other as follow:

 “The Thraustochytrids, most notably the species of Auranthiochytrium and Schizochytrium genus, are particularly relevant for this type of application, mainly due to their high content in n-3 LC-PUFA [6, 7, 8].”

Not all Schizochytrium species had their genus name changed. So, it makes difficult to keep track of them, especially when species is not known and strain as Schizochytrium sp.

- Line96: Other use the heterotrophic fermentation in this experiment but did not state in the review why? Please add the reason in this part.

- Why author use 15% of them to instead the fish oil? Please add the reason in the introduction part as well.

 

To have a significant impact on fishmeal and fish oil substitution, we targeted 15% as a first step. This statement and justification are included in the introduction and discussion as follow:

(Introduction) “The applicative objective is to partially replace fishmeal and fish oil ingredients generally obtained from the small pelagic fish industry in fish feed with 15% of microalgae biomass. It is meant to be a first step towards higher replacement percentage.”

(Discussion)“The Sea bass juvenile experimental feed included 15% of non-hydrolyzed biomass. Inclusion level of 15% was targeted to have a significant impact on fishmeal and fish oil replacement in fish feed. The Seabass larvae experimental feed included the same level (15%) of A. mangrovei biomass but in a hydrolyzed form as protein hydrolysates were previously shown to enhance larval growth and/or survival performance of European sea bass Dicentrarchus labrax larvae.”

 

 RESULTS
- Fig.1: I suggested to use the line graph or bar graph for this figure, this will help reader know the exactly amount of the data. Also, how’s about the replication and its statistical value? 

 

Although data points are closed to each other, they are two values obtained from two cultures performed concomitantly in two separated 800 L cylinders. The biomass of these two cultures were then used as ingredient for the dietary fish trials. Fig 1 caption has been clarified as follow:

“…At each sampling, the presented two values were obtained from the two 800L cylinder cultures performed concomitantly..”

- Same comment for Fig.3 Please add the statistical value to clarify the significance of the treatment.

In Figures 2 and 3, mean and standard deviation were obtained from “analytical replicates” as samples were taken at different levels within the feedbag. Thus, we could not perform statistic comparison on these data. Analytical variability was high in Figure 2 but still allowed visual comparison. Variability in micro-diet (Figure 3) was lower. Overall, it supports the observation that the microalgae diet and micro-diet had a slightly higher level of MDA than control diet and micro-diet.

- Fig2,3: What’s difference between Total and free MDA? How’s they represent to lipid oxidation?

Total MDA is the sum of free MDA and MDA bound to proteins.

We clarified the sentences as follow: in section 2.6.5

“Free MDA or, after a hydrolysis step, total MDA (including protein-bound MDA and free MDA) concentration was determined in all samples through spectrophotometry, by measuring the absorbance of a MDA-chromogenic agent adduct at 586 nm”,

and in section3.3 “Total MDA (including MDA bound to proteins plus free MDA) and free MDA”

- Table4: Why the survival rate is too low? Is it normal? For control, why the survival rate of each tank is too much difference? Please check the data and clarify this point.

Survival was lower and more variable than usually encountered in our experimental facilities. We addressed this point in further details above as reviewer 1 rose the same question. Thus, as requested by reviewer 1, we added few additional information although we have no definitive answer. Our best explanation is that the larvae batch we bought was not as good quality as we were expecting.

 DISCUSSION

- Line676-677: Why author use microalgae for the juveniles but using the hydrolyzed form for larvae? Please describe and state on the text as well.

For both experiments, we were limited at 6 tanks (3 replicates per condition). Thus, as hydrolysates were expected to favor larval growth and/or survival performance of larvae, we decided to test hydrolyzed Thraustochytrid biomass on larvae. For the Juvenile experiment, we used non-hydrolyzed biomass because we did not have the capacity to produce large quantity of Thraustochytrids hydrolysate (20 kg for the juvenile experiment). We recently have the opportunity to use larger biomass processing and experimental capacities, which allowed comparing hydrolyzed biomass, non-hydrolyzed biomass and control conditions all together on small sea bass Juveniles (2.5 g). Analyses and data treatments are currently under process.

As suggested by reviewer 2 we added some additional inputs on these experimental choices at the beginning of the discussion,

“Inclusion level of 15% was targeted to have a significant impact on fishmeal and fish oil replacement in fish feed. The Seabass larvae experimental feed included the same level (15%) of A. mangrovei biomass 15% but in a hydrolyzed form as protein hydrolysates were previously shown enhancing larval growth and/or survival performance of European sea bass Dicentrarchus labrax larvae. “

and at the end of the introduction

“The applicative objective is to partially replace fishmeal and fish oil ingredients generally obtained from the small pelagic fish industry in fish feed with 15% of microalgae biomass. It meant to be a first step towards higher replacement percentage.”

- I suggest author describe why author measure both total and free MDA? Please add/discuss more how the level of lipid oxidation occur in this fish affect to other parameters like survival rate? Growth performance?

As secondary oxidation may result in different MDA responses in protein-bound and free compartments depending on tissue for instance, we decided to measure total MDA (protein-bound and free MDA) with hydrolysis step and free MDA without hydrolysis as it is generally done in the literature dealing with this assay. We agree that it may has been useless here as results revealed that protein-bound and free MDA varied in a parallel manner but we could not know before. This would save some of these expensive kits. The higher level of MDA in the diets mirrored in juveniles tissues and larvae but not in a significantly manner. Overall, it appeared having no fitness consequences on growth and survival for both juveniles and larvae

- Why the fish feed with control diet still have the low survival rate? Please clarify this point.

 

As reviewer 1 rose the same question, we tried to clarify this issue both in our response to reviewer 1 and in the text (See above for our detailed response to reviewer 1)

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors have fully and carefully addressed the comments raised, and the manuscript is now suitable and acceptable for publication in the Special Issue.

Reviewer 2 Report

The authors conducted the required revisions as suggested before.