sYnThOmAtIc (Hive Addict)
07-17-03 13:09
No 448087
      Anybody have this reference to c.paspali culturing     

Type of paper: Paper in journal

Title: Biosynthesis of Ergot Alkaloids by Immobilized Mycelium of Claviceps paspali

Matošić, Srećko (71284)
Mehak, Milena (51414)
Ercegović, Lidija
Okwor, James
Journal: Prehrambeno-tehnol. biotehnol. rev
Number: 2
ISSN: 0352-9193
Volume: 30
Year: 1992
Pages: from 83 to 88
Number of references: 18
Language: hrvatski
Summary: The effect of the selected parameters on the biosynthesis ofergot alkaloids during semicontinuous cultivation of immobilizedmycelium of Claviceps paspali F-2057 was investigated. Comparingthe effect of various substrate mass in the me of biosynthesis ofergot alkaloids medium, it was established that the mediumcontaining 6% mannitol and 5% ammonium succinate yields moreergot alkaloids. For the biosynthesis of ergot alkaloids, 4.25g/L 6 days old mycelial inoculum appeared to be most favorable.Investigation of the effect of alginic acid concentration usedfor the immobilization, showed 4% alginic acid concentration tobe the mest favorable for alkaloid biosynthesis. Insemicontinuous process, the immobilized Claviceps paspalimycelium produced alkaloids over a period of 60 days (6reincubations). Productivity of the process in each of thereincubations with immobilized cells is 75 to 120 % higher thanin corresponding free cell cultivations and the totalproductivity with immobilized cells is two times higher than thatof free cells. The results obtained here show that immobilizationof mycelia forming microorganisms, might be effective inbiotechnological production of complex metabolites, such as ergotalkaloids.

Yes, That pic really is me!
(Chief Bee)
07-17-03 14:21
No 448100
      Anyone speak Hrvatski around here?     

Language: hrvatski

That doesn't sound promising...
07-17-03 14:28
No 448101

hrvatski is Croation.

alginic acid, (C6H8O6)n? chemfinder lists that as pentane.
(Hive Bee)
07-17-03 20:01
No 448159
      Immobilizing Claviceps or Other Fungi Cells     

is a royal pain in the ass! You have to isolate the mycelium under sterile conditions, harvest mycelium on sintered glass funnel, then process the cells with liquid B-glucuronidase/arylsulfatase lytic enzyme(obtained from Helix pomatia), incubate the mycelium protoplasts, centrifuge them, wash them, then re-suspend in growing mixture. All of this is done in very small volumes (ml's). There may be higher alkaloid production from protoplasts, but not exactly the best procedure for large scale cultivation....
(Hive Addict)
07-21-03 11:53
No 448858
      Yea? so what?     

Well lets put it this way, I would rather do the initial steps than to have to do the workup and isolation at the end. Do you know long it takes to filter out 200liters worth of cells then dry and extract them and centrifuge and extract the 200l of fermentated broth? A long ass time when you only have a bench top centrifuge that only holds 10 50ml tubes. I believe that the extra work is well worth the effort.

Yes, That pic really is me!
(Hive Bee)
07-21-03 14:03
No 448882
      I've Done Submerged Culture of Claviceps paspali     

(2 & 3 liters) and rather than centrifuging, I just decanted off the liquid media after letting most of the cells settle out overnight. Then filtered the media broth through fine mesh, then cloth, and finally twice through fine & extra fine filter paper and got what is almost a completely cell free liquid. Average weight of cells (dry)is 15-17 grams per liter. The strain I use produces product only in the broth - the mycellium doesn't contain anything and is thrown away!
Granted a large Sharples centrifuge pushing 15,000 G's would be nice someday, however a filter press like those used for wine making would work pretty good.
I can't say that I've ever seen anywhere a research paper or patent where they did large scale cultivation of Claviceps protoplasts. You have to start with regular cells anyway, so where's the advantage? Creating protoplasts is typically done for experimental purposes on a very small scale to study the biosynthetic pathways, enzyme and alkaloid production, etc.
(Hive Bee)
07-21-03 14:08
No 448883
      The Original Paper On Making Claviceps Protoplasts     

is by:
Stahl, Neumann, Schmauder,and D.Groger
Biochem. Physiol. Pflanzen 171, page 363 1977.

Sorry I don't have a link..
(Hive Addict)
10-15-03 10:21
No 464820

Thanks for the reference.

Whats the advantage???

I could purge and fill then ferment up to ten liters of broth in a couple days every five days up to ten times without creating huge masses of cells and mess to work up.

Do you know how bad emulsions I get using chloroform without centrifuging? Anyway not to mention better yields are had when they are based and extracted separatelyfinding that the cells extraction provide almost nothing measurable, but anyway back to the point. The point is that a sintered glass plate could be fixed with these cells and then placed in to a custom glass adaptor for a tapered neck then insert the adadtor into a three neck flask and will allow you to purge and fill the flask with the cells producing alkaloid broth with no extra cellular production. With just a pump and sterilized media I can empty and fill the flask then immediatly base and extract versus having to conatantly provide the new cells with suffieient amounts of air while keeping a sterile enviroment in order to keep high yields. While this method yields even higher without such an elaborated setup just a tedious prepartion? Got any good info or suggestions for read on this type of method of cell preparation and such? You seem to know a bit about it. I would like to know your sources or good reference material to study. Thanks, Sorry I forgot about this post untill looking for information from another post.

Yes, That pic really is me!
(Hive Bee)
10-15-03 15:24
No 464853
      I Hear You About The Chloroform Emulsions
(Rated as: good read)

There are ways around that too, like absorbing the raw finished liquid media on activated carbon, then extracting the carbon with ammoniated methanol. (Sandoz got a patent for that!)
No doubt if one were able to get Claviceps protoplasts and then re-use them again and again by adding more media, it would be a good thing. However the keyword here is IF!
These sort of things sound good on paper; however in practical use it's a different story! It's hard enough to keep everything sterile, supply sterile air, etc. for just one fermentation for one week. The longer these fermentations go, the more chance there is for contamination, etc. Also, as an example: when Penicillin fermentation was begun, thought was given to re-using the cells and also continuous fermentation where media was added at the same rate as it was removed. Neither methods proved practical. Not do-able, mind you, practical.
However, I wish you the best of luck and any knowledge or experience gained will help us all. So since I'm such a nice guy frown here are some references to help you out:

"Synthesis of Ergot Alkaloids by Protoplasts of Claviceps Species" James E. Robbers, etc. Journal of Natural Products Vol. 42, #5, pg.537-539 1979
"End Product Regulation of Ergot Alkaloid Formation In Intact Cells and Protoplasts of Claviceps Species, Strain SD58" Li-Jung, James E. Robbers, et al Journal of Natural Products Vol. 43, #3, pg.329-339 1980
"Long Term Production of Ergot Peptides By Immobilized Claviceps Purpurea in Semicontinuous and Continuous Culture" Dierkes, W.- Applied and Environmental Microbiology Vol.56, 2029-2033 1993
"Production of Lysergic Acid Derivatives with Immobilized Claviceps Mycelium" Rozman, D. Applied Microbiology and Biotechnology, Vol.32, pg. 5-10, 1989.
"Biosynthesis of Ergot Alkaloids by Immobilized Mycelium of Claviceps paspali" Matosic, S. Food Technology and Biotechnology, Vol.30, pg. 83-88, 1992.

Also, could someone possibly find a link or post this paper for me:
Chemical Abstracts 77:P156333n (Describes a method to obtain lysergamides from a fermentation culture)
(Chief Bee)
10-16-03 23:51
No 465128
      Abstract to one of the above articles     

Long-Term Production of Ergot Peptides by Immobilized Claviceps purpurea in Semicontinuous and Continuous Culture
W Dierkes, M Lohmeyer and H Rehm
Appl. Environ. Microbiol. 59(7), 2029-2033 (1993) (


The semicontinuous and continuous production of pharmaceutically useful ergot peptides with immobilized Claviceps purpurea could be demonstrated. A key aspect was the presence of high concentrations of CaCl(inf2) (96.9 mM) to give marked prolongation of the productive phase, and cultivation in a bubble column reactor became possible. Restriction of the phosphate supply avoided an otherwise problematic massive increase of outgrowing hyphae.
10-23-03 12:26
No 466355
      Culturing sclerotia     

I don't know what people are referring to with "protoplasts" -- a protoplast is an organelle.  There are many steps in the lifecycle of claviceps pupurea, but usually when I've heard of people using it to extract ergotamines it was with the sclerotia of the fungus.

Culturing it will be easy, but yes -- you need to be sterile and have good tissue culture skills.  As long as your sample is alive (sclerotia should keep for a while by itself) you can wipe the outside down with alcohol, get a sterile scalple, and cut a live, sterile section from the inside to cuture in liquid media or whatever.  Watch out -- it will probably be kind of hard to cut.  To form sclerotia, make an aqueous suspention of mycelial cells from your culture in a syringe and inject it into a sterilized mason jar of solid substrate, such as cooked brown rice (use water at 3/4 of the recipe on the bag.)  Put the cooked rice into the jar, trying to keep it a bit on the airy side, poke 2 or four holes in it and put it in a 15 psi pressure cooker for 1 hour, starting at the time the pressure has built up.  After this, inject your hyphae into the jar and simply let it sit on a shelf for a half a year.  If it didn't get contaminated you should have a jarr full of c. pupurea sclerotia.

Non-nutritious substrates like white rice are a lot better at forming sclerotia, but I don't know if it has the nutrition requirements for growth of this species.

If you're using liquid media, PDY broth should work well and you should be able to add antibacterials like gentamycin, if you need.

(Hive Bee)
10-24-03 06:45
No 466508

Protoplasts are cells (plant, fungal or bacterial) that have had their cell walls removed. See:
You're technique for working with sclerotia is fine, however
NO species of Claviceps will form sclerotia by trying to grow them on Brown Rice! Claviceps fungi are NOT mushroom fungi. There are many good published papers on Claviceps - Rhodium's web site has many good links.
10-24-03 10:48
No 466548

That would be my bad, then -- I thought it was the nucleus wo/ a cell or membrane because a lot of people do gene splicing via protoplast fusion lysing cells in PEG.  Never worked on it myself though; just read some stuff a while ago.

I'm well aware claviceps is a smut, but if you can culture the myc. in a liquid culture you can do it on solid culture.  The reason sclerotia forms (in any fungi) is because of adverse conditions (nutrition, weather, etc.) and it's usually pretty easy to simulate these conditions (unless c. pupurea is an exception, but I don't think so.  I think I have a textbook with the info in it, though, I can look it up if I have a chance.)

I guess my point is I still think you could grow the sclerotia.  It's not difficult like fruiting mycelium -- it's just a stage of encapsulation and inhibited growth spawned by adverse conditions (as opposed to specific benificial ones.)

(Hive Bee)
10-26-03 09:41
No 466872
      unless c. pupurea is an exception     

All Claviceps species, C. paspali, C. fusiformis, etc. are an "exception". The sclerotial form of Claviceps fungi is what's known as Ergot. There is still much debate over what induces the sclerotial form, but in Claviceps in vivo (in plants) it seems to be a normal part of the fungal life cycle, and not induced per se, by abnormal conditions.
For 50 years reserchers have been trying to get Claviceps to form sclerotia in vitro (submerged fermentation) and the techniques usually used are creation of mutant strains via chemical or UV exposure. Some strains will produce "sclerotia-like" forms, but not true sclerotia, and these "sclerotia-like" forms are then put into submerged culture to produce ergot and Clavine type alkaloids.
If one were successful in getting an alkaloid producing strain to make sclerotia on non-liquid media, that would surely be a scientific and commercial breakthrough!
10-26-03 14:45
No 466912
      Sclerotia (ergot)     

First of all ergot is the sclerotium.

You're right -- it might not form sclerotum right away, looking at its mechanism of action, but it seems like all you have to do to get alkaloids (like you were asking, I think) is grow it in a 30% sucrose medium.   But it might form sclerotia in a rye grain solid media -- has anyone tried this?  I'm not sure.

This is quoted from "The Fungi" second ed. (Watkinson + Gooday) 2001:

p. 522 Fig 8.17 caption:  Ergot of rye.  The ovary of rye (Secale cereale) is infected by Claviceps pururea, and a slerotium (ergot) instead of grain develops....

p. 524:  A very long time elapsed between the pure culture of Claviceps spp.  and the achievement of alkaloid production in such cultures....  Alkaloids are synthesized  only when the hyphae are composed of roughly spherical cells resembling those in the naurally occuring ergot.  Such a morphology is obtained only in the presence of a high osmotic pressure, produced by including in the medium a high concentration of a slowly utilized carbon source, for example 30% sucrose.  Akaloid fermentation was obtained.... with less difficulty with C. paspali and C. fusiformis....

** Think here -- if I remember correctly, table sugar is sucrose.  If you got some jars and put septum plugs in the top of them (the kind that close up after a needle goes into it) and used syringes and rubbing alcohol it would be fairly easy to do it this way.  I'd use PDY broth for the media (with 30% sucrose, of course.)

Also from p. 171:


Sclerotium production is widely distributed in Ascomycetes, Basidiomycetes and mitosporic fungi, and is common in species that attack herbaceous plants.  Sclerotia are usually approximately spherical in form and from about a millimeter to a centimeter in diameter, depending on the species....  Sclerotium production is initiated by the onset of starvation conditions or other circumstances unfavorable for continued mycelial growth.

Cheers, and good luck!

10-27-03 17:03
No 467156

Sorry -- bubble -- did you edit your post?  If you didn't I misconstrued it in the first place.

(Hive Addict)
10-27-03 21:31
No 467207
      This is from a fungi 201 style course at ...     

It is my understanding that the sclerotia will only form in dry and nutrient limiting conditions, but as mentioned above in terms of adverse conditions such as the high osmotic difference this may encourage formation.  I'd be surprised though; you're most likely just to find regular ascocarp development.

This is from a fungi 201 style course at Kansas State University, in pdf form.  Well done and humourous (check out the Discomycetes).  It discuses the life cycle Of C. purpurea

Here's an assortment of references that may be worth checking out, at least one journal is Russian but I believe the guys in the Hyperlab might be able to help out with that.  A couple are german, I believe there are several bees who may be able to access and translate those.  Apologies about the lack of links and associated articles, but my school only carries so many online journals and most date back only a few years.

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids
AU: Kren,-Vladimir; Pazoutova,-Sylva; Rylko,-Viktor
SO: Applied and Environmental Microbiology v 48 Oct 1984. p. 826-9
PY: 1984
LA: English
DE: Ergot-; Metabolism-Carbohydrates; Fungi-Metabolism; Mycology-Cultures-and-culture-media
DT: Feature-Article

Stereochemistry of the isoprenylation of tryptophan catalyzed by 4-(c,c-dimethylallyl)tryptophan synthase from Claviceps, the first pathway-specific enzyme in ergot alkaloid biosynthesis
AU: Shibuya,-M; Chou,-H.-M; Fountoulakis,-M
SO: Journal of the American Chemical Society v 112 Jan 3 1990. p. 297-304
PY: 1990
LA: English

Title: Optimization of conditions for storage and cultivation of the fungus Claviceps sp., a producer of the ergot alkaloid agroclavine.
Author, Editor, Inventor: Boichenko-L-V {a}; Zelenkova-N-F {a}; Arinbasarov-M-U {a}; Reshetilova-T-A {a}
Author Address: {a} Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Oblast, 142290, Russia; E-Mail:, Russia
Source: Prikladnaya-Biokhimiya-i-Mikrobiologiya. [print] May-July 2003 2003; 39 (3): 335-340.
Publication Year: 2003
Document Type: Article-
ISSN (International Standard Serial Number): 0555-1099
Language: Russian; Non-English
Abstract: Conditions of agroclavine biosynthesis by the mutant Claviceps sp. strain s 106 were studied. The content of agroclavine was maximum (1.5-2 g/l) on days 15-16 of cultivation in the complex medium T25, containing sucrose, citric acid, and yeast extract. Agroclavine was the major component of the alkaloid fraction (90-95%). Storage of the culture at -70degreeC in T25 supplemented by 7% glycerol provided a stable level of alkaloid formation.

DE: Ergot-; Tryptophan-; Isoprenoid-compounds; Alkaloids-

Boll Chim Farm. 2003 May;142(4):187-90.  Related Articles, Links 

Better biosynthesis of ergot alkaloids.

Okide GB, Ajali U.

Department of Pharmaceutical Chemistry, University of Nigeria, Nsukka-Nigeria.

Different culture media containing different effectors for production of ergot-alkaloids were prepared. The culture media were inoculated with equal mycelia of Claviceps purpurea and some were incubated for 22 days and some incubated for 45 days. The medium that contained a mixture of glucose, glycine and tryptophan showed highest yield of the alkaloids and reached the maximum period for production of agroclavine faster.

Adv Biochem Eng Biotechnol. 2000;68:1-20.  Related Articles, Links 

Progress and prospects of ergot alkaloid research.

Mukherjee J, Menge M.

Institut fur Technische Chemie, Universitat Hannover, Germany.

Ergot alkaloids, produced by the plant parasitic fungi Claviceps purpurea are important pharmaceuticals. The chemistry, biosynthesis, bioconversions, physiological controls, and biochemistry have been extensively reviewed by earlier authors. We present here the research done on the organic synthesis of the ergot alkaloids during the past two decades. Our aim is to apply this knowledge to the synthesis of novel synthons and thus obtain new molecules by directed biosynthesis. The synthesis of clavine alkaloids, lysergic acid derivatives, the use of tryptophan as the starting material, the chemistry of 1,3,4,5-tetrahydrobenzo[cd]indoles, and the structure activity relationships for ergot alkaloids have been discussed. Recent advances in the molecular biology and enzymology of the fungus are also mentioned. Application of oxygen vectors and mathematical modeling in the large scale production of the alkaloids are also discussed. Finally, the review gives an overview of the use of modern analytical methods such as capillary electrophoresis and two-dimensional fluorescence spectroscopy.

Publication Types:
Review, Academic

PMID: 11036684 [PubMed - indexed for MEDLINE]

Appl Microbiol Biotechnol. 2001 May;55(4):411-6.  Related Articles, Links 

Application of oxygen vectors to Claviceps purpurea cultivation.

Menge M, Mukherjee J, Scheper T.

Institut fur Technische Chemie der Universitat Hannover, Germany.

The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.

PMID: 11398919 [PubMed - indexed for MEDLINE]

100% Canadian Bullshit
(Hive Addict)
10-27-03 21:40
No 467209
      Sorry missed one     

Back to the protoplast aspect here:

Activation of ergot alkaloid biosynthesis in prototrophic isolates by Claviceps purpurea protoplast fusion.
Didek-Brumec M, Gaberc-Porekar V, Alacevic M, Milicic S, Socic H.
Biotechnol. 1991 Oct;20(3):271-8.

Intraspecific protoplast fusions were carried out with active ergocornine-ergokryptine and inactive ergocristine Claviceps purpurea strains and vice versa. The isolated prototrophic strains from both types of crossings produced all three alkaloid types, showing that biosynthesis of distinct alkaloid was activated in an inactive partner strain. The prototrophic isolates were stable on minimal medium but they segregated by subculturing on complete medium. In comparison with the original partner strains, differences in morphological and cytological characteristics were also established.

Medline (PMID=1367573)

100% Canadian Bullshit
(Chief Bee)
10-28-03 07:37
No 467288
      proper medline PMID linking     

Can you please use proper medline PMID linking (see the FAQ).
(Hive Addict)
10-29-03 01:59
No 467487

Apologies Rhodium, but I checked the FAQ and I'm not certain how my postings of PubMed articles contravened this linking aspect.  As I assumed many people here don't have PubMed access, I simply gave the absracts and any other pertinent info for obtaining the listed articles.  If there is a way I could better present this info than having been done, I again apologize and ask that someone inform me specifically about how to better do so.

100% Canadian Bullshit
(Chief Bee)
10-29-03 11:57
No 467619
      How to use the MedLine tag
(Rated as: good read)

Post 340100 (Lilienthal: "New [medline] markup tag", The Server Room)

The proper PMID can be found in any single Medline record, and may be listed like PMID: 1367573 [PubMed - indexed for MEDLINE]

It can also be deduced from the URL of any PubMed page:
(Chief Bee)
11-15-03 13:14
No 471102
      Ergot references (retrieved by lugh)
(Rated as: good read)

As mentioned in Post 465128 (Rhodium: "Abstract to one of the above articles", Tryptamine Chemistry)

Long-Term Production of Ergot Peptides by Immobilized Claviceps purpurea in Semicontinous and Continous Culture
W. Dierkes, M. Lohmeyer, H-J. Rehm
Applied and Environmental Microbiology 59(7), 2029-2033 (1993) (../rhodium/djvu /dierkes.djvu)
____ ___ __ _

As mentioned in Post 464853 (Bubbleplate: "I Hear You About The Chloroform Emulsions", Tryptamine Chemistry):

End-Product Regulation of Ergot Alkaloid Formation in Intact Cell and Protoplasts of Claviceps Species. Strain SD 58.
L-J. Cheng, J. E. Roberts, H. G. Floss
Journal of Natural Products 43(3), 329-339 (1980) (../rhodium/djvu /cheng.djvu)

Synthesis of Ergot Alkaloids by Protoplasts of Claviceps Species
James E. Robbers, et. al.
Journal of Natural Products 42(5), 537-539 (1979) (../rhodium/djvu /robbers.djvu)
(Hive Addict)
12-20-03 00:46
No 477966

Damn I thought this thread was dead after the three months it took me to get back to it!

Yeah And I hear you on that sterility!!! Of course serial dilution and propogation of diluted suspension of cells can lead to the isolation of sterile cells but take alot of time and resources. Sterile air supply is not hard. Lab supply stores sell hepa-cap inline hepa filters .3 micron. They even sell .1&.2 micron hydrophobic inline filtes for resonable price. Though sterility is easier to maintain than to achieve. Once you get a good clean room and glovebox and proper equipment it is no problem again. And a small cleanroom can be build in your home rather cheap. The most expensive part of course is the the ULPA filter/fan modular ceiling assembly. 5' deep x 8' long x 8' high clean room can be built for under two hundread bucks and will allow for two  gloveboxes with bidirectional airlock, under cabinet area can be used for holding large 10gal drums for culturing, above cabinets can be used for small fridge/freezer for stock cells storage and incubator, Enough room for over 200liters of buckets 200lites for a neatly spaced divided sections. Plus ten liters per box with enough room to do innoculations and cloning and such.

But sterility has never been an issue of late, and if it were compromised there are ways to get is back. I should suggest a book "the preservation of living fungi" lots of useful info on how to keep parent stocks of vigorous strains and maintain them and recover them when contaminated. The biggest problem I have have notices with the isolation of cells from contaminated culture mediums is their dramaticly lower yield of products. Not sure what is happening here maybe jsut aging of the cells through all the subculturing and shit required to isolate clean cells. Production drops well over a third. I'm positive they are clean, just old I guess. Or maybe parasitized by some kind of bacteria that isn't seen to be growing in colonies other than the mycelium? But more likely to be just the ammont of cellular growth during the process just like every other mycelium I've seen grown.

Thanks so much for the reference earlier here bubbelplate!

And LUGH haha, Documente huntore.  I wish I had access to even the simplest sources of information.. I guess it's time to move again ehh??

I'm glad to see this thread sprung to life again somewhat. I had hoped that there was some interest in this subject...

By the way anybody know where to find information on all these referenced "mutation by known methods". I'm sure I could devise soething but would like more detailed information on how this is carried out and stuff like that.

It would be nice to be liberated from the cell banks if something should happen to the parent stock. 

And Thanks again very much, two mods for the iput..and bubbleplate.. Now to find a country that has scientific journal access and legal ergot culturing law.
12-20-03 01:52
(Rated as: insignificant)