PREL "BINARY EXPERIMENTATION IN THE DEVELOPMENT OF NATURAL FOOD
ANALOGUES FOR CULTURE OF DETRITIVOROUS SHRIMP
Steven Y. NeweII and Back %. Feil
june, l975 Technical Bulletin No. 30 Price $3.00
Library of Congress Catalog Card Number:
This cwork is a result of research sponsored by NOAA Office of Sea Grant, U. S. Department of Commerce, under Grant /I00-5-J58-10. The U. S. Government is authorized to produce and distribute reprints for governmental purposes notwith- standing any copyright notation that might appear hereon.
Inf orm ation Dissemination University of Miami Sea Grant Program l54I Brescia Coral Gables, Florida 33I24 TABLE OF CONTENTS
LIST OF TABLES ~ e ~ r ~ ~ ~ e ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
LIST OF FIGURES ~ ~ ~ ~ ~ Yl
ACK NOW LEDGE MEN TS ~ ~ jx
ABSTRACT ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ e X
INTRO DUC TIO N ~ ~ e
1. Studies of formulation of artificial feed for shrim 1
2. The formation of lant detritus in estuaries 2
~ a 3 Use of fun al biomass as feed 5
MATERIALS, METHODS, RESULTS 9
5. ~ffiator of an artificial detrital feed ro'ect . ~ ~ e 9
6. Culture-screenin on wheat bran and ba asse
~ i I d ~ ~ ~ ~ ~ 23
~ ~ ~ 25
9. Feedin tests 1979 . 05
10. Cultur~creenin on deli nified ba asse and straw 59
DISCUSSION 71
ion of wheat bran...... ,.... 71
l2. Effect of simultaneous modification of fermentation con dj.tlons ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ e ~ 76
l3. Feedin of fermented wheat bran to naeid shrim ~ ~ ~ e 82
14. Cultur~creenin on deli nified ba asse and straw 91
15. Future directions 92
16. Ran e of a licabilit ~ - 97
REFERENCES CITED ~ ~ ~ 98 LIST OF TABLES
Table 1. Frequency 96! of occurrence for the prevalent fungi which colonized
materials used as artificial detrital feeds in mariculture of penaeid
shrimp.
Table 2. Results of the first culture-screening intended to detect strains
of fungi optirn ally able to convert wheat bran and bagasse into
a mycelial biom ass.
Table 3. Results of the second set of culture screens intended to detect
ra Ins
lillIJI tel m wheat bran fermentations might be optimized to determine how
;in production. with regard to pro i96 confidence intervals of nitrogen percentage Table 6. Mean values with protein content of fermented wheat bran and change in crm designed to determine how the bran fermentations from an experimel with regard to protein production. Results might be optimize iaritima RZ3l2, for which all experimental ncentration, salinity, and nitrogen source! treatments bran ~ between and among them produced significant and all interactior
5! effects on both nitrogen percentage and P, Type I error <0 e. See Figures 2 and 3. crude protein char Table 7. Mean values with 9596 confidence intervals of nitrogen percentage
and change in crude protein content of fermented wheat bran
from an experiment designed to determine how the bran fermentations
might be optimized with regard to protein production. Results
for Lulworthia sp. SC73, for which bran concentration, salinity,
nitrogen source, and the interaction between nitrogen source
and bran concentration producedsignificant P, Type l error< 0.05!
effects on nitrogen percentage, and bran concentration, salinity,
and the interaction of bran concentration and salinity produced
significant effects on crude protein change. See Figure 4.
Table 8. Mean values with 95% confidence intervals of nitrogen percentage
and change in crude protein content of fermented wheat bran
from an experiment designed to determine how the bran fermentations
might be optimized with regard to protein production. Results
sp. SC87, for which concentration of bran, salinity,
and the interaction of bran concentration and nitrogen source
producedsignificant P, Type I error ~0.05! effects on nitrogen
percentage, and bran concentration and the interaction between
bran concentration and salinity produced significant effects on
crude protein change. See Figure 5.
Table 9. Mean values with 9596 confidence intervals of nitrogen percentage
and change in crude protein content of fermented wheat bran
from an experiment designed to determine how the bran fermentations
might be optimized with regard to protein production. Results
far Trichoderma viride SC5l, for which bran concentrations, salinity,
the interaction between bran concentration, salinity, and nitrogen lv '!!!!!!!!!!!!!!!!!!!!!i!!!!!!!!!!!!!!ihWi'8!rjwilifiiiltlijiPriaiiiiiiii'iiiiisiiiiiijijijiiaiiiirrrrrrirrriiriiiiiirrjj!I!Jiiiri!!!!Ni!!!!
significant effects on crude protein change. See Figures 6 and
7.
Table LO. Control values for the 3 x 3 x 3 ANOVA experiments designed
to determine how the wheat bran fermentations might be optimized
with regard to protein production.
Po ~ ' w I ~' Feaerr~s=?~j:9~~ IIII!lilt!tlat!II!!IPjiI! II!!J!IIWII!KIIKIIL!!II!jijj!II I ! IIIiN I lllllllllllllllllllllllllllllllllllllllllllllllllllllliiiiiiijijiijjjiiiiIi~a!IiiliIIIRNIIIL.lggglg IIIHlllllllllllllllllllllllllllllll
Table l2. Results of shrimp-feeding experiments in the F74 Series: F74C,
with white shrimp Penaeussetiferus! and F74D, with pink shrimp P. duorarum!. Treatments are identified in Table 13. See Figures
10 and ll.
Table L3. List of treatments used in shrimp-feeding experiments of the
F70 Series. Compare with Figures 8-ll and Tables ll and 12. Except
where otherwise indicated, feeding levels were equal on a dry
weight basis within experiments.
Table LO. Results of a culture screen designed to find strains of fungi capable of efficiently converting "delignified" alkali-treated! wheat straw
and bagasse into fungal biomass.
Table L5. Results of studies of fungal protein production from amyloid and/or
simple-sugar substrates comparabLe to the present wheat bran
a f er mentation results.
Table l6. The eff ectiveness of types of nitrogen source in stimulating fungal
production processes. -~~"'severa nv6 -"' amor= - +~ LIST OF FIG UR ES
Figurel. Distribution of screenedfungi by size classof: A. nitrogencontent
N%! of fermented wheat bran. B. percent crude protein change
d CP96! of fermented wheat bran. See Tables 2 and 3 and text
for further details.
affected by treatments P, Type I error <0.05!. The interacting effects of branconcentration, nitrogen source, and salinity S o/oo! on nitrogencontent N%! of fermentedwheat bran. See
Table 6 for further details.
affected by treatments P, Type I error <0.05!. The interacting effects of bran concentration, salinity S o/oo!, and nitrogen source on crudeprotein increment hCP, mgs!. SeeTable 6 for ~ CP96.
Figure 0. Lulworthia sp. SC13. Meanswhich were significantly affected by treatments P, TypeI error < 0.05!. A. Theeffect of salinity
o/oo! on nitrogen content N96!of fermented wheat bran. B. The interacting effects of branconcentration Bran96! and nitrogen sourceon nitrogencontent. C. The interactingeffects of salinity
~nd.bran..cansercraf3on.on :t[Kfe.Qtotein iosroNeE15 ACR~.m~s! ce intervals and 96k CP. See Table 7 for confick
Meanswhich were significantly aff ected error < 0.05!. A. The effect of salinity by treatments P, Typ<
:ent N96! of f ermented wheat bran. B. S o/oo! on nitrogen co
>f bran concentration Bran 96! and nitrogen The interacting effect
.nt. C. The interacting effects of salinity source on nitrogen con and bran concentration on crude protein increment hCP, rngs!.
See Table 8 for confidence intervals and % ~ CP.
Figure 6. Trichoderma viride SC5l. Meanswhich were significantly affected
by treatments P, Type I error < 0.05!. The interacting effects
of bran concentration, nitrogen source, and salinity S o/oo! on
nitrogen content iV%! of fermented product. See Table 9 for
further details.
Figure 7. Trichoderrna viride SC5J. Meanswhich were significantly affected by treatments P, Type I error < 0.05!. The effects of bran concen- tration Bran %! and nitrogen source on Lossof crude protein h
CP, mgs!. SeeTable 9 for confidence intervals and %h CP.
Figure 8. Average final fresh weights, with 95% confidence intervals, of
pink shrimp from feeding experiment F74A. Feeding treatments:
l-no feed; 2-unfermented wheat bran; 3-autoclaved wheat bran;
0-Purina shrimp chow; 5-wheat bran fermented by Trichoderma
viride SC5l dried!; 6-treatment f75, 90%, /l0, l0%; 7-J0%level, treatment b4; 8-treatment //5 undried, cold-stored!; 9-wheat
bran fermented by SC87 dried!. Bars at haseof graph join those
means which could not be shown to be significantly diff erent from
sais, of Figure 9. Average final fresh weights, with 95% confidence in atm ents: pink shrimp from feeding experiment F79B. Feedinl ited by l-no feed; l0-regular fertilization; JO-wheatbran fer
:necrlrmap. =-92;arranarleal Caro-Stereo!; =-unrermenlea wliesr "> l ' n V'" ' ' ' ' ' ' " Q
ALJmean weights were significantly different from one br another P, Type I error < 0.05!. Details in text and Table 11.
Figure 10. Averagefresh weight gains,with 95%confidence intervals, of white shrimp from feeding experiment F70C. Feeding treatments:
ll-regular fertilization; 2-unfermented wheat bran; l2-wheat bran
fermented by Pestalotia sp. SC38 dried!; l3-treatment /k3, 50%,
treatment 82, 50%; ll-wheat bran fermented by Curvularia sp.
SC86 dried!. Bars at base of graph join those means which could
not be shown to be significantly different from one another P, Type I error > 0.05!. Details in text and Table l2.
Figure ll. Average final fresh weights, with 95% confidence intervals, of pink shrimp f rom feeding experiment F7QD. Feeding treatments:
2-unfermented wheat bran; 0-Purina shrimp chow;15-wheat bran
f ermented by sp. SC87 undried, cold-stored!, 50%,
.iWaatia& ~, '$3'<8 Qvkeai PKan~arran need 4' ';-.rmgmdorru a,.v 6de
SC 51{dried!, 57%, treatment 2, 03% total ground through 20-
mesh screen, Wiley mill!: 17-like treatment 5, but ground through
60-mesh screen. Bars at base of graph join those means which
could not be shown to be significantly different from one another
{P, Type I error > 0.05!. Details in text and Table 12.
detrital aquacultural process. ACKNOWLEDGEMENTS
Weare particularly grateful to the followingparties for theirassistance during the tenureof ourproject: for financialsupport--the Coca Cola Export Corporation Dr. L. 3. Greenfield,Mr. M. H. Farnsworth,and Dr. R. P. 3oslinwere especially helpfulin the acquisitionof support!,the Officeof SeaGrant U. S. Dept.Comm.!, theDenius Foundation, Bio-Marine Research Inc. Dr. P.Bowman!, and the University of Miami SeaGrant Program;for guidance,advice and/or library or technical aid- Drs. E. 3. Healdand D. C. Tabb guidanceand advice!, Mr. l. M. Master advice!, Messrs3. P. Norris,R. Hammer,and 3. Klinovsky aquacultural aid andadvice!, Mr. 3. Carnuccio laboratory technical aid!, Ms.A. M. Duerr library aid!, andMs. A. S. Tallman manuscriptaid!; for offers of exchangeof information and/or materials- Drs. W. D. Gray, E. B. G. 3ones,R. R. 3ones,S. P. Meyers,A. E. Reade,G. L. Soiomons,and 3. Worgan.We wish to expressour sincereregret to somemembers of this last groupof gentlemen E.B.G. 3ones, A. E. Reade,G. L. Solomons,and 3. Worgan!that we wereunable to carry out proposedcooperative projects. ABSTRACT The fungi are the primary mediators of the microbial degradation of vascular plant debris to particulate detritus in aquatic and estuarine ecosystems. Several estuarine animals of commercial f ood value including penaeid shrimp are known to be nutritionally dependent, at least in part, on the microbiaJ-detrital complexes resulting from degradation of vascular plant debris. Thus, fungi form a natural part of the diets of these animals.
Recent extensive research has demonstrated that the fungi are very attractive as agents of the manufacture of nutritious microbial protein on an industrial scale.
The cost of feed is one of the factors which yet prevents the commercial profitability of aquaculture of penaeid shrimp. If, as seems reasonable, agricultural by-products can be converted inexpensively into fungal materials simulating the naturally eaten microbial-detrital complexes, effective low-priced feeds may be developed. This technical bulletin describes a 3-year preliminary effort at testing of this hypothesis. Fungal fermentations of agricuJtural by-products were conducted and attempts were made to optimize fungal protein production. Resultant artificial detrital feeds were tested on a proto-commercial scale in outgrowth of penaeid shrimp. Yields of shrimpranged from very poor to encouraginglyhigh, consideringconditions imposed. Screening was begun of fungal capability to degrade the least expensive types of agricultural by-products, when t hese were modified to reduce Lignin-associated refractoriness. Perhaps the most significant of the conclusions to be reached is that successful developmentof artificial detritaJ feeds is not Likely to be realized without a major commitmentof time, expertise,and money. Suggestionsare presentedfor future research directions, and range of applicability is briefly discussed. PRELIMINARY EXPERIMENTATION IN THE DEVELOPMENT OF NATURAL
FOOD ANALOGUES FOR CULTURE OF DETRITIVOROUS SHRIMP
INTRODUCTION
J. Studies of formulation of artificial feed for shrimp le unrealizedpotential of shrimpmariculture as a viable commercialindustry nt frgm cost of the feed necessary to bring about the growth of satisfactory, marketable crops of penaeidand Macrobrachiumshrimp is one of the major obstaclesto the emergenceof a profit-producing shrimp mariculture industry Andersonand Tabb, 197J;IdylJ, l973; Shang,J974; Webber, 1973!. The profitable shrimpculture industry of japan is dependentupon the high market price of shrimpin that country Bardachet I'p p!, but evt'.o,,~n.>m< f~-mum ace--o"esmWr"ateseh Jn~ 9-'.~~'-66..1>Jfoe~ .~ ~! researchinto artificial feedpreparations is underwaythere Deshimaruand Shil l972!. In the UnitedStates, where shrimp prices are considerablyJower, profital P has eludedthose businessorganizations which have sought to achieve it in sI. farming. and research into formulation wf itm~sive!vr ts ~tiveLm&- is. or the steps being taken to overcome this problem Neal, l973!. Results of feed formulation research have varied among researchers, conclusionsare difficult to draw. Thereis disagreementregarding the optimal Je of major and minor components;for example,Deshimaru and Shigeno J972! indic that artificial feeds for Penaeus~rnfcus should contain at least 6096proteins more, while Andrewset al. l972! concludedthat 28-32%protein producesthe 1 growth of penaeids. Shigenoet ai. J972!found a positive correlation of pro content between 60% and 80%! with feed efficiency, and Balazs et ai. l973! belie that %LairMta Mmr.vztm~ thai t& rIriijmai:~fair +veri' ~M= ~~e~ rr0%,but Shewbartet al. J973!found that the optimal protein content lay betw 22.5% and 30.5%. Zein-E!din and McGaffey J976! found what is probably part of the reason for these apparent conflicts; their work showed that percent protein of over 50 gave the best growth results when one protein source was used, but wHen a different source was used, 32% protein produced better growth. They suggested that protein quality affected the relationship between shrimp growth and percent protein in the diet. Other feed component requirement levels about which there is seeming disagreement in the literature include aliphatic lipids and their fatty acids, carbohydrates, sterols, glucosamine or chitin, and binding and attracti+ agents. The clearest conclusion to be drawn from examination of the above and other recent studies of shrimp ration requirements Andrews and Sick, l973; Balazs et al., 1915; Deshimaru and Kuroki, J970a-c, 1975; Forster, J971, 1973; Forster and Gabbott, 1971; Hysmith et al., 1973; Kanazawa et al., 1970, l971; Kitabayashi et al., J97la-c; Meyers and Zein-EJdin, 1973; Shudo et al., 197l; Sick and Andrews, 1970; Sick and Harris, 1975; Venkatararniah et al l975; Zein-trldin and Meyers, l979! is that the ~interactin effects of protein, lipid, carbohydrate, and other components in shrimp diets make the optimal formuiation of artificiai feeds very difficult. Taub l913! has noted that one means of finding satisfactory diets for aquacuitured animals is to simulate their natural diets. The natural diets of penaeid and Macrobrachium shrimp may include sizable quantities of the plant detritus producedin the estuariesand bordering waters which these shrimp inhabit Bardachet al., 1972;Condrey et al., 1972;Cook and Lindner, l970; Costello and Allen, 1970;Dali, l967; Ling, J969; Odurn and Heald, 1972!. One approach to determination of an adequatediet for these animals is to simulate this natura/ ~!ant detritus. 2. The formation of lant detritus in estuaries llILI !L84% jI! !N!I Florida estuary, finding that the food web of this shrimp nursery ground Kutkuhn, 196@mw<>aserL|yes'w~y. aw.~. prJMlvrd.vnnK'g"==a.r@~q pg~+'e~ of- deb .-hr,m g".aves~~. This plant debris was found to undergo microbial biodegradation to detrital particles which were higher in protein content than the original material Fell et al., in press; Newell, 1975!. These microbial-detrital complexes served as a food source for meiofaunal and larger detritivores of the estuary. The pattern of dependence of food we..hs~aarLmcnab~ v..rniczahi3 t .prod~r tie.n. pppLie~ emmet fly .i= ~-attic.eczyxMem with relatively large ratios of shoreline length to water-surface area Mathews and Kowalczewski, 1969!. Recent studies have demonstrated that the phenomenon of protein enrichment of low-protein submerged plant debris is related most directly to the activity of fungi 8'arlocher and Kendrick, !974; Gessner et al., 1972;Kaushik and Hynes, 197l; May, 1974; Triska, 1970; Willoughby and Redhead, 1973!. Fatty acid enrichment due to Recognition of the fact that conventional methods of production of protein for human consumption are not meeting the present-day need, and that this situation will continue to worsen, has led to a great deal of research into development of more efficient meansof production of high protein material A. 3ones,1974!. Much of this research has dealt with the production of microbial or single-cell protein SCP!, and it is reviewed by Mateles and Tannenbaum 968!, Kihlberg 972!, and Porter 974a, b!. Substrate sources for the fermentation processes involved in the production of SCP have included high-starch-content agricultural products e.g., cassava! and a wide variety of agricultural and food-industry wastes. Although filamentous fungi are not single-ceiJ organisms, they are generally included among agents of SCP production. Reviews of the use of fungi as producers of protein, food and feed are given by Cooke 973!, Gray l970!, Hesseltine J968!, Litchfield 968!, Solornons 975!, Thatcher 954!, and Worgan 968, l973!. The prin- cipal advantagesin the use of filamentous fungi in SCP processesare economic. In submergedculture fermentations,because of their growth form, these fungi can be more easily harvested, by basket centrifugation or other coarse filtration methods, and the resultant product is more easily processed into textured food or feedstuff than those of single-cell organisms Spicer, 1971,i973; Worgan,1973!. Perhapsthe greatest economicadvantage of filamentousfungi lies in their amenabilityto a less conventional in the West! type of fermentation, viz. solid state fermentations. This point will be further exploredbelow Section15! ~ Recent reports of filamentous fungal protein production research include substrate in parentheses!: Barnes et al. l972!, Biodeterioration Inf ormation Centre of the University of Aston in Birmingham,England waste paper!; Bednarskiet al. 970, 1971!,Agricultural Universityof Olsztyn,Poland milk whey!;Brook et al. 969!, TropicalProducts Institute, England cassava!; Chahal and Gray 970! and Chahai et al. l972!, Punjab Agricultural University, india wood pulp ar< rice!; Church et al. 972!, North Star Research and Development Institute, Minnesota corn-canningwaste and soybeanwhey!; Imrie 973, l975!, Sekeri-Pataryaset ai. 973! and Christias et al. l975!, Tate and I.yle Research Center, England, Greece, and Belize, Central America carob beanextract and citrus waste!; Pooieand Smith 'iV 4 ' v' Center, Ohio lignoceilulosicwastes!; Spicer 971, l973!, Solomons 973, 1975!, Solomonsand Scamrneil 974a, b!, and Anderson et al. 975!, Lord Rank Research Centre,England several carbohydrates!; Thanh and Simard 973!, Laval University, Canada domesticsewage!; Torev l973!, Bulgaria,where industrial-scaleproduction of polyporemyceliurn for useas humanfood "sausages"!is now operating according to Von Hofsten,l975!; Updegraff{J97l!, University of Denver,Colorado wastepaper!; Von Hofsten and Von Hofsten l974!, University of Uppsala, Sweden cereals and cereal by-products!; Wiener and Rhodes J97rr! and Griffin et al. {J974!, Northern Regional Research Laboratory, Illinois {feedlot wastes!; Worgan J973; and 3. Sci. Fd. Agric., in preparation!, National College of Food Technology of the University of Reading,England {food processingwastes!. Evenwaste plastics are being used,with some success,as substrates in. the oradursiao af fi>ameoto!j&gpzqj, ototei~ {Bmwa et al., J97rr!. Perhaps the most concerted effort has been that of the Lord Rank Research Centre, which has progressed to the point of attempting satisfaction of international food-safety regulations with its fungal protein product, having proven the nutritive value and non-toxicity of their fungal protein product for rats, chickens, pigs, calves, and baboons Duthie, l975!. Use of fun al biomass as f eed Many of the above researchers have tested their experimental protein products as feed for laboratory animals rats, mice, chickens! and positive results have been obtained with regard to growth potentiaJ. For example, Reade et al. J974! reported that a protein efficiency ratio of 2.3 was achieved when their ~Aser iius product was f ed to rats. Solornons l973! reported that true protein digestibility by rats! of one of his filamentous fungal products was virtually J00%. This same product gave growth results and non-toxicity! equal to that of a casein diet with baboons Duthie, J975!. Church et al. {J972! reported equal growth rates among rats fed a casein-protein diet and rats fed a fungal Trichoderma viride! - protein diet. Evidence for the utility of fungi as a feed source has also come as an outgrowth of research intended to pinpoint the causes of feed quality reduction or toxicity by moulding; positive effects on growth were discovered in some farm animals chickens, sheep, pigs! and laboratory animals mice, rats! when specific moulding conditions physical and chemical fermentationconditions and fungal strains! were used Chahet al., l973; Fritz et al., l973; Marasasand Smalley, J972;Richardson et al., J967;Sharda et al., l97l; Thomke, ice and rats with the mycelial by-product of penicillin production used as results in t for soybeanand casein fractions of diets. See Anon. J97J!for a replacemt of further reports of this kind. bibliograp lrowth-promoting qualities of fungal feeds are not surprising if the attritivequality of fungal myceliumis taken into account. The Readeand potential is productsmentioned in the foregoingparagraph contained 08% crude! the Solom 5096 true, = total amino acids! protein respectively. Although most protein ai roteins are low in methionine content relative to the requirements of the microbial which they might be fed, the range of methionine contents of filamentous animals t< ies above that of the FAO minimum requirement level for humans fungi rea .t al., J975;Rhodes et al., 196l!.Nonetheless, as Churchet al. l972!and Anderson l972! point out, methionineaddition is commercially feasible since this Shack lady is now inexpensive and easily obtainab/e. Furthermore, Kanazawa et al. amino aci Sick and Anderson J970! have found that soybean meal, which is l970! an i low in methionine content," served better as a protein source for penaeid "notoriou. i some animal proteins. Other than the sulfhydryl amino acids, fungal shrimp th generalcontain nutritionally satisfactory levels of essentialamino acids, proteins i a wide range of specific amino acid contents, This is true even among and exhib .hesame genus Rhodes et al., l96l!. Therefore,if onewere to searchfor a species o1 facehaving a givenamino acid balance,it is likely that a speciesor strain protein s< auld be found possessing that quality. of fungus ps mostnotably important in this regardis the tractability of the protein t'ungi. Levi andCowling J969!,Litchfield et al. l963!, Merrill andCowling content o l969! demonstrated that fungi were able to adapt their nitrogen and protein contents to the C/N ratios of their substrates. Their reports, along with that of Graham l97l!, show that the increase in nitrogen reflects an increase in protein content, not in glucosarnine or chitin! or cellular ammonia. Alteration of other physical and < hemical conditions of culture can also be used to adjust protein and amino acid "ontent, including methionine content Chebotarev and Zemlyanukhin, l973; FJorentino and Broquist, J974; Graham, l97J; Pinto, I963; Solomons, l973; Tkachenko et ai., J972; Verona et al., l973!. In addition, the chitin content of cell walls of higher filamentous fungi varies greatly from species to species, and the chitin percentage of rnyceliaJ biomass varies greatly with age of culture Blumenthal and Roseman,J957!. The wide range of contents of nutritively valuable components of fugal myceiiurn together with the manageability of the relative amounts of these components in a given species, validate Gray's l970! "optimistic view that if one searches far enough and widely enough,he can often find a fungus to perform the particular synthesis or serve the purpose! in which he is interested." Three studies of the use of filamentous fungi which relate directly to the potential use of fungal feeds for shrimp are those of BKrlocher and Kendrick l973, J975!, Joshi J970!, and Nikolei J96l!. These are reports of the use of fungal tissue in the culture of arthropods which feed naturally on dead, fungal-decayed plant material. The Barlocher and Kendrick studies were intended as sequeJsto the studies of Kaushik and Hynes l97l! and Triska 970! Section 2! who had shown that the fungi were responsible for the major portion of the protein increase in decaying plant debris, and that detritus-feeding crustaceans definitely preferred fungal-invaded material over steriJe material. B'arlocher and Kendrick fed leaves with a minimal microbial population, leaves richly colonized by microbes, and fungal rnycelia JQ species!, separately to cultures of a detritivorous garnmarid amphipod. Some of the fungi proved to be excellent growth-promoting food, better than the leaf material, while other fungi produced little or no growth. 3oshifed fungal meals of differing types to a rnycophagousthysanopteran; he foundthat the lengthof the larval periodand adult fecundity were markedlyaffected by the speciesof fungus usedin preparation of the meals. Nikolei found similar results in his search for suitable fungaJtissue to serve as a culture mediumfor three speciesof cecidomyiidflies. He tested 67 speciesof fungi and seven strains of Trichoderma viride. Fifty of the species were unsuitable for culture of the fly larvae and of the seventeen which were suitable, only four were suitable for all three fly species. There was considerablevariability amongthe T. viride strains in nutritive capacity which ranged from highly positive to distinctly negative. Also, as Barlocher and Kendrick had found or suspected, culture conditions andage of the fungi hadmarked effects on their growth-producingpotential. Again, Gray's l970!"if onesearches far enough..." viewpointseems to be borneout. Although the present authors know of no pubiishedreports of the feeding of fungal rnyceiiato postlarval or juvenile commerciallyvaluable shrimp, the following reports indicate its plausibility. R. R. 3ones J974! has demonstrated that, as in the caseof other detritivorous crustacea Fenchel, J972;Hargrave, l970!, natural detrital materials are nutritively utilized by youngjuveniles of Penaeusaztecus with high protein assimilation efficiency. Even the cell walJs of the fungi probably serve as sources of shrimp nutrition. The cell waits of the higher filamentous fungi are chitinous Le3ohn, l97l!, and Hood and Meyers J974; and unpublishedcommunication! have shown that penaeid shrimp have, in addition to a hepatopancreal chitinase, a bacterial gut flora which is 85% chitinolytic and which increases in size with addition of chitin to the diet of the shrimp. Minami et al. J972! have shown that the size of the chitinolytic bacterial population of fish guts is positively correlated with their efficiency of utilization of petro-yeast. The requirement of penaeids for a chitin component in their diets has been examined, but disagreement concerning the nature of this requirementexists compareDeshimaru and Kuroki, 1970b;Kanazawa et al., l970; Kitabayashi et al., 197ia!. The following reports are also of interest in this connection. Ewaid 965! used marineyeast a Rhodotorulaspecies! as a componentof the food whichhe usedin the earfy successful work with raising of larval Penaeusduorarum. Forster 973! found that the nitrogen in a yeast-proteincomponent of a palaernonidshrimp ration was assimilatedat 95% efficiency, and Andrewsand Sick 973! found that yeast-protein labeledwith carbon14 was incorporatedat a high rate into penaeidshrimp muscle tissue. Meyerset al., 970! found that youngfreshwater crawfish grew "encourag- ingly" when fed a diet consistingof 39% spray-driedyeast. In a very important personalcommunication, Dr. E. B. G. 3ones PortsmouthPolytechnic, England! reports that he and his coileagueshave found that commerciallyculturable shrimp ~wittin est fungal ntycetia, and that "reasonably good" yields have resulted. MATERIALS METHODS AND RESULTS 5. Histor of an artificial detrital feed ro'ect ecsAi.~1-$r~~<. ak 94m~ tn4- »iIiiiiiiiiiiiiiiiliIglijjijIjijj!jiijijjlltIjI II!I]i'pj I !Ii! ! II I I1 !I II! IIIIIIII Il!I!t i IT jtjlli~ee! !l l! I IAjijjlig~iiiiii~ii jliljiljTjjjjj!Iiji~ijjjiSIjjjtjjjl~iIijjij i!i IJllijjtlllliIJll !IlllIIIIJIIIQIIIIIIlIIIIgllI!Jll IIIIIIIIIIIIIIIIIIIII ! IK'I I I ii II I II!III!Il[lllklllllllllllllllllllllllllIIIIIIIIIIIII lll 10 As an adjunct project, we unpubEsheddata! conducted a microbiological and grossbiochemical survey of the artificial detrital materials from the shrimp tanks over a periodof four weeks. Litter-bag samplingmethods similar to thoseof Newell l975! were used. One major deviation from those methods erasthe added emphasis placed on results from incubation of pieces of the materials in dishes of sterile seawater l5 o/oo! with antibiotics penicillin andstreptomycin, Oa05%!. The survey showed that, although distinct fungal florae had inhabited the different detritai material Table l!+, nitrogen enrichment of the materials did not take place. During the four weeks of sampling fungal colonization increasedas bacterial colonization decreased,and the materials lost about half of their original nitrogencontent. The biodegradationphenomena which Caillouet and Tabbhad hoped for did not take placein their tanks. Themajor reasons for this wereprobably: l! that significant increasein nitrogencontent of iignoceHulosicmaterials due to fungal biodegradationrequires more time than was allowed; ! that the conditionsrequisite for permitting the fungal biodegradatoryprocess were not supplied; ! little or no natural inoculum was present note { Table l ! the marked differences in fungal occurrencepatterns in the tanks as opposedto the adjacentmangrove pond!; and ! unavailability of rnacronutrients and buildup of fungal growth inhibitors may well have beenbrought about as a consequenceof the standingcondition of the water of the tanks. Yield of shrimpin the experiment,rather than being related to extent of + The following are points of note from Tablel, The bagassehad the heaviestfungal inoculufn at the outset. Further colonization was most extensive on bagassein the tanks, but the difference in extent of colonization was less marked in the mangrove channel~ Somefungi presentinitially on the substrateswere not reducedin frequency of occurrence by submergence e.g., Trichoderma viride, S nce halastrum racernosum!while others were eliminated Chaeto:niumspY, or reduced A!tamer!a s i« i** * * i i «* Is~~I' otherswere equallyfrequent in the mangrovechannel Trichortermaviride, ~tthia sp. on straw!. The obligate marine fungi Lulworthia sp., Zalerion varium, g 0 cv 0 0 0 6 4h '4 % 0 0 00 0 X Cl fdg N ~ eq co r 0 0 0 0 0 r g C4 0 o Cl V Cl 0 O 0 4 O 0 0 O 0 O V R V~ W U ch 0 r 0 O O H W O 0 P 0 0 X CV ~ 'cl Cl Cl ClW 4 0 0 0 0 0 0 O 0 0 td Pl CV Cl 4J pl Cl Ch 0 O 0 0 0 0 0 0 0 0 0 0 Kl V Clw 4W V 4 V C' n w cC 0 W 0 0 0 0 4 4 Ch 4 0 0 0 0 ~ 0 0 0 0 0 r4 0 0 0 0 V ~ ~ Cl48 Cl cC 0 O 0 g M cc4 0 a 0 a 0 0 o 0 0 0 o P cC 0 C4 W 8cC C4 C4 g ~ QJ ~ Cl 0 4 ml 4 cC m Cl 0 g IJ Cl Cl V A V IJ cC g Cl O N C IJ CN 9! Ql 0 cC g IJ IJ 0 IJ OcC Cl W Cl 0 V Cl 0 C Cl 0 g '0 N 0 0 12 V O Ch Pl N Ill CV 47 + cV Pl 0 0 0 0 0 CV I4 O O O e 0 0 CV O rl O r O 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O 0 O O O 0 0 0 O O 0 0 O 0 0 4 ~ ~ g Ch 0 4 4 0 4J 4ql Ol ~ IO Ill 4 Qe lll Q al CP pl 4J Ill C4 C V ~E V C CI III ql kk pl V8 e Cl OIll 0 V 00 Ill OIj 13 cb I 4I «i 0 «d Cl Cb cl 'cd cd dl 'tj W 0 O C O b0 '0 0 Cl X cd cd O Q a Cl Cl Cl 4I W CI jd 4J 4J Cl cd CI «j jd cd O 4J cd I aa w a CI O O V cj V 'cd Cl cd Cl 0 4 j CI W4 lJ '0 0 jjj I W bO bd jc CI 0 ~ I O '0 Cj cd CI Cl W 4I 4J cd M cd CI 4I Cl j5 A Cl 4J 4J W CI a W ~ Q O 0 lc a 0 cd O O «j bd v 0 cd cd W 4J «j cd cj A cd~8 cd O O dl 6 IJ W4dj 0jd V '4 p 4I W 4I dj 4J 4J u 0. CI ~ 4J j«I I W CP CI 8 cl awcj Q cd CI Cl CI 'rl A A I '0 e CI «: WO jj W W cd 0 ~ 0 0 0 4J «4 cd Cl 0 4I 0cd 4 Cl H A. CI jd V H jd «c I CI O W cC! vl Cj U «j g cd X ~ ~ a bb 0 W CI 0 Q Q u cd 0 ~ 0 u cd bb Cl cd 4I W C4 bd cd ~ CV 0 «' 4I 0 kk cd cj cd W W a CI jd O o8 W cd p ~ CI + O 4J 4J g Icb I 0«l Cl b0 4J cd Cl jd F4 cd 0 cd & «c '0 CV 5~ cj cl W CI C Cl W dl 0 4 4J 6 0 'cj 0 CI dl 4 CI aj cd 0 ~ A«j dl N A uU cd Cl 4J 4l cd Cl CI W X lJ cb bb be 0 W 0 cd 5 W 0 CI 4l 0 4J 4 0 td «j 0 M Cl C4 «j «c N 0 V S Cl d cd 0 0 Ce cjl cj 4k 4l 0 0 0 Cl 4I 0 W 0 g4 'cd CI 4l g QaC4 4 IR Pr W A4J 4l CI Cl W Cl K cd p 0 W CI g «c WCI 0 lJ 0 Cl 4 cj 4J 4J Pl «j Cj 4I 'R cd a 8 Cl JC biodegradation, was positively correlated with the original nitrogen content of the materials; shrimp fed wheat bran N% = 2.7! were on the average over twice as large as those fed the nearly purely Jignoceliulosicstraw and bagasse N% = OA!. 6. Culture-screenin on wheat bran and b asse Background. The next logical step in the development of artificial detrital feeds was an acceleration of the natural biodegradation process. This we hoped to accomplish by moving the process from the shrimp tanks into separate containers in which the biodegradationcould be conductedas controlled, submerged-culture,fungal fermen- tations. We plannedour fermentation experimentationfor use of diluted J5 o/oo! seawater because: J! penaeid culture is most economically accomplished as a coastal process; ! fresh water is one of the earth's threatenedresources; ! Gray et al. J963! had shown that use of seawater in fungal fermentations could produce enhancementof protein production; ! the estuarine-adaptedfungi form a portion of the natural shrimp diet Section 2!; and ! both terrestrial and obligately marine fungiin generalcan proliferate in partialseawater ones et al., l97J!. Materials and methods. We beganby conductinga culture screenof 29 isolates of fungi and ~ mixed cultures of fungi for their ability to increasethe proteinaceousmateriaJ in wheat branand bagasse. A diversityof fungalspecies was used Table2!, includingfungi restricted to marine-estuarineenvironments e.g. Lulworthia spp.!, fungi isolated from the plant materialsduring the CailJouet-Tabbshrimp-feeding experiment e.g. materials in local estuaries, someof which had been indicated as potentially efficient proteinproducers e.g. Trichoderma viride by Churchet al., l972!. Shakeflask fermentationswere performedas prescribedby Solomons J969! and by adaptingthe methodsof Sguroset al. l962!. A cylinder cm2 diam.>was 15 removed sterile Pl4 cork borer! from the growing edge of a colony of each of the test fungi on cornmeal Difco! agar CMA! made with l5 o/oo seawater. The cylinder was placed in the bottom of a presterilized Monel Metal Waring microblender in 25 mi of ..., Sp~]e J5>/yp seawater and Mmoaeniz~for 6I| qqconds at low,~a~ Qqe~,pf thg ., homogenate was added, in a l25 rnl DeLong flask, to 25 ml of the inocutum-growth medium:glucose-i%, NH seawater. The flasks were capped with Belco metal closures, sealed around the lower edgesof the caps with clear tape to inhibit contamination!, and incubated at 280 C on a temperature-controUed reciprocating shaker 0 cycles/min. through 8 cm distance!. These inoculum-growth flasks were harvested at approximately the early stationary phase to 9 days!. As was the case with Wanget al. J910!,only subjective determinations of growth phasewere made due to the difficulties inherent in determining quantities of mycelia mixed with solid substrate residues Calarn, l969b!. The sieve-separation method of Borzani et al. J972! was not attempted. The entire content of each flask was homogenized f or 60 seconds, and 2 ml of homogenate was added, in each of two l25 mi final culture-screen fiasks, to 25 mi of the f oHowing media: one of 5% ground bagasse 0-mesh! and one of 5% ground wheat bran, with the remainder of the media the same as that of the inocuium-growth medium, with the omission of the cellulose and pectin. These final flasks were incubated as were the inoculurn-growt h flasks. Ideally, the inocuia would have been washedand their dry weights standardized. This was avoided for the following reasons: impracticability in view of limited time and personnel; severe increase of contamination risk; ineffectiveness of this method of standardization of inocuium potential. Our work Table 5! has shown that equal times of homogenization produce large variations among different fungi in numbersof viable myceiiai elements per unit dry weight. This can have a marked effect on 16 inoculum potential e.g. Graham, J97J!. The mycelium exhaustion steps of the inoculum preparation of Sguroset al. 962! were not used in the present experiment, as this would certainly not be a part of production-scale fermentation processes,in which optimization of vigor of inoculum is desired. The work of Sguroset al. 962! and Ward and Colotelo l960! on systematically divergent fungi both showed that amounts of inoculum of dry weight greater than 2 mg into 25 ml of medium! do not have marked effects on final yield of mycelium. We calculated from the papers of Sguroset al. listed in Sguros, l973! and Meyers reviewed in Meyers, 1971!that 2 ml of hornogenateproduced as above wouJd contain at least 2 mg of mycelium. Upon inoculation of each culture screen flask, a second, control 2 ml of homogenatewas vacuum-filtered on a tared glass filter pad Whatman GF/A!, washedwith 50 mi deionized water, and dried at 55 C. The range of inoculurn 38!. In order to determine the effect of unequal inocula, a regression was performed, of final nitrogen percentage of f ermented wheat bran on inoculum weight. Significance was detected p = 0.00l! with a slope of 0.ll and the coefficient of determination r 2 ! = 0.25; those fungi which produced heavier inocula tended to produce lower finaJ nitrogen percents in fermented wheat bran, and 25% of the variation in percent nitrogen was due to inoculurn size. This was probably primarily a function of the positive correlation between inoculum size and growth rate: the fastest-growing forms produced larger inocula, and were likely to be harvested at a point in their growth cycles beyond early stationary phase. Harvesting of fermented product was done at approximately earJy stationary growth phaseby coarse 2 N m!, double-cloth filtration, with tap water washing until soluble nitrogen was removed equal volumes of tap water, with intervening through resuspensions!. The products were then dried at 550 C and ground through 60-mesh Wiley mill screen. Nitrogen content was analyzed using a Perkin-Elmer Model 200 17 ElementalAnalyzer, and protein figures givenare for crudeprotein N x 6.25!. Further screens of fungal isolates for their capacity to convert wheat bran into fugal protein were conducted using a second standard set of fermentation conditions raising the total numberof isolates screenedwith wheat bran to 52. The medium used in the second set of wheat bran screens was: wheat bran, unground - 596, NH~N03- 0.8696, KH2PO~ - 0.09%, yeast extract - 0.0196,in 15 o/oo seawater. Inoculation of 50 ml of medium in 250 ml Ehrlenrneyer flasks was by addition of 2 ml m were m~d-ea-ez- ~+~ -I -. «m".n'r'axz!re%"-mavr sr%~.daerqf~armt m~ioo=errv~ permitted calculatedon the basisof approximatetheoretical maximal conversion of wheat bran and maximal potential content of rutrogen and phosphorusin rnycelia; several references were used in this calculation, principally Burnett, 1968;Cochrane, Results. Resultsof the culture screensare given in Tables2 and 3 and Figure l. The figuresare for the solidend products of the fermentations,fermented plant particles plusfungal rnycelium. Althoughonly oneof the wheatbran products was lower in percentprotein than the original material, the rangeof crude protein incrementswas Table 3!. The medium used in the first bran culture screen was 3.6 times lower in inorganicnitrogen than the mediumused in the later culturescreen. Probablyas a partial consequence,there weremore losses in crudeprotein observedin the first wheat bran screenthan in the second Fig. 1!. Bagasseconversions Table 2! were muchpoorer than with wheatbran. Althoughthere were somehigh increments in Table 2. Results of the first culture screening intended to detect' strains of fungi optimally able to convert wheat bran and bagasse into mycelial biomass. FUNGAL STRAENSb WHEAT BRAN BAGASSE CPRc ACP% CPX aCPR Lulworthia sp. SC73 28.9 +16.9 2.9 -12.8 SC73 6 SC51 27.9 +15.4 2.9 -15.3 25.8 +14.5 2.7 -15.3 25.4 + 3.3 2.9 -13.0 Zalerion varium SC21 24.1 + 2.9 2.9 -12.8 Zalerion varium RZ264 26,4 + 2 ~ 2 3.1 7.9 Trichoderma viride SC51 24.8+ 2.1 3.3 5.2 f Drechslera hawaiiensis RZ17 24,5 + 0.1 Luluorthia medusa v ~hisca nia RZ 281 26.1 0.1 5.3 +53.7 24.4 ] 5 23.9 2.1 Zalerion varium RZ394 23.8 3.5 3.3 - 0.5 Culcitalna achras ora SC24 23.0 4.8 23.4 6.0 4.0 +14.5 25.3 7sl 4.6 +22 ' 2 Lulcitalna ~achras ora RZ327 22.4 8.9 3.8 +15.5 22.1 -12.2 3.6 + 4.2 Cirrenalia seudomacroce hala RZ280 22 ' 8 -12.4 2.8 -17.0 SC47 6 SC70 23.0 -13.8 3.5 + 2.2 Festalotia sp. SC38 20.4 -14.8 4.0 +20.7 Zalerion varium RZ393 21.7 -15.8 3.2 6.2 22.4 -17.1 5.5 +45.0 22.6 -17.2 6.3 +68.9 Ascotricha chartarum SC44 20.0 -18.4 2.5 -26.1 SC73 6 SC62 21.8 -19.3 2.8 -19.0 ll alostach botr s sp. SC74 18. 7 -24.9 4.7 +40s6 Nia vibrissa SC72 16. 9 -25.6 3.6 + 8.6 SC47 & RZ387 18.9 -28.9 2 ' 6 -21.4 H alostach botr s sp. SC62 19.9 -29.4 3.5 1.0 19 FUNGALSTRAINSb BAGASSE CPXAcpXd CPX ACPX SC47 6 RZ393 18.4 -32.5 4.4 +28.8 Robillarda sp. SC75 17.3 3 3 ~ 5 2.1 -34.7 SC47 t SC73 17 ' 3 -37.4 3.3 4.7 S nce halastrum racemosum SC47 15.1 -46.5 2 ' 2 -34 ' 7 ~oendr t>iell" salina SC77 2.8 -17.0 Processed controle 14.3 -44.4 2.6 -22.9 Untreated material 16.7 2.8 Medium used: 5% wheat bran or bagasse ground through a 40-mesh Wiley mill screen!, 0.24XNH NO , 0.006XKH P04, 0.01X yeast extract, in 25ml. 15 o/oo seawaterfn 125mi DeLongflasks. With culture collection accession numbers. Percent crude protein ~ percent nitrogen x 6.25! of fermented product wheat bran and mycelium!. Crude protein of final product/crude protein of original material, x 100, Flasks of medium taken through sterilization, fermentation and harvesting process, but not inoculated. Not determined. 20 Table 3. Results of the second 'et of culture screens intended to detect strains of fungi optimally able to convert wheat bran into mycelial biomass. FUNGAL STRAIN WHEAT BRAN CPX hCPX H~roth..cium sp. SC87 34.3 Skelton'222aSecedena SC104 29.8 +40.6 Curvularia sp. SC86 33.4 +37.9 ~Ztliia sp. SC20 32.5 +35.9 Chaetomium sp. SC97 31.7 +34.8 Sco ulario sis ?! sp. SC8 31,0 +20 ' 3 31.0 +19.6 Lulworthia sp. SC73 29.9 +19,3 Nelanos~ora sp. SC94 27.6 +17.9 Curvu'aria tubercul ata S 91 26 ' 3 +10.1 ~Assr 'lies ~ni tr SC90 27. 7 + 7.7 ~Cehalo~s orion sp. Scl 28. 3 0 ~ 3 22.9 1.1 sp. SC102 22.0 2s3 Geotrichum candidum SC117 26.4 2.3 Alternaria ~lon isslma SC122 24. 4 5.7 B tr haeria sp. RZ 254 25.5 7 ~ 3 ~Z thia sp. SC7 21.2 -1] .8 Dendry~l>ie11'. salina SC77 26.4 -12.0 Blakeslea tr~is ora SCSS 20.6 -15.3 ~Sororstia sp. SC100 23. 7 -15.9 21. 7 -20.9 Tr ichod erma v ir id e SC51 33.1 Pestalotia sp. SC38 32. 9 Pestalotia sp. SC89 32.5 21 Table 3. continued! Mediumused: SXwheat bran, 0.86XNH4N03, 0.09X KH PO<, 4' 0.01X yeast extract, in 50 ml lS o/oo seawater, in 250 ml Ehrlenmeyer flasks. With culture collection accession numbers. Percent crude protein ~ percent nitrogen x 6.25! of fermented product wheat bran + mycelium!. Crude protein of final product/crude protein of original material, x l00. Not determined. C 0 v 9 + 0 C bOv 0 C U U bO C 0-9{O v 0 C W g CP ~ ~ W 0 c p+ U { {O 8 Q " a.v! CQ C D saw C~ C!e v { nj ep {O 0 {O ~ V! w{Oa v 0 {Og 0p Q - w CJ ~O 0 bO z O 23 %~bi i,»., d~il maritima, 68.9%!, the highest finaJ crude protein content of the end productswas only 6.3%, considerably lower than the requirement of penaeid shrimp Section i!. 7. Production of ex erirnental feeds Background. Before the crude protein analyses of the first culture screen could be completed, an opportunity arose for preliminary testing of fungal-fermented products as feed for pink shrimp. Three fungi from the culture screen were used to ferment wheat bran in 5 gallon glass carboys, using the same conditions as in the first culture screen. The praducts memDacvewted.as befm~.. clued aP.R..r,s~iod.tbay.sh o~ a .6O- mesh Wiley miU screen, and fed to postlarval average initiaJ fresh weight - O.JJgm! pink shrimp in the concrete tanks described above. It was unfortunate that the crude protein analysesfrom the first culture screen were not available during the design of this experiment, for the three fungi chosen were among the poorest performers of the ~5', i i 'i, ~l' ll di mum final crude protein content f rom the carboy f ermentations was J8.6%, correspondingto a net loss of 57.0% crude protein. Shrimp yield from aJJthree fungal feeds was l.6 times or more lower than that from unfermented bran, though all three fungal feeds gave average shrimp yields 7-8 times higher than unfed controls. Materials and methods. During the fall of J973,we obtained a JO-liter laboratory fermentor Microferm, New Brunswick Scientific Co.!, with dissolvedoxygen and pH controls, for the purpose of feed production for the J974 shrimp-growing season. Only the warmer months when temperature does not fall below 20 C are suitable e.g. Kilgen and Harris, l975!. We completed analysis of our culture screens, and identified those isolates apparently best able to convert wheat bran into a more highly proteinaceous product. Fermentor runs were planned using these fungi in order to determine how closely scaled-up fermentation processes would correspond to the flask-level fermentations, and to produce feed material for shrimp-feeding experimentation in 1974. We soon encounteredthe difficulty discussedby Solomons 969!, Underkofleret al. L907!,and Weismann 970! with fermentation of particulate substrates: contamination due to the resistance to conventional steam sterilization of spore-forming bacteria, when these are protected by starch or protein componentsof particulate media in large volume. Rather than solve the problem by purchase of a stirring autoclave, as Weismann 970! suggests,or by usingin-place steamsterilization impractical in our case!, we found that slit,ht modification of the autoclave and s~earate autoclave steritization of all fermentation components bran, seawater and inorganic nutrients, apparatus!was effective in preventingbacterial contamination.The autoclavemod- ification involved venting from near the bottom rather than the top, permitting saturated steam to displace air Ernst, 1968!. Becauseof the potential contaminationproblem, we usedfast-growing andthus contamination-resistant! fungi as fermenting agents in our production of test feeds. Other considerations here were the necessity to produce large quantities rapidly, and tn rsa3s,tee the nrniPr tfarfrqstt nf. !argyleus'r aide nnyrati.nn g j< b t'fatal 'QP i Je~gtb Qf I,glLLIJ I tk!l< lurn eras produced Calam 969a, b!, Rowleyand Bull 973!, and Solomons 969!. Ino rere performed as as follows: 250 rnl Ehrlenrneyer-flask fermentations of wheat bran vvested at early in the second set of wheat bran culture screens. These were seconds as in the stationary phase, the entire 50 ml contents homogenized for 6 arne medium in a culture screens, and the entire homogenateadded to 450 ml of the =!her&:Fu.d I mq, gggAfrn'Fernjm~h M,Kr Xb.;= I'he fermentation and added aseptically to the sterilized medium in the fermentor. 25 was then immediately begun agitation, sparging, pH, foam, and temperature controls 4 .et. i o2zpeez [ ~ ass pre@~!~i ro ziti l III indicated by decline in oxygen uptake and rise in pH all runs were with ammonium ion as the inorganic nitrogen source; see Prokop and Stros, 1970!. Harvesting and analytical methods were similar, though on a larger scale, to those used in the culture screens. ln addition, solids which passed the double-layer, 42 u rn nylon-cloth filters were collected by continuous centrifugation in a Sharpies Tl Super Centrifuge which was obtained at mid-year, 1974. Results. The four fungi which were used and the results of ferrnentor runs, the products of which were used in subsequent shrimp-feeding experiments, are given in Table 0. Range of content of crude protein for the four fungal products was from 25% to 32%, and increment in crude protein from -3!% to +25%. S. Attem ts to o timize crude rotein roduction Materials and methods. During summer, !970, we performed four flask-scale experiments designed to determine the extent to which wheat bran fermentation results could be improved by manipulation of fermentation conditions. The experimental design was a 3-way, 3- level, 2-replicate, factorial analysis of variance ANOVA!, and each experiment tested the response of a different one of the fungi from the bran culture screens. Statistical methodology used in this experiment and the others of this communication was der'ived:ro!r.:Sle' a !956!;r'oned'e'd'or mh! ochrah iI!96? i,~56M"~ aihK konlf l969! 26 an an 3 aaI an N acr In ja 6 W W CII arj Cja N Caa N N r + + + + l + + + + r l l UTBZCjld epn1 J Cca N g N Ccj N W g g an e r aca > ara N ral M M N M M Ial M N N N N 0 0 3$5ag 1@g r M w e s o co pa 0 r 0 m g g Cj arl caj" cn ' 81$ UPTQSXBQ W cra rh an Cra acr N ccj N H M r m N m 8 era ara M W aca cja an era Cra W M Cal M M arj '4 0 Q Ugajj/'SQI UQ xB ca 6 N N N N N N N N N N N 4 N I Iaj 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 g A Na @dan 0 0 o an an0 0 an an0 0 In an0 0 an ano o an ro r 0 o 0 gj 0 ~ W aj c ~~ 0 0 M cra 0 w 0 0 0 N N pl ccj 0 p 'pu peag aag j 0 N a-I ga kJ P I g ao .prl u aj u ' ale e P ' PPUJ N N N aP ae 0 aca 0 N N N aP ICa cca crj aajg Cj p cra N N cra cv w w In cra cra W j4 p CJ v < 0 o 0 0 0 0 0 0 0 0 0 0 ~a]7 jjjn7n Ups ~ i ~ ~ ~ ~ t ~ ~ ~ ~ ~ ~ ~ 0 0 0 ac4 ja W 0 "e ala 0 p CL Cj 'Cj an In an In an In an an In an p 4J ONCET ~ m ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ M M O 0 0 O O O O O 0 0 0 0 0 0 0 UB1g QBeqN $ an an an an an an an In an an an an carr c4 r W r W r r ap Crr CC> W r r U U U U U U U U U U U U U U U car CA crj caj cQ caj nfl crj CA Ca rij ch Crj crj crj 1eqmnN Ung cc O W N e an ~ r CCaCra ~ N < an aca N N N N N N N N N M Pl 27 Table 4. continued! ungal strain used, given as culture collection accession numbers. ,ground >he percentage of wheat bran used, w/v. hlheat bran was ough 20-mesh except for runs 35 and 36, for which it was pre-ground ia; runs I,'iley mill screen. Runs 24-36 employed 10.5 liters of 19-24, 11 liters ded to the '".e age at which inoculum cultures were homogenizedand fermentation vessel. system fn The amount of 2N KOHutilized by the automatic pH contx maintaining the pH above not at! 4.0. The revolutions per minute of the fermentor impeller. The air flow into the fermentation vessel. fermentation. The length of time between inoculation and harvest of t initial dry Final dry weight of product mycelium and residual bran weight of bran, x 100. Percent nitrogen of product x 6.25. 100. Crude protein of product/initial crude protein of bran, k pH maintainedat 4.5 throughoutfermentation. grata lost. NH4! SO4. and Zar J974!. Two fast-growing fungi were used Trichoderma viride SC 5J and » g, salinity, 5 o/oo, l5 o/oo, 30 o/oo; bran concentration w/v!, 2%, 5%, 8%; type of inorganicnitrogen source w/v!, high NH sources were, for high NH NO: 0.30%, 0.86%, and J.38%; for Jow NH NO: O.J7%, 0.43%,and 0.69%; for NH> SO<. O.J0%, 0.34%, and 0.54%. The C/NH<-N ratio was usedfor standardization,rather than C/N, becauseof the probability that the fungi would take up the ammonium ion in preference to the nitrate ion Nicholas, l965! and the fact that the ammonium was probably present in large excess of requirement for maximal theoretical nitrogen content of myceliurn and maxirnaJ theoretical conver- sion of bran Section 6!. Methods of preparation, incubation, and analysis were similar to those of the first bran culture screen, with two important exceptions: l! inoculum level was lower than in the culture screen Table 5; 2 rnl of each homogenate were added to each 125 ml experimental flask! and the inoculum for SC 73 was less than 2 mg/flask; ! all fermentations were terminated at 7 days, rather than at subjectively determined early stationary phase. Due to the large numbers of flasks having to be harvested at the same time, all flasks of a given experiment were removed from the incubator at the same time, and stored at 20 C. They were then harvested on a random basis over a period af 5 days. -.. +~~get"~~ veriprnfme4 ru'. ohio.pntgde aeoton-ie-, .=in'"scdr@~:-.~r==dgsgs-:-'-, and slope -0.003! was not significantly different from zero r = -0.005, N.S.!; the storage period did not bring about loss of protein from the fermented product. 29 Table 5. Inoculum data for the 3 x 3 x 3 ANOVA experiments designed to determine how the wheat bran fermentations might be optimized V.E.c FUNGAL STRAINS a 48 Lulvi. rthia sp. SC 73 0.' 210 ~Yirothecium sp. SC 87 /ml Trichoderma viride SC 51 2.. With culture collection accession number as inoculum for each b Dry weight per unit volume; 2 ml were us experimental flask. =he same one ml pipette, c Number of viable elements in one drop fr! determined on cornmeal agar spreadplates d Too manyto count; inestimable. 30 Results. ANOVA results of the four experiments are given in Tables 6-9 for nitrogen content N%; arcsin transformation! and crude protein increment a CP! of the fer- mentation products, and treatment means ar e graphed in Figs. 2-7. In each case, the treatment means are graphed only if the ANOVA indicated that the treatments had si nificant T e I error < 0.05! effects on them. Values for controls no fun ai llllllllllllllllll1lImllHIIII IIIII11IIIIIIIIIIII III!IIIIIIIIIIIIIIIIIIII IIIIIIIIIIIIIIIIII I IIIIIIIIIIIIIIIIIIIIIII II llllllllllllllt IIII given Iri iriocu ationi no lrIe',gaf! fc nitrogen.a .tiara~.ping;n! yce rom sucrose '..gf Table 10. from 30 Two general patterns emerge from Figs. 2-7: l! decrease in salinit low o/ooto 5 o/oobrings about increase in N%and L CP,and ! highNH N% and NH OA, Fig. 5 CP. Thesepatterns, however, are clear in only a few instances e.g. Fig. th other 3, 8% bran!. Bran concentration was involved in significant interactions i In most treatments in every case but one Fig. 7!; no generalizations are possible. eralized cases, the interacting effects of two or ail three treatments prevent gi conclusions e.g. Figs. 2,3, and 6!. yin 8% f .' and 3! ~ bran,at 5 o/oosalinity and with the highconcentration of NH>N03 Figs. 'able 3!, The 6 CP% 0! was near the highest of the bran culture screen results culture and the 5 CP, mg/ml, A! was higher than that of the highest of the br s in the screen results .3!. This was in contrast ta the performance of this funl e lower first bran culture screen N% = A.i, a CP % = i0.5; Table 2! at concentrationof NH 0 Ql 00 O QJ 4 cd c0 0 Ql 4 '0 4 O'0 Q crI O Ch c0 Ql Y0 Ql 0 0 Ql c0 QJ 0 cfi col cJ+rx Ql QJ Ql QJ'0 cQ U QJ c0 A QJ 00 0 QJ JJ 60 cl 0 Lh Crc crI QO0 Ql H Ql 0m' 4 O QO 4 J ccrc i'll QI 00 O U Ql 0 0 04 4 QJ O cJ Crl 00 Crl 0 0 4' oc 0 0 tEl Ch Crl Ql nl g al 4 C 8 cJ cQ 0J 0 QJ 0 H Ql ~oC M 0 4J 00 4 O I I Cl 4 I P~ RM 32 33 rn 'V ba,< gv nj CJ Q ec>~ IA Q v~ ~ c 8 yv RE s ~ C a! c 0 tU~ < ~ ~w 8 0 0 8 ~g lO - c QO. ~ e E + c8~ C dJ O~ Cl U L lj SID~ bC~ E 0 v rg,~ L. c~ ~ ~ ~ 0 A5 O 0 Q + +0m CV 4P bO O M IR e= l~ c~ I 'v~ cu I e g 'x I Z C-y 6 V o: C C 0- ~ c 0qj C '~ 4 v ~~ 0 h. 0 CJ v 0 tl o O C 5 0 CP bG tZ O + O+ O O s8m 'ggV 35 Table7. Meanvalues with 95Xconfidence intervals of nitrogen percentage and change in crude protein content of fermented wheat bran from an experiment designed to determine how the bran fermentations might be optimised with regard to protein production. Results for Lulworthiasp. SC73, for whichbran concentration,salinity, nitrogen source, and the interaction betweennitrogen source and bran concentration producedsignificant P, TypeI error < 0.05! effects on nitrogen percentage,and bran concentration,salinity, and the interaction of bran concentration anc salinity produced significant effects on crude protein change. SeeFigure 4. H INN LONN 4.60 + 0.13 2 4.68 + 0.31 4.67 + 0.41 4.20 + 0,40 b S 0/Go 4.27 + 0.18 BX 5 4.19 + 0.64 4.22 + 0.33 4.17 + 0.38 30 3.90 + 0.15 4.08 + 0.33 4.04 + 0.30 4.06 + 0.40 hCP S o/oo 15 30 ll. 0 + 2. 6 3X! 4.3 + 9,9 X! 3.0 + 4.8 X! 33. 0 + 13. 9 5X! 16.8 + 13.5 8X! -8.3 + 6.7 X! 30.2 + 16.0 9X! -9.7 + 13.4 X! -34.5 + 13.9 0X! a Nitrogen percentage b Salinity c Bran concentration d HINNhigh NH N03 concentration, LONN low NH NO LONS low NH4! SO e Changein crudeprotein in mgs.,with percentagechange in parentheses. 4.75 36 4.50 Z 425 3.75 15 5 SK BRAN % +40 +20 0! E 0 V cl -20 -40 15 S%o Figure 4. Lulworthia sp. 5C73. Meanswhich were significantly affected by treatments P, Type l error < 0.05!. A. The effect of salinity S o/oo! on nitrogen content i%%!of fermented wheat bran. B. The interacting effects of bran concentration Bran 96!and nitrogen source on nitrogen content. C. The interacting effects of salinity and bran concentration on crude protein increment d CP, mgs!. See Table 7 for confidence intervals and 96d CP. 37 Table 8. Mean values with 95X confidence intervals of nitrogen percentage and change in crude protein content of fermented wheat bran from an experiment designed to determine how the bran fermentations might be optimized with regard to protein production. Results and the interaction cf bran concentration and nitrogea source produced significant P, Type 1 error < 0.05! effects on nitrogen percentage, and bran concentration and the interaction between bran concentration and salinity produced significant effects on crude protein change. See Figure 5. N X H INN LONN LONS 5 4.41 + 0.13 2 4.71 + 0.24 4.59 + 0.16 4.33 + 0.11 b S o/oo 15 4.23 + 0.13 BX 5 4.37 + 0.28 4.25 + 0.21 4.37 + 0.18 4.40 + 0.13 8 4.11 + 0.25 4.11 + 0.30 4.29 + 0,10 hcp S o/oo 15 30 16.5 + 8.4 9X! 17.3 + 8.0 1%! 12.7 + 8.1 5%! -7.5 + 23.0 %! 1.8 + 8.8 X! 9.8 + 7.6 %! 0.3 + 20.5 %! -27.2 + 22.0 8%! -15.3 + 11.1 %! a Nitrogen percentage b Salinity c Bran concentration d HINN= high NH4N03 concentration; LONN~ low NH4N03,. LONS~ low NH4 S04. e Changein crude protein in mgs., with percentagechange in parentheses. 38 4.75 4~ Z 4.25 0 5 15 30 0 2 SXo BRAN X +20 +10 gl E 4 10 0 CI -20 -30 0 15S Io 30 by treatments P, Type I error < 0.05!..A.! The'effecT of salinity S o/oo! on nitrogen content iV%!of fermented wheat bran. 8. The interacting effects of bran concentration Bran 9b!and nitrogen source on nitrogen content. C. The interacting effects of salinity and bran concentrationon crude protein increment hCP, mgs!. See Table g for confidence intervals and 96 aCP. 39 Table 9. Mean values with 95X confidence intervals of nitrogen percentage and change in crude protein content of fermented wheat bran from an experiment designed to determine how the bran fermentations might be optimized with regard to protein production. Results for Trichoderma viride SC 51, for which bran concentration, salinity, the interaction between bran concentration, salinity, and nitrogen source producedsignificant P, Type I error < 0 ' 05! effects on nitrogen percentage, and bran concentration and nitrogen source produced significant effects on crude protein change. See Figures 6 and 7. 2% B 5% B b S o/oo S o/oo S o/oo 15 30 15 30 5 15 30 HliVN 3.94 3.59 3.72 3.95 3.82 4.00 4.07 4,04 3.96 LOiVN 3.79 3.94 3.95 3. 90 3. 57 3.72 3.87 3.73 3. 99 LOVS 3.56 3.54 4. 04 4.17 3.84 3.89 4.18 4.02 3.84 hCP -17.9 + 3.2 LX! HINN -32.8 + 7.7 5%! -37.0 + 5.7 7%! LGNN -40.1 + 11.2 l9%! -49. 3 + 9.3 4%! LONS -31.3 + 8.0 5%! a Nitrogen percentage b Salinity c Bi an concentration d INN high NH4N03c~n~~~t~~ti~n; LONN low NH LAYS- low NH4 S04. e Ctrange in crude protein in rags., with percentage change in parentheses. f Confidenceinterval not computed; these means are for tworeplicates only. O U r58 w ~w 8~ ~Vu Qe eC ~a~ C~ g ~ v V Cg VJ U U W~ e~ C oR, C V C 6 t5 W 0 0 g w C 0 8 E0Urd 08 -15 -20 Il 30 C 0 g -35 -40 +45 -50 5 BRAN HI NH4 NO3 LO NH4 NO3 LO NH4!p SO4 N ITROGE N SOURCE Figur. 7. Trichoderrna viride SC5l. Meanswhich were significantly affected by treatrnents~P, Type I error < 0.05!. The effects of bran concen- tration Bran%! and nitrogen source on loss of crude protein ~CP, mgs!. See Table 9 for confidence intervals and 96' CP, at:he 296level of branconcentration, where N% produced with NH At the optimal salinity level of 5 o/oo, 296 bran gave lower ~ CP than 5 and 896 bran. I ' I ' C culture screens Tables 2 and 3!, gave much lower N% and hCP in its 3 x 3 x 3 ANOVA experiment. Its highest significantly affected mean N% was r!.7 at 2% bran andthe higherNH and there appeared to be little effect of salinity at this bran concentration though it had marked effects at the 5 and 896 bran levels. Nitrogen source had no effect on 6 CP. As in the case of sp., Trichoderrna viride SC 5l Fig. 6! exhibited much lower N% maximum mean of 0.2%! in the 3 x 3 x 3 ANOVA experiment than it had in the second culture screen .3%; Table 3!, though its response in the first culture screen rr.096; Table 2! was lower than its ANOVA experiment maximum. ln mostcases, the higherNH 5% and 30 o/oo - 8%. At all three bran concentrations, highest N% was given by NH< SO<, this was at 30o/oo salinity with 2% bran, and at 5 o/oowith 5 and8% ijji!jjti!jr!»iitiiiiYii»!i!!!!!!!»iiii»7iiiI!ii»jI!ijl!gglj'ijijLtjjijjj!ip y'hajj!i!Ilitjjjjtj>jijig!Ijj>jjigijijigigiijgjtjjjjjjilqjjIi thaneither the high NH f ollowing. Visual observations made during harvest of the flasks included 1 ches from conidium formation by SC 5l as the percentage of bran decreased the v, .nc reased, the 2% bran fermentation were bright green!. As the percentage of brar th size of the mycelial pellets formed by SC 87 decreased markedly. Since neither sp. nor Trichoderrna viride performed in a fashion corresponding to its bran culture screen results, it is possible that these discrepancies were due to the standardized harvest time days! of the 3 x 3 x 3 ANOVA experiment. Both of these fungi are fast-growing Table 5!. As can be seen from Table 0, maximum 8, CP% for Trichoder ma viride from Microferm runs was +25%, at a bran concentration, nitrogen source and level, and salinity which gave negative 6 CP > l0%! in the ANOVA experiment. %hen the set of conditions indicated as optimal for N% and h CP by the ANOVA experiment were tried in a Microferrn run with T. viride, results quite comparable to the ANOVA results were obtained N% = I.J and L CP = -3J%! Tables 0 and 9 and Figs. 6 and 7!. This suggests that the ANOVA experiment showed best results for that combination of treatment levels which causedthe fungus to grow Jessquickly into the Jater stationary phase when conidium production conidia are not efficiently harvested by 02 p rn filtration and other loss of cell nitrogen takes place!. Comparisonof runs 35 and 36 Table 0! with Table 8 shows another distinct lack of correspondence, but in this case, the cause is not clearly a time-of-harvest problem as with T. viride; Microferrn runs 22, 25, and » |' either Table 3!, so that scale-up difficulties of some sort appear to have been involved with this fungus. Likely candidates as sources of these difficulties are differences in autoclaving of materials and in availability of dissolved oxygen during fermentation. The control figures for N% and a CP Table lO! appear to show that the productsfrom flaskswith NH 8 X 15 o oo No Fun i HINN LONN LONS NON 2.91 3.04 3.16 3.15 -106.0 i-31%! -93.0 -27%! -77.5 -23X! -77.5 -23X! 8 X 15 o/oo Non Lulworthia s . SC 73 X N 3.10 8CP -ee.s -20%! 2 %Sucrose, l5 o/oo, 0.45 X NH4N03 RZ 312 SC 73 SC 51 SC 87 3.12 3.10 4. 31 7. 38 Flasks treated like all others in the experiment, except that no fungal inoculum was added; medium of 8% wheat bran and 15 o/oo salinity; other components as in all other flasks. b HINN~ 1.38XNH NO ; LONN~ 0.69XNH NO ; LONS~ 0.54% NH ! SO Non no added nitrogen- c XN nitrogen percentage in washed and dried product. d Changein crudeprotein from original mg.!with percentchange in parentheses. e Culture collection accessionnumbers for fungi used; see text for binomials. feed at all F74A, F74B!. The feeds were alwaysthoroughly wetted with tapwater beforethey wereadded to feedingtanks, so that theywould immediately sink and b"comeavailable to the shrimp. All fermentedfeeds were prepared in the Microferm fermentorand processed as describedabove Section 7!. ln mostcases, fermentation productswere dried at 550 C andground either by mortarand pestle or in a Wiley mill. Whenthis wasnot the case,products were harvested and washed as usual,but then refrigerated rather than dried. Results. Theresults of the F74Experiments are givenin Tablesll andl2 andFigs. 8-ll. Averagefinal fresh weights AFFW! were analyzed by l-wayANOVA followed by a f~aehrNb'wrrraa wK x~~ 3iQlW'id~~--~;jgaQ;~D t say e.miffed.gf ro>sLtipl~ rerpyaeisan amongmeans, with confidencelevel = 95%. Logarithmictransformation was necessaryfor F74Aand F741, in whichvariance was inhomogeneous Bartlett's test, p = 0.05!.AFFW was approximately equal to averagefresh weight gain in experiments F74A,B, andD dueto the verysmall average initial weightsof theseexperiments. Freshweight gains were used in thestatistical analysis of F74C,since the average initial freshweight in that experimentwas much larger than in the others. Oneof the clearestconclusions to be derivedfrom theseresults is that there v eredifferences from tank to tank not due to intendedtreatments. Theseoften causedmarked, statistically significant differences in shrimpyields and AFFW. For this reason,means are not calculatedfor combinedresults of replicatetanks. Rather,replicate results are shown separately, sothat the range of meanresults is perceivable.Two major possible reasons for theunintended differences among tanks aredifferences in nutrientretention among tanks following previous experiments, and differencesin the rneiafaunaand microbial flora autotrophicand heterotrophic! whichdeveloped along with the shrimp,due to inoculumdifferences and nutrient retentiondifferences. YieLd values in Tablesll andl2 are fresh weights,and feed Table ll. ResuIts of shrimp-feeding experiments in the F 74 Series: F 74k and F 74B, with pink shrimp Penaeus duorarum!. Treatments are identified in Table 13. See Figures 8 and 9. 4 Ol 8Ql W4 Duration, days 58 60 Salinity range, ojoo 32-38 35-38 0, Temperature range, 0 26-31 27-3 Stocking density/m 15 15 Ave ~ in1 tre 1 wt I g 0. 010 O.G05 Tres tmi nt 0,29 4.1 93 C.35 3.6 70 0 17 2 2 83 0.21 2.2 70 1.19 16,1 90 9 0.94 10.8 77 14 0.92 12.9 93 11 1.27 14.6 77 10 0 72 108 JGG 13 0.48 6.2 87 23 j.35 20.2 100 7 1.27 15.2 80 10 0.54 7.2 90 2G 0.40 5 ~6 93 26 0 76 ll 3 100 13 0. 60 8.7 97 17 0.46 6.9 100 21 0.47 7.0 100 21 0.29 2 5 57 30 0.29 2.9 67 50 0.31 4.6 97 32 10 0,62 7,5 80 20 0.43 6.4 100 23 10 1.02 11.7 77 13 1. 50 1. 25 E Z 1.00 T0 0.75 T lh LLI K C90. 50 0.2518 98 91 75 57 33 26 4 6 2 4 TREATMENT NUINBERS Figure8. Averagefinal fresh weights, with 95% confidence intervals, of pink shrimpfrom feedingexperiment F74A. Feedingtreatments: l-no feed; 2-unfermentedwheat bran; 3-autoclavedwheat bran; 4-Purinashrimp chow; 5-wheat bran fermented by Trichoderma viride SC5l dried!; 6-treatment 735,90%, $34,l0%; 7-l0% level, treatment f/4; 8-treatment k/0 undried,cold-stored!; 9-wheat bran fermented by SC87 dried!. Bars at baseof graph join those means which could not be shown to be significantly different from oneanother P, TypeI error >0.05!. Details in text andTable ll. 1.50 1.2 1.0 x 10.72 10 10 TREATMENT NUMBERS rior~- - "- 4".aa~aif' rz kmrhee'qh p~.~ii+l'A5%.ronf~rim~e.iotas~~~s. nf pink shrimpfrom feedingexperiment P74B,--Feeding treatments: K undried, cold-stored!; 2-uniertnented wne'at bran. AH mean weightswere significantly different from oneanother P, Type I error<0.05!. Details in text and Table ll. 50 Table 12. Results of shrimp-feeding experiments in the F 74 Series: F 74C, with white shrimp Penaeus setiferus! and F 74D, with pink shrimp P. duorarum!. Treatments are identified in Table 13. See Figures 10 and 11. Duration, days 60 Salinizy range o/oo 35-41 33-45 Temperature range, C 21-26 15-24 Stocking density/m 7 ' 5 A~crage initial wt, g 3.27 0.017 ?. eatr,-nt 0. 63 2.5 80 ll 0 73 2 9 53 0.54 2.7 100 ll 0.97 1.5 20 2. 59 9.1 70 0.81 5.3 87 16 1,85 9.2 100 Lost 12 1.26 6.3 100 12 1.23 8.0 87 11 12 l. 03 5. 2 100 14 l.ll 6.6 80 13 13 2.07 9. 3 90 15 0.79 5.1 87 17 13 1.39 6.3 90 12 0.84 4.2 67 20 14 0.74 2.9 80 26 16 0. 68 4.1 80 24 14 0.45 2.3 100 33 16 0.57 4. 0 93 24 17 0.79 3.5 60 28 17 0.67 4.0 80 24 5l 300 2.50 Cl 2.00 Z0 rM 1.00Lu K0.500-1411 11141212 1322 13 TREATMENT NUIHBERS Figure l0. Average fresh weight gains, with 9596 confidence intervals, of white shrimp from feeding experiment F10C. Feeding treatments: ll-regular fertilization; 2-unfermented wheat bran; l2-wheat bran fermented by Pestalotia sp. SC38 dried!; l3-treatment II 3, 50%, treatment kI2, 50%; l4-wheat bran fermented by Curvularia sp. SC86 dried!. I%areat baseof graph join those rneansawhici could not be shown to be significantly different from one another P, Type I error > 0.05!. Oetaiis in text and Table l2. 52 1.2 E 1.0 ZULU Z 0.7 0.5 0.4 16 17 i6 iS TREATMENT NUMBERS Figure ll. Average final fresh weights, with 95% confidence intervals, of pink shrimp from feeding experiment F70D. Feeding treatment".' 2-unfermented wheat bran; 0-Purina shrimp chow; l5-wheat bran fermented by M rothecium sp. SC87 undried, cold-stored!, 50%, treatment 3, 50%, l6-w eat bran fermented by Trichoderma viride LlljclL = ~379 Jlci=- [l>Ulll~ ri..vi!". z A- zs- eaotmaek-.- 5 S-.total arete6 .hxe}eh C' g I! ',Q[ QQ J~~ [$ :!'.-'~gz=~~cedrmzTJR~?i Qgj,gqo~ry':~ "g~lighl pJ,I~ ~~+~5 I IJQII~~IJ JJJJI~IJjJJIJI[[Q+y, ++7++ ,9 ~".191 P, Yseof graph ioin those means whii,r 60.mesh screen, Bars -at gnificantiy different from one another could not o'e'shown to b'e .tails in text and Table l2; P, Type I rror> 0.05!. l 53 List of treatments used in shrimp-feeding experiments of the F74 series. Compare with Figures 8-11 and Tables 11 and 12. Fxcept where otherwise indicated, feeding levels were equal on a dry weight basis within experiments. Ro f eed iny, or f er tilizat ion. Unfermented wheat bran. F74A, F748: not ground; F740, F74D: ground Wiley mill! through 20-mesh screen. Paean CPX 16,7. Wheat bran, autoclaved, washed, and dried as ir. fermented wheat bran treatments, but not inoculated or fermented; ground in mortar and pestle. Nean CPX - 18.9. Purina 'shrimp chow' ' Experimental Marine Ration 20'!; fed as thick flakes as received. Ingredients include f ish meal, soybean meal, ground wheat, brewer's yeast, dried whey, soybean oil, dicalcium phosphate, iodized salt, vitamins, minerals. CPX 21. 8 . Wheat bran fermented by Trichoderma viride SC 51. Dried at 55oC and grouna by mortar and pestle. See text for CPX and ACPX. Treatment I/5, 90X, and treatment 84, 10X. Treatment r'/4, at 10X of its feeding level. Treatment !l5, but refrigerated rather than dried. 55oC and ground by mortar and pestle. Treatment b9, but refrigerated rather than dried. Ses text for CPX and ACPX. Fertilization by daily addition of a commercial fertilizer 8-24-16! such that daily addition of nitrogen was equivalent to that of the most nitrogenous of the other feeds in an experi- ment; pre-dissolved. Wheat bran fermented by Pestalotis sp. SC 38; dried at $5oC, ground Wiley mill! through 20-mesh screen. See text for CPX and BCPX. Treat'ment /f12, 50X, and treatment i/2, 50X. Wheat bran fermented by Curvularia sp. SC 86; dried at 55 C, ground Wiley mill! through 20~esh screen. See text for CPX and ACPX. Table 13 continued! 15. Treatment 10, S0%, and treatment l2, 50%. 16. Treatment 85 but ground Wiley mill! through 20-meeh screen!, S7%, treatment I/2, 43%. Fed 13X more/day than other treatments in F74D. See text for CPX and dCPX of fermented portion. 17. Like treatment 5'16, but the fermented portion ground Wiley mill! through a 60-mesh screen . Fed 13% more/day than other treat- ments in F74D. See text for CPX and 6CPX of fermented portion. 55 efficiency = total dry weightof feed ~ total fresh weight of animals.The treatments used in the four feeding experiments are identif ied in Table J3. ExperimentF70A was begunin 3uneand harvestedin August Table ll!. Wheat as feed for postlarval pink shrimp, The fermented materials came from Microferm runs l9-22 and 20-26. As Table 0 shows, these materials varied in N% from 28.3 to 3l.8 and in 5 CP% from +5 to +25 for SC 51, and in h CP% from +5 to +ll for SC 87 with N% = 31.6.The SC 5l material wasfed both dried mortar andpestle ground! and refrigerated. Other feeding treatments were unfermented,unground wheat bran, autoclaved,washed, dried and groundwheat bran, commercialflaked feed "shrimp chow," Purina Experimental Marine Ration 20!, a combination treatment of 90% SC 5l fermented bran and l0% commercial flakes, a commercial flakes treatment at l0% of the feeding levei of the above flake treatment, and a no-feed control. The experimentalfeeds were fed at 5 gmsper day on a dry weight basis. The commercial flakes performed best in AFFW in both replicates Fig. 8!, althoughthe higher of the unfermentedwheat bran replicates was not significantly different from those of the commercialpellet replicates lower than the highestone by a factor of only 0.9!. The autoclavedand washedbran replicateswere both tower in AFFW and total yield than the unfermented bran replicates, although one of the replicates of each fell in a group of three means which were not significantly different from one another. Thus, autoclaving of wheat bran does not, in itself, impart higher nutritive capacity to bran. The higher AFFW of the replicates of the dried T. viride fermented-bran feed "'I sp. product significant difference was not detected between them!, and both AFFW's were lower than the higher of the unfermented bran AFFW's by a factor of about 0.5. Both replicates of the refrigerated T. viride feed were as low in AFFW as the higher 56 of the no-feed replicates, lower by a factor of 3 than the unfermented bran replicates. In total yield, the refrigerated T. viride feedswere lower than the higher of the no-feed controls, due to the markedly lower survivals, indicating a possible toxicity of the refrigerated T. viride material. It must be noted here that refrigeration problemsapparently occurred during storageof this feed material, for bacterial spoilagewas quite noticeableby the time of harvestfor F7' A. The addition of I0% of corn merci al flakes to the dried T. vi ride feed produced marked improvementin AFFWand total yield; the higherof the AFFW'sof the mixed- rnaterial replicates was significantly different from and i.rr times greater than that of the higher of the unmixedreplicates, although it was still smaller than that of the higherof the unfermentedbran replicates by a factorof 0.6. Thatthis improvement was not due to the commercial flake fraction alone, is shown by the .fact that the AFFWof the higherof the replicatesof the diet which consistedonly of commercial flakes at J0%of the daily feeding level of the other diets! was significantly lower than the AFFWof the higherof the mixed-dietreplicates by a factor of 0.6!. In 3uly, experimentF7rrB was initiated. Experimentalconditions were the same as thoseof F7%A Table ll!, with one important exception: the averageinitial fresh weight mg! of the postlarvaJpink shrimpused was one-half that of F74A. Since other experimentalconditions were little different betweenthe two experiments,it wasprobably this factor that led to the reducedsurvival observed in F746. Three productwashed and refrigerated!; unfermented, unground wheat bran; and a no-feed control. Feedinglevels were the sameas thoseof F7%A gm/dayon a dry weight basis!,and experiment duration 0 days!was approximately the sameas that of F74A 8days!. The fungalmaterial came from Microferrnruns 27 and28 Tablerr!, the productsof which were very similar in N% and 5 CP96!to those of the Microferm *. 57 The meansof all treatmentreplicates were significantlydifferent from one anotherin F708.The AFFW's and total yieldsof the unfermentedbran replicates of F748were similar to thoseof F70A{Fig. 9!, andthe samewas true af the no-feed controls;in both cases,total yieldswere slightly lower in F748. The mostmarked ii * TheAFFW of the higherof the two replicatesof the refrigeratedfeed F701!was higherby a factorof 2.4than that of thehigher replicate af thedried feed F7VA!.lt wasalso lower than the higherAFFW of the unfermentedbran in F708by a factorof only 0.8. Similarfigures applied to comparisonsamong the sametreatments with regardta total yield. Theseresults represented a marked improvement over the dried feed with respectto comparisonon a higher-replicatebasis ta the unfermentedbran treatments; the dried-feedAFFW of F74A was smaller by a factor of 0.05 than the AFFW of unfermented bran. ExperimentF70C was begun in August Table l2!. Salinitiesranged higher and temperatureslower than in F70Aand F74B, but the major differences in experimental conditionswere that a differentspecies of shrimpwas used white shrimp, Penaeus setiferus! and averageinitial fresh weightwas muchlarger valuewith 95% confidenceinterval = 3.27+ 0.2lgrn!. Forthis reason, average fresh weight gain AFWG!and total fresh weight gain TFWG! were compared among treatments. Also, stackingdensity was lower on the basisof numberof individualsper m 2 !, butmuch higheron the basis of freshweight per m 2 8.2grn,as compared to O.lgm far F70A!. Feedinglevel was 2.5 grn per day on a dryweight basis approximately I5% of body weight at the beginningof the experiment!. Feedingtreatments again included unfermented bran, which for thisexperiment wasground through a 20-meshWiley mill screen.Rather than a no-feedcontrol, an j ra~rrw~r~ ~:+ca aaI ~ ii kgbI I .rc-'al . erti[i er was aaQed asm rate tnat ~ ~ ' %tvf gelt- 'Il1YVgtf! QQAKOL was Usec.::a con !f the mast nitrogenous af the other made nitrogen addition equivalent to thi feeding treatments. Two fungal-fermented feeds were tested, one produced by Pestalotia sp. SC 38 Microferm runs 3l and 32! and Curvularia sp. SC 86 Microferm run 29!. In both cases, N% and ACP% were lower than had been the case with the b ~ > g F74C were dried and ground through a 20-mesh Wiley mill screen. One combination tern: ~r't warn ~acth eo dG~:-mxttjre m' ~~ibtta=- mp. XC ¹ ahern < rrmWih ~ unf erm ented bran. The highest AFWG was that of the higher replicate for unfermented wheat bran Fig. IO!, but it was closely followed by that of the higher of the mixed-treatment replicates lower by a factor of only 0.8, and not significantly different!. The TFWG of the higher of the mixed treatment replicates was higher than that of the higher of the bran replicates. The higher replicate of the unmixed Pestalotia-fermented material had an AFWG which was smaller by a factor of 0.6 significantly different! than that of the higher of the mixed-treatment replicates. The Curvularia replicates fellI in a group of means not significantly different from one another, including the inorganic nitrogen controls, and the higher replicate of the Curvularia feed had an AFWC which was only l.2 times higher than the higher inorganic nitrogen control. In TFW4, the Curvularia results were virtually the same as those of the inorganic nitrogen controls Table 12!. Pink shrimp postlarvae were again used in experiment F74D, begun in September Table l2!. The average initial fresh weight l7 mg! was larger than that of F748, but percent survival was again lower than in F74A, probably due to the combined effects of higher salinity range and lower temperature range. Stocking density was one-half that of F74A and F748, and feeding level was similarly reduced .6 gm/day on a dry weight basis!. As in F70C, an inorganic rutrogen control and a 20-mesh unfermented wheat bran treatment were applied. The commercial flake treatment used in F70A was also 59 applied. The three fungal-fermented bran treatments were all mixtures with 20-mesh ~l»' .SCI i ig mixed 50-50% with unfermented bran. Trichoderma viride SC 5l material was dried and ground through two mesh sizes, 20 and 60. Each of these two sizes served as a separate feeding treatment, mixed at 57% fungal material to 43% unfermented bran. The mixed Trichoderma feeds were fed at 3 gm/day. The fungal materials were produced in Microferm runs 26 and 34-36 Table 4!. Runs 34-36 were lower in N% and markedly lower in a CP% than previous runs with the same fungi. ln addition, the I' '1 F74D, and by the end of the experiment, it had suffered noticeable spoilage. Because survival was markedly different among treatments and even among replicates, and because there was an apparent marked effect of survival upon AFFW Table l2!, only those replicates with greater than 80% survival were compared by ANOVA and SNK Fig. ll!. As in F74A, the commercial flake replicates were both higher in AFFW than those of the unfermented bran treatment; in F74D, the higher flake replicate was I.S times larger in AFFW and total yield than the one bran replicate and significantly different from it!. The unfermented bran AFFW and total replicate with survival greater than 80%. The higher Trichoderma+ bran treatment replicates with survival greater than 80% gave lower AFFW though significant difference was not detected! and poorer feed efficiencies by a factor of 1.4! than the E ' '"" ~ " P' ' PP between the two Trichoderma + bran treatments with different particle sizes. l0. Culture screenin on deli nified b asse and straw Background. Plant waste materials of minimal acquisition cost are, of course, the eventual target of our feed-development program. The agricultural wastes of least value are 60 thosewhich do not readily serveas productivefeeds for terrestrial farm animals,i.e., those which are high in lignified cellulose Pigden and Heany, 1969!. The lignin portionof thesematerials acts on a molecularlevel as a physicaland/or chemical barrie' to cellulosedigestion, and thus devaluesthe materials as feed for ruminants. Bagasseis a goodexa'nple of an agriculturalwaste material of this type. 1t is the remainsof the sugarcane stalk after crushingto removesucrose, and on a dry weight .J~ts,: err+~~=AsN.Z'lt.."eJI!>J ra-> bs.roiwllukx.o.-3K%. Jjpnin,.anxf 2% sili~>, Scinivasan andHan, l969}. Materialswith lignin andsilica contentsas highas theseserve largely as ine-t fiJJersor roughagein feedingof ruminants Van Soest,1969!. Wheat straw is also oie of the lowest quality forages; like bagasse,it is very low in nitrogen content .4%}, and addition o" urea in itself hasno effect on its digestibility for ruminants Pigden and Heany, l969!. JYheatstraw and bagasse,then, are two typical lignocellulosic,low-value agri- cultural by-products. In addition,both are easilyobtainable in SouthFlorida, and bagasseis readilyavailable in the warmtropical countries where shrimp mariculture develipment is taking place Gross, 1913;Webber, 1975}. Therefore, these two materials were chosen as substrates for a fungal culture screen. The results of our earlier culture screen on bagasse Section 6! had demonstratedthe m arkedresistance to f ungal decompositionof this material. Other workers had showna similar responseusing cellulolytic fungi and bacteria Chahal et al., 1969;Srinivasan and Han, l969!. As stated above, wheat straw is degradedonly to a small extentby rumenmicroorganisms. However, in the caseof both materials,physical and chemical treatment of the materiaJswhich alters the lignin-cellulosephysical/chemical relationship can greatly improve their degradability Callihanand Dunlap,1971; Toyama and Ogawa,1972; Wilson and Pigden,J964!. Therefore,we applied a delignification this wordis usedhere to meanalteration of the lignin-celluloserelationship, rather than complete removal of lignin!treatment to the bagasseand straw which was to be used in the culture screen. 61 ~AJaterials and methods. ,'he treatment was a "dry" method and involved a combinatian of conditions used in several other delignification studies Ghose and King, l963; Han and Srinivasan, 1968; Pew and Weyna, J962; Toyama and Ogawa, l972; Wilson and Pigden, J964! The oven-dried 5 C! materials were ground through a 20-mesh Wiley mill jjiIII|IIijijjjijI!Iiiji!I!jI'"""" " iii!jljiITiji"!jii jji!jjliijjijjjjijjjjjjji]IjijIIIi i is closer to a wet solid than w/v! 'laOH solution in deionized water. This cornbin; >edwith tapwater over a 205 a JiquiI suspension.! Whencool, the materials were i became clear {J2 liters were v m n; Jon filter cloth until the liquid passing the fil then resuspended in 800 mls requir~d for each of the materials !. The materials v, :h tapwater until constant pH of 5% acetic acid, stirred for 60 seconds, and washed about l0 hters in each case; of the resuspendedmaterials was reached this requi >oth bagasse and straw, the final pH: straw, 5A; bagasse, 5.2!. In the case ~analdry weight. This was in delign fication and washing resulted in a 62% loss of rough the coarse filter cloth part due to the Joss of fineJy divided solid materia t 55 C and ground through a during washing. The materials were then oven-drie 60-mesh Wiley mill scr mn. : availabiJity, it was deemed Due to constraints of time, funds, and equipn m the earlier bagasse screen. desirable to use a different culture-screening method J by the agar-plate growth It was decided to try an experimental plan insp 67!, Trinci l969, l97l!, and measurement methodology developed by Pirt l966 gal biomass measurement by Morrison and Righelato J970!, and the methods of {197l!. Their methods were direct observation of Warnock J97l! and Nagel-deS to degrade and convert the adapted to yield indices of degree and rate of abi bagass= and straw substrates. The fuji .chosenfor screenipewere selected for one or more of the following reason.: high frequencyof occurrenceon bagasseand/or straw before or during the shrimp-feedingexperiment described in Section 5; description in the scientific literature Sections 3 and 0! as highly cellulolytic and/or of high potential or indicated nutritive value; high nitrogen content produced in wheat bran fermentation or relativ ly high nitrogen content produced in bagasse fermentation during earlier culture screening Section 6!; isolation f rom estuarine or maritime cellulosic substrates and availability in our culture collection. Many of the fungi chosen were not taken all the way through the cultur~creening procedure. In some cases, this was du to poor growth on the inoculurn or test media, but in others, time constraints were t m cause. 'Ihe experimental procedure was as follows. All cultures to be tested were inoculatedonto fresh plates of cornmealagar CMA! madeup with l5 o/oo seawater. These and aH following plates were sealed with masking tape and incubated in darkness at 25 C; as soon as ampl= vigorous growth was produced on these CMA plates by each fungus, transfer was effected onto plates of I/O-strengthCMA with additional agir to bring it to 2% I/O CMA +A!. TheseL/4 CMA +A plates were of uniform agar media depth 0 ml/plate, LOcm diameterplates! and were preparedat the sametime and usingthe samebatch of l5 o/oo seawateras were ail the following plates. The transfer onto L/0CMA ~A andall following transferswere doneuniformly as follows: a sterile Ill cork borer mm diameter! was used to remove a cylinder of agarfrom the plate to be inoculated;a cylinderwas then takenfrom 5 mrnbehind the grwwirM.~ of. the inocuiumplaty. inverted. and placedin the hole on the plate to ~ I~ ...,hogg,-' ting}4-.,TbqL @sr'~ "cneJ!4 CXAA,0-< la~ms az jntr ei~ddnJ~>/to.inner < of exc.ssive endogenousreserves. The fungi were permitted to grow on these until they h~d reached the edges of the plates or until growth slowed. They were then 63 inocuia>ed onto plates of agar media with incorporated deiignified material. These ate of KH2PO,,O.OJ% yeast extract, 2.0%agar, in JSo/oo seawater. One epared delignified straw medium DSA! and one of deiignified bagasse DBA! wa.' ates or for each test fungus. Again, these were allowed to grow to the edges of thi >U.@qadi grew very poorly or not at all and were eliminated from further testing. .'d to From these inocuium plates of delignified media, the fungi were transf !eA. the culture screen piates. Six media were used, two of which were DSA ai DSA Corresponding plates of media with non-delignified materials were prepare< water!, and one of plain agar CMA only Difco cornmeaJagar added to the JSo/oo lia except bagasse or straw. medium PA! having all the ingredients of the test .d in the centers of culture- Three cylinders from the inoculum plates were juxtai g rrtrn Pu . A~g .Yrlf ~ ~=--- ~~pg|6Ãug@ QQ ~The hgg@%f=4Pi4$.i+gCQ!gf1 Ac ' inoculu~ plates. The PA plates received 2 DSA and one DBA cylinder. The CMA plates received 2 DBA and one DSA cylinder. This rather large inoculum including a portion of the substrate to be fermented was intended to be analogous to the procedures of the fermentation process to be derived from the culture screen results. Each test plate was read once daily after inoculation. Colony radial growth extent and rate mm/hr! were read for three radii on each plate. Each of the radii bisected a sector which contained one of the inoculum cylinders. Plates were read untiJ growth of 2 of the 3 sectors reached the edge of the plate, or until growth rate of the 3 sectors slowed by a total of O.l mm/hr over a 24-hour period. When this occurred, the plates were first stained with trypan blue in lactophenol, then flooded with a killing and fixing agent FAA: ethyl alcohol 95%! - 50%, glacial acetic acid 5%, f ormaldehyde 0%! - J0%, water - 35% Sass, l958!!. The stained and fixed plates were used to make a determination of biomassof fungusproduced. A cylinderof agar wasremoved from 5 rnmbehind the leadingedge of each of the 3 sectors on each plate. A 2 mrn thick slice was taken from the top of each cylinder and placed on a microscope slide. An eyepiece with photographic reticle Wild! having a transecting line was used to measure hyphal density. The eyepiece line was positioned so that it was perpendicular to the majority of hyphae in the field, and ali surface hyphae crossing the line were then counted and recorded as hypha~per mm. Cover slips were then placedon the slices and the diametersof 5 randomly selected hyphae from each of the three sectors were measured. The 15 hyphaldiameters and 3 densitieswere averagedto yield valuesof each for each of the test plates for each fungus. iJsing these two rneasurernents,an indicator was calculated of the fungal biomass produced, sirnp1y by multiplying mean hyphal density by mean hyphal diameter for both control PA! and test media, and subtracting the value for PA from that of each of the test media. This is given as biomassindex B1!in Table 14for those !'ewfungi for which analysiswas completed, alongside an indicator of potential ~ jiiitv.| in ths f~rmeetatinn nrnr e cs ty,gq,PqygJpped UI. = B1x maximumerqp$grqg!. stationary phaseis also given in Table 14, but not included in Growth extent at earl idices although it might logically have been! for this was the the calculation of the .nedof the growth parameters measured. least accurately deteri Results. >r which completeanalyses were conducted Table 14!,the 2 i!f the 15 fungi iC 14 and 134! and Chaetomiurn sp. SC 6 gave the highest Bl's species of Drechslera 578 respectively!,and Chaetomiumsp. SC 6 gave by far the on DSA 28, 658, ani Surprisingly, only 4 of the 15 fungi produced higher 11 on highest Bl 29! on D.' these, marked increaseswere shown by Chaetomium sp. SC 6, DSA than on NDSA. C iC 125, and Trichoderma viride SC 51. This is likely to have ~Pcno rus 'neu 65 Table 14. Results of a culture screen designed to find strains of fungi capable of efficiently converting "delignified" alkali-treated! wheat straw and bagasse into fungal biomass. KlX. GROWTH MEAN MEAN GROWTH EXTENT HYPHAL HYPHAL FUNGI Sb BSTRATE RATEc CLASSd DENSITYe DIAM.f BIg UI PA 0.17 9.0 2.1 0.34 58.0 3,4 197 67 SC 6 0.40 74.5 4.1 305 122 DS 0.40 121.9 5,4 658 263 0. 34 36.6 2.7 98 33 DL 0.4l 91.3 6.9 629 257 PA 0. 29 11. 4 2.1 0.37 58.3 2.6 128 47 SC 8 U. 35 42.6 2.8 95 33 DS 0.33 18.6 1.5 0. 31 25. 2 1.8 21 DB 0. 31 12.6 2.0 PA 0. 31 9.3 3.1 0. 35 70.9 4.1 262 92 SC 14 0.31 305.1 4.2 1253 388 DS 0.33 302.7 2.5 728 240 0. 37 36.6 4.5 136 50 DB 0.30 87.1 3.6 284 85 PA 0 ' 25 23. 4 2.2 0.36 78. 1 3.4 214 77 SC 20 0.37 45,6 3.3 99 37 DS 0.28 43.2 2.1 39 0.34 22.8 3.4 26 DB 0.30 3A 30.6 2.3 66 'i able 14. continued! MAX . GROWTH MEAN MEAN GROWTH EXTENT HYPHAL HYPHAL PUNGI SJBSTRATE RATE CLASS DENSITY DIAM. BI~ UI PA 0.61 8.7 4.5 0.83 74.5 5.2 289 SC 51 0.79 58.9 4 ~9 249 197 DS 0.80 106.3 3.9 375 300 0.71 20 ' 4 3.7 26 0.75 3* 82.3 315 236 PA 0,34 5.7 3.7 0. 32 27.0 3.5 73 23 SC 77 NDS 0.34 96. 7 4.2 385 131 DS 0. 36 53.1 4.3 207 75 0.35 35.4 4.5 138 48 DB 0. 30 33. 6 3.5 97 29 PA 0. 25 5.3 2.4 0.50 3* 18.6 4.8 77 38 sc 90 0.44 30.0 3.7 DS 0.35 20.4 2.5 38 13 NDB 0.31 6.3 3.5 DB 0. 32 13. 5 3.3 32 10 PA 0.37 58.9 4.9 0.39 120.1 4.2 216 84 sc 98 0.43 77.5 5.1 107 46 5 '! DS 0.41 97. 3 3.5 21 0.37 93. 7 3.3 0.38 141. 2 3.2 163 62 67 Table 14. continued! fAX. GROWTH MEAN MEAN GROWTH EXTENT HYPHAL HYPHAL FUNGla SUBSTRATE RATEc CLASS DENSITYe DIAiM.~ BIS UI PA 0.18 24. 9 1,6 0.24 38.7 2.3 12 SC 100 NDS 0. 30 9l. 9 2.1 153 DS Lost 15.6 1.5 NDB 0.24 33.6 1.7 17 DB 0.14 20.7 1.4 Q. 06 9.0 2.3 0.19 39.9 2.7 87 17 SC 101 0.45 75.7 4.9 350 158 0.43 51.6 4.5 212 91 0.41 27. C 5.4 125 0.45 54 ~ 7 3.9 193 87 0,19 17.4 2.1 0.19 91.3 128 SC 114 NDS 0. 25 94. 6 1.9 143 36 DS 0.22 104.5 2.0 172 38 NDB 0.21 22.8 3.4 DB 0.19 69.7 3.7 221 42 PA 0. 28 9.9 5.7 0. 13 60.7 4.0 186 SC 122 0.30 111.7 4.6 457 137 DS 0. 28 48.6 3.8 128 36 0. 35 36.3 4.7 114 40 DB 0. 29 28. 8 3.5 44 13 Table 14. continued! MAX. GROWTH HEAN MEAN GROWTH EXTENT HYPHAL HYPHAL FUNGI SUBSTRATE RATE GLASS DENSITY DIM. BIg 0.22 70.2 3 2 0.26 303.9 2.8 626 163 SC 125 0.25 193.4 2.5 259 0.25 217.4 2.9 406 101 iNDB 0.21 91.0 3.5 94 20 0. 24 164. 6 2.5 187 45 PA 0.54 25.5 2.1 0.40 69. 7 2.6 128 51 Sc 133 0.35 94.9 2.2 155 DS 0.38 80.5 2.0 107 41 0. 36 34.8 1.9 13 DB G.38 54.7 1.3 18 PA G.20 9.9 2.6 0.20 73.0 4.5 303 61 SC 134 iNDS 0.31 158.6 6.3 973 302 DS 0.30 133.3 4.6 587 176 NDB 0.26 7.8 2.3 DB 0.25 45.1 3.7141 35 Fungal strains listed by accession culture! number; SC 6 Chaetomium sp.; SC 8 ~Scoulario~sis ?! sp.; SC 14 Drechslera sp.; SC 20--~tthia sp.; SC 51 prichoderma SC 98 Pasta lotia sp.; SC 100 ~Soxormia sp.; SC 101 Chaetomium sp.; SC 134 Drechslera spe 69 'lable 14. continued! PA plain agar control!; CMA cornmealagar Difco!; NDS non-delignified straw; DS ~ delignified straw; NDB non-delignif ied bagasse; DB delignif ied bagasse. All media 2.0X agar Difco! except CMA!, 3.25K NH4NG3,0.09X KH2P04,0.01X yeast extract Difco!, 2.9Xtest substrate except CMA!, in 15 o/ao seawater. Maximumgrowth diametric extension! rate observed over a 24-hour period, in mm/hr. 0 0-10 mm; 1 = 10-20 mm; 2 20-30 mm! 3 ~ 30-40 mm; 3* rate of growth not declining when growing edge had reached plates edge. Places were flooded with fixative FAA! whengrowth rate declined by an average of 0.03 mm/hr or more over a 24-hour period. Across a 1 mm-long transect made perpendicular to the growing hyphae 5 mmfrom the growing edge. In pm, average of fifteen randomly-selected hyphae. BI biomass index mean hyphal diameter MHDI! on teat substrate x meanhyphal density MHDE!on test substrate! MHDIon plain agar x MHDEon plain agar!. UI ~ apparent utility index BI x maximumgrowth rate. 70 been an effect of excessive alkali treatment and thorough washing having removed soluble nutrients, including otherwise available hemicellulose %'ilson and Pigden, l964; Garcia-Martinez et al., J974!which may have positive effects on cellulolysis and growth Basu and Ghose,1960; Nilsson, l974!. Of the 11fungi which produced higher Bl on NDSA than on DSA, the highest values and most marked differences were shown by the Drechslera spp. SC L4and l34! and Alternaria ~ton'ssima SC l22. To the contrary, ll of the l5 fungi producedhigher Bl on DBA than on NDBA, highest values and most marked differences being shown by Chaetomiurn sp. SC 6, Trichoderma viride SC 51, and ~Aser 'llus terreus SC litt. Of the four which did not, only Alternaria ~fon'ssima SC l22 produceda markedly higher Bl on NDBA than on DBA, and the other three had Low BI's on both media. Thirteen of the L5 fungi had higher Bl valueson the straw agars than on the corresponding bagasseagars. Of the two whichwere otherwise, Pestalotia sp. SC98 hadhighest BI on CMA, and ~Aser 'llus terreus SC lftr was the only test funguswith highestgl on DBA. Maximal radial growth rates were much Jessevidently affected by the test substrates than hyphal diameter and especially hyphal density, so that within a fungal test group, UI valuesreflect Bl values. However,because of its very rapid growth rate approximately0.75 mm/hr, 24-hr mean,on all media except PA!, Trichoderma viride SC 5l producedthe highestUl on DSA 00!, followed by Chaetomiumsp. SC6: 263, and Drechslera sp. SC l4: 240, and was secondonly to Chaetomiurn sp. SC 6 on DBA SC 6: 257, SC 5L: 236; next highest was 87 for Chaetomiumsp. SC JOL!. HighestUl on NDSAwas 388 Drechslerasp. SCl4! followed by Drechslerasp. SCJ34 at 302, and the next highest was Chaetomiurnsp. SC JOlat l58. The values of Ul and Bl for CMA relative to these values for the other test substratesvaried amongthe fungi tested. Somefungi exhibited highest values on CMA fe.g. ~pennprus ~sanuineus SC l25 and Pestalotiasp. SC 9g!, while others 71 exhibited valueson CMA as low or lower than thoseon NDB, which was, in general, SC» dCI i .S DISCUSSION ll. Fun al fermentation of wheat bran Wheat bran was not chosen as a substrate for our initial fungal fermentations becauseot its potentialfor scaled-upcommercial production of feed. Its cost is substantiallyhigher timesor more!than that of the nearlyentirely lignocellulosic agriculturalby-products since Jts relativelyhigh soluble carbohydrate 3%! and protein J7%!content give it valueas a feedfraction and bakery product. Rather, we choseit because,of the four plant materialstried as artificiaJdetrital materialsby Cailiouet et aJ. J975b;Section 5!, it wasthe only one which: ! supportedmarked growthof shrimpwhen fed without treatment Caillouet et al., 1973,1975a, b!; and, - '-mv<'~zN.JI Zknlpvixt~m.-eke@ mrmvth~44~iim mtgoer~edfepqmtation-.. We chosewheat bran in order to determineon a preliminary basis: ! whether tb conceptof feedingof fungalfermented materials was a practicableone; and whetherthe yield of shrimpproducible with untreatedwheat bran could be improved by fermentation,beyond the sizesatisfactory for a commercialbait-fishery concern. Our culture screen results indicate the extent to which the protein content o wheat bran can be changedby fungal fermentation under the specific condition imposed! Tables 2 and3!. Conditionsof the two screensdiffered in severaJrespect Section6!, but perhapsmost importantlyin that phosphateconcentration an especiallyinorganic nitrogen concentrationwere markedlyhigher in the secon screen. Probablyas a consequence,the percentageof nitrogen in the product wa pushedhigher in the secondscreen Fig. 1!; the mutabilityof nitrogencontent c fungalmycelium as influenced by theC/N ratio of thegrowth medium is well know e.g.Cowling, 1970; Lilly, 1965;Section 3!. It is probablyfair to suggestthat th 72 highernitrogen contents reflect increases in true protein content, for Leviand Cowling l969! and Graham l97l! found that decrease in the C/N ratio of growth mediumleads to increasein non-nucleicnitrogen of myceliurn,but doesnot resultin "arrr,acres' aikido mr ng'~ahaL~==w~e.nc a~mn~ia There was a lesser effect on the percentagechange in crude protein; the " grates-frac~ os' ooaho figures derivedby one methodto those derivedby anotherare impossible. ! Differing methods of presentation of results have been used biomass/carbohydrate utilized, protein/carbohydrate utilized, protein/volume of fermentation Jiquid, etc.!. ! Although wheat bran is a cornrnongrowth rnediurn usedin the production of fungal enzymes Codner, l969; Ghandi et al., l974; Underhofler et al., 1947;Wang et al., J974! we kiiow of onJy one very recent report of its use for the production of fungal biomass Von Hofsten, J974!. With regard to item !, it is perhapsbest in our particular case to use total nitrogen x 6.25, since the major part of the error in calculating true protein from total nitrogen lies in the contribution of chitin nitrogen. At least a portion of the chitin may be nutr itively valuable to shrimp Section 4!. Results of several studies of protein production by fungal fermentation of largely-starch or simple-sugar substrates are given in Table 15. When substrates such as these are utilized for production of fungaJ biomass, and only the resultant iIiilriiiIiiiiiiiriirrrirrrlrrrriiriliTiiiiiiriirrrrrrriAiriirrrrjitilillIlririirjrrtrrisjgjiiiissgliiiisrnInisilfil ~y~g]iiqg ~yp~l~teg the ~iir4 ~r~t~i~ wnrttanf nf,t+ ~spiv t~~g lian8eltiliiIIiIIljiiIIilOlsl ~>go, gh isil since included in the fermentation product, the upper end of the range is lower 8 !n of non-nitrogenous, unfermented plant structural materials some cornbir if our cellulose, hemicellulose,lignin, pectin, residual starch! remain. The resul s, are wheat bran fermentations, the products of which include undigested bran fl other much the same in terms of range of crude protein content as those usi Table substraies in particulate suspension. The values for protein production giver n and l5 are dependent upon concentration w/v! of substrate used in the ferment .rn to upon ability of the fermenting microorganism s! to efficiently convert plurne microbial biomass at those concentrations. Thus, they are related tc s/unit production efficiency biomass produced/unit volume of fermentation rr time!, though the figures in Table 15do not include growth-rate information. other The wheat bran values and probably the value f or most of t 74 O 43 N ch N Pl 0 P4 m + lA + + + + + + + + + CQ 0c CO Ch QD 0i O N ~ O 4 cw 0 Ã N co N I g1 cd 44 O I I N cd ck 0 k ~ ~ 0 d4 Coo N Cl O cd 0 4 jj 44 cd cd N + H O vl Q cd cc 9 4 Pl Pl Pl Pl 0 tJ 0 G dl 0 VI 44 U P '0 ccl N clJ 0 N N N N C4 0 g 44 dl QJ 0 4 cd Cc k qj Oa dj cd g Ql dI 00 0 g dc k IU k O W d4 'cd N CV d4 dl W 44 C cd CII 44 X Qj 44 dj k cd w ccd 0 5 C co 0 co I 4-I gj 44 cd I Qa44 W O OQ V4g O ccj ch Cfi ~ g g O N 0! jc a 0 dc Cdj ~ I 44 A a ccj cd cQ lJ cd cd cd cQ ~ dl a k cd dl QI 0fL ccj M 4 cd cd CC4 cd Q cd cd dj Il dl 0 0 cd cd 44 44 44 CII s r4 U U A dl dl cd 0cj dj g W dl cd cd cd dl cd 0 dl cd 0 dl cd 4 00 fj co DO cd '00d4 N '00dj P4 I a 44 I I I Icd r-I dI I r w! O n 0 A Vdj S cd dl cd A dl 'rt 44' 44} dl 0 Dd g 44 cd W 0 jj G cd m cd 'Cj 0 rd 0cd k 0 cd jj o A cd '0 44 0 cd cd 0 cd k 4 cd 4 k k ccl ccj O CCI D U 75 cd W 0 c» W 0 0 0 Ql QI 8 JJ 0 9 0 QI JJ 0C4 th JJ I W ~e W JJ 0 bD Ql 4J ql qj 5 0 0 W O Ql V '0 Ql Ql QI 4J e 0 JJ qj qj C 0 4J 4J O JJ 0 Ql qj V C4 0 II O Ql 4 0 qj 0Ql '0 0 Ql C JJ e 4 Ql QI W Qj qj 0 Ql V Qj JJ u 0 O Ql 0 Ql S 04 CL JJ dj g 0 qj QJ Ql S4 e W C 0 0 jd jmQj Ql 4J 0 JJ 8 Cl W CQ JJ e JJ JJ 0 W aj Ql JJ 0 Q Cl a aj JJ Ql O 0 JJ e qj 8 0 g3 JJ m 8 Ql Qj O JJ WQI Ql JJ QI Qj Qj g C O4PI 0 Ql H jd sl 0 Ql V ql Ql e qj 0 0 4J JJ 'rl Qj qj 0 00 Ql 0 V C4 0 '0 Ql '0 O C JJ Ql Ql QI cd JJ 4J JJ 8e D0Ql Cl JJ Cl JJ JJ Cl 00 ~ jdW WO W 0 0 4J Ql 'rl Ql 0 0 Rj M c4 W 76 particulate-suspensionf ermentationsin Table 15! are likely to include protein remainingor convertedfrom original substrateprotein in addition to that produced from ~norganicnitrogen. Therefore,production of protein over quantitiespreviously prese it is given in parentheses. Since someof the original protein is undoubtedly lost from the solid phaseduring the fermentations Section8!, andthis wasnot measured, the ai tual protein production values are not known. The highest values fc-. fungal protein production over that originally present in wheat bran are as high as the highestof the other valuesof protein productiongiven in Table15, except for thoseof Bednarskiet al. 971!. Highervalues not yet reportedmay well havebeen achieved in the r'cent extensiveresearch projects of Readeet al, l974!, Solomons 975!, and Worgan report duefor publicationin 3. Sci. Fd. Agric.!. lhe protein productionfigures of Bednarskiet al. in Table 15are an order of magnitudehigher than mostof the othersgiven. Prefermentationof milk wheyby bacte;.ia,followed by mold fermentation, is reported to have been the reasonfor these high values,but confirmation of these results is needed: the concentrationof easily fermentable sugarsand conversionefficiencies necessaryto have achieved these results would have to have been quite high; the production results of El-Akher et al. 974!, workingwith yeastfermentations of milk whey,do not approachthose of Bednarskiet al., washingof the fermentationproduct is not reportedby Bednarskiet l2. Fff ect of simultaneous modif ication of fermentation conditions Comparisonof the crude protein contentsand especiallyprotein production valuesfor the four phasesof the presentproject shownin Table 15give evidenceof .k"~~".N'og. fe,.eh'r4 a!ME 4'. u of fermwot&i~mrtifia~~~ao..improve,pcodhK+i.on performance.The high crude protein contents reported by Falangheet al. 960!, Grah-m 97l!, andSolomons 973! werealso achieved by this means.Graham 971! and Pinto 963! amongothers had demonstratedthe remarkableextent to which inorgariicnitrogen source and nitrogen content of fermentationsubstrate could affect 77 proteiii content and ainino acid balance of fungal biomass. Pinto's work clearly demon peated tow fermei ted wheat bran. Gray et al. l963! and 3ones and Irvine l97l! had shown that salinit> of the fermentation medium could have marked effects on myce!ial and protei i yield, so we examined this phenomenon. Finally, we examined the effects of conceiitration of substrate, which is one of the most direct determinants of volume productionefficiency Sectionll!, and thereby one of the most important factors in econor,iic evaluation of any developmental fermentation process Caliihan and Dewlap, l97l; Gray, l97l!. She results of our comparison of nitrogen sources with regard to their effect,venessin stimulating protein production Section 8! reflect the findings of other investigatorswho have examinedthe questionof optimal nitrogen sourcefor fungalf ermentations Table 16!. Interaction is commonamong the effects of type of nitrog=n source and several other variables type of carbon source, concentration of carbon source, medium pH and medium buffering, other medium constituents, and, especiJly, species of fungus used!, Also, optimal nitrogen source may be different dependingupon the goal of the developersof the fermentation process i.e. whether optimized nitrogen percentage of final product, optimal protein production, or optim:0 biomass production is desired!. It is interesting to note, in the case of two a O a a O M R Mld N dI a dl A O gl a RO O OR 4J N R~ N ~R R AA V Fl N O 4 O U O O O O O CQ M O O N A A N ~ A A A 140 N N O O N a a a a O ~! ~9 O O Fi O N N 4 Cl RR R-, gj A 11 A A A Z N +g. < O W O A A A O 0 G O F4 Q O O Q O O Cfl Vl Ch K O N N A N N N N N N A g A A a o 0 qf I~ td 4Ql 4 V g dI 0 V dl 0 4 M QOi VdI 1d V4 V V 1ll dl 0dd V4 0 Cl 4 rd cd c5 0 4J dl I LI V V I UI dI ld 0 0 dI 0 0 0 1d 0dd V I-1 LI Qc bd 0V 0rd 4 ld Cd VI 0 0 0 0 0Vdd i4 Qc C4 ! 0! 0 I I I 4a C4 . 0 a a O O a 4 dl gl QI JJ IN nl 4J Ae I a ql 0 4J 0 A '4 dl ~9 V! O Q cJ! O CV 79 %R A A O O A O O O O O a O 44jIN O A A VCV 0C4 4 N RIe A O CV O O A Ojl OO 044 EV IN N . 4 4J A /II g O CZ * II 0 RR A O u O Yl A A A 0 0 N 'K ?- 0 O O Y> Z 0 hl g A II R O A .r A clj O O C 6 O V 444 O O O I A O Z g O OU A N hl CV ccl hl Pl A N A A O O c~~ 0 IN N R N O Oclj R clj 0 R y. 4J 44II ~6 ccl 4J 4J apl clj 4J JJ 4III Ct 4j ljl 1 Ijj 4J JJ 0 444 0 444 JJ 0 444 W0 clj 0N 0 4J 444 '0cljcl A 0 Qe 44j 4J 0 0 4l 4J 0 Cl 0 ccj 444 0c JJ 0 cb '04 0 '0 ~ R g 444 4II 444 0 44 4J I 0 0 gl ce 4 0 5 IJ 0 'lljr I 4J CJ W 0 0 I 0 ccj d III W U 0I JJ 0 0 clj N0 $ II44 0 Cl 44II A 0 I I 0 4 '044j 444 cll R I-"I 44 44j ii U ~4 H0 Ch ~ 4 04444 A cfi 89 that although nitrogen source had a marked effect on final percentage of nitrogen in the fermented product, there was no detectable effect upon the a/solute change in crude protein, meaning that the deficiencies in production of a highly nitrogenous product were balanced by difference in mycelial biomass produced or original material lost. Clearly it is of little value to determine optimal nitrogen source for fungal fermentations without examining simultaneously the effects of other potentially interacting variables. However, two generalities which appear upon examination of the references cited in Table l6 are: I! as 3ones and Irvine l97l! point out, when urea, NH2 CO,has been tested, it hasoften performed as well as or bet~=rthan other simplenitrogen sources; ! as Nicholas'review 965! and Sgurosand Simrns' J963! work show, when ammonium salts are to be used, it is advisable to use buffered media. The present study did not include examination of applicability of these generalities to wheat bran fermentations~ it is quite possible that higher levels of protein production might have been achieved had urea been usedas a nitrogen source, or had better buffered media been used with the ammonium salts used. Final percentages of nitrogen and crude protein yields of the present experimentappeared to dependmore heavilyon type of nitrogen sourcethan on C/N concentration, when the lower C/N gave markedly higher finaJ nitrogen percentage and crude protein increment. That C/N had no effect on final nitrogen percentage of the Lulworthia products was to be expected, since this fungus had shown nearly equivalent results in the two culture screens, which had utilized media v.:ith much different C/N ratios f in culture screen I and JVin culture screen ll!. The two C/N ratios used in our experiment Jb and 28, considering only ammonium nitrogen see Section 8; these values fall to about 7.5 and 10 when the bran nitrogen is included!lie in or near the range -25! given by Litchfield J968!as 81 optirr al for fungi in general with regard to fungai protein production. The C/N ratio must '.>einterpreted with caution in media including complex substrates such as wheat bran, especiallywhen ammonium and nitrate ions are present;not all of the organic carbcn Von Hofsten and Von Hofsten, 1970!or nitrogen of the wheat bran is available to fi.ngi, and ammonium ion uptake depresses or prevents nitrate ior uptake Nicholas, t965!, so that the ratio of available carbon to available nitrogen is not known but is certainly different from the ratio of carbon Dresent to nitrogen nresent. rrrrrrrrrrriirrrirrrrrirrri!rrrrrIrriirrriiirririiriiirriiiiiiiiiiiiiiirIIiiriiririirtrlmrrr!mmrrrrmmrrrrrrrrrrrrrrrrrrrrrririrrr!regara tO Salinity'S eff.eCtSOn prOtein rOdQQtianar e in !line ~urI!! ixinaingsIrrrr!rrrrrrrr u ! ii!!!ii!i! i!! jrrIr, I!!Ir!!j!Lrklllgj,II!IIIIII[lj!j jII!II!IIIIIIIIIII IIIIII IIIIII I IIII IIII IIII IIII III I IIII IIII IIII IIII IHI lI I III III !!H HH! iiili!'iiii!iijiii!iiiiii iI'iI" i"iNiiI iijj it in both studies, the obligately marine fungi showed the interesting to note stein production as salinity decreased from 30 o/oo to about 5 clear" st increases ir :em was clearer than the one observed with type of nitrogen o/oo. This general, here too, salinity had significant interacting effects with the sourc.. Section 8!, I es in five of the eight possiblecases, considering b ith final other treatment var I crude protein increment!. nitrogen percentage i were detected for the modified concentrations of wheat bran Signif icant eff ~Ptof eight!, and significant interaction was detected in seven in every tested case y general conclusion which can be reached, and this applies to of these cases. The nt variables examined, is that in experimentation de igned to all three of the trea editions for a given developmental fermentation, ine must deter!mine optimum opendencies exist among conditions which can be varied, and deter mine where int .rn together, as many at a time as is practicable, if one is to syste natically alter a production levels for given fungal strains. For example, if discover optimal pn we h id examined th source at 296 bran and 30 o/oo salinity, we would have varying only nitrog SO salinityat 2%bran with NH< SO<, we would conclude that 30 o/oo permitted best perfarr.ance. Finally, varying bran concentration at 30 o/oo and with NH< SO<,we would -onclude that bran concentration had little eff ect, and miss the peak perforn.ance at 896bran with NH NO3and 5 o/oosalinityl If apparatusfor continuousculture is available,the recently reportedmethod of media 'ptimization of Matelesand Battat 097%!might be adaptableto improvement of fung!I fermentations. 13. F edin of fermented wheat bran to enaeid shrim ~ ~.<~der!ee2~d r! at. W: +.-..-edstdxva..es ~~0,~".ki«en.Ansz'eao=! Ps~hqh.w+aot~~l i i e~g4 g~Q~> IIII 5 eke !IJNNI98l!'Illgllk"... '...... agONC" >-.0. I Central and northern South American countries where commercial mariculture developmentis now centered %'ebber,1975!; ! wheat bran is composedof both easily! .rmentablematerial starch andprotein! andhighly refractile material {native cellulos,'cs! Caillouet et al., 1975; Yon Hofsten and Von Hofsten, 1974!. Caillouet et a.r ~pm-.= ~pVaxerna.-===.k!any«r.=-'a! snM"L@e"" .~-5 8 m'-'k'=orachy4 dna'm oofyWanrl 11%"fit er," but the Von Hofstens report a 5696cellulose + hemicellulose content. The diff ere!!ceprobably lies in the excessivesolubilization of hemicelluloseswhich takes placei i proximateanalyses {Von Soest, l969!. In any case,some portion of the cellulosic material is probably potentially available to some fungi, i.e., not so heavily lignified that fungal enzymescannot degradeit. Yet even this portion is probably protect.d to someextent by the high starch and protein content of wheatbran, since f ungal<.ellulolysis is of ten inhibited by the presence!f more easily degradablecarbon 83 sources Basu and Chose, 1960; Bravery, l968; Hueck and Hazeu, l969; King and /veins l973, Zeltip. 3970;..andeypeciailv Ta~ulatov and Teslinoya. I97rr!. Item ! f rom the above paragraph has an important bearing on interpretation of the results of our shrimp-feeding tests. Becauseof the relatively high starch wheat starch was shown by Forster and Gabbott l97l! to be very effectively assimilated by prawns! and protein content, wheat bran, fed directly to penaeid shrimp with no treatment whatever, will support the growth of shrimp to a size and at a rate satisfactory for a commercial bait fishery Caiiiouet et al., l973, 1975a, b!. Performance of wheat bran, in terms of yield of shrimp produced, was often only slightly lower in both Cailtouet's experiments and in the present ex;-eriments,than commercial or developmental commercial! pelleted or flake feeds Glencoe peBets, Q 17&M~ - J+k.rh, g~U~ - 6 Era+aa.@tpwumrnfz ' ~ INa~ = 8~m = 20! In =~miUaee-+.'s experiments, yields with wheat bran ranged from 69 to I00% of yields with commercial feeds. Any fermentation of the bran will necessarily reduce the available energyper unit of original starting material Yon Mofsten,l975!, and since much of t k Jjpaif ~/ ~m]Gibson mad, hrmr~lbdaa" msilI ant ~ ~~rrrvm"~ ul it4mt~mtvwxtrrt~t severalreports in Could,l969; Rogersand Spino,l973!, this fraction will be higherin the fermented product than in untreated bran. The cellulosic fraction of the bran is no more available as nutritive matter to penaeid shrimp than it is to the fungi. Cellulosehas beenused in shrimpfeeds as an inert fiBer-e.g. Andrewset al. l972!; Forster and Gabbott l97l! found that calories of even a finely ground purified ceBulose pere assimilated by prawns to only a small extent. Therefore, on a unit dry weight basis, fermented bran will have a lower quantity of nutritively valuable material and available energy than untreated bran. Since the feeding treatments used in our feeding studies were applied at equivalent rates of dry weight per day, the anirrrals receiving fermented bran were receiving less nutritive material than those receiving untreated bran. Our I&liter fermentations of wheat bran Table 0! were conducted largely fez. production of m aterial for f eeding to shrimp, as was the case in the paper fermentation and chick-feeding study of Crawford et al. l973!; also in parallel with their work, the large-scale fermentations yielded lower quantities of protein per unit weight of original substratein most cases,than had similar small flask! scale fermentations, due to greater loss of dry weight with respect to the original. Loss of efficacy in yields of fermentations due to scaling up is common and to be expected in developmental-studiesof this kind Gray and Abou-EJ-Seoud,1966a; Hishinuma et al., l972; Solomons,1969!. It is due to subtle differences in preparation and sterilization of media, in preparation of inoculurn, and in oxygenation and other transport phenomena Solomons, I972!. Systematicanalysis of the variablesinvolved in the Ib- liter fermentations was not conducted and more than one variable was often changed from run to rm. Therefore, reasonsfar scale-up problems were not pinpointed. It is important to note that where crude protein producedwas less than in small-scale fermentations, andmore of the original weight waslost to respired gas on solubili- zation, the remaining product would contain a greater fraction of the cellulosic material not nutritively valuable to penaeid shrimp. The results of our feeding experiments exhibit the lack of replicability among feeding treatments which is to be expected when experimentsare conductedin outdoor facilities under semi-natural conditions. Under these, conditions, interacting. uncontrolled variables such as development of microbial and meiofaunal populations, weather effects, development of parasite and pathogen problems, etc., are not preventedfrom imposingmarked and differing effects on shrimpgrowth. Problemsof this sort are discernible in the results presented for other studies.of this kind, for example,by Caillouet et al. l973! andParker and Holcomb l970!,.andZein-Eldin and Meyers 910! encounteredand discussthe problem. Until these natural variable factors can be better controlled, it is the higher levels of productior achieved which should be noted, for these represent indications of levels attainable when some measure of control over experimental variables is accomplished; for this reason, we did not combine means for replications of treatments in our feeding experiments. The range of responses to fungal-fermented feeds in our experiments ranged from very poor yields of shrimp due to a possible effect of toxicity, to slightly higher yields than unfermented bran. The yields using cold-stored Trichoderma viride material seemed to indicate toxicity, for they were lower than yields when no feed was supplied, due to heavier mortality. Cases of toxicity have been reported for T. viride e.g. Mirocha and Christensen, l974!, but so have casesof goociperformance of T. vinde mycelium as feed material e.g. Church et al., l972!. Improper refrigeration certainly could have been the factor responsible for the poor performance of this particular feed; when the T. viride material was dried, excessive mortalities did not occur. The dried T. viride product gave poor shrimp yields relative to unfermented bran feed efficiency > 20! except when combined with commercial flake material 90% + l0%, respectively!, but best feed efficiency was still only 0.< that of '*' ' P and Curvularia sp., aiso gave feed efficiencies greater than 20. It is quite possible that the drying process used was at least in part responsible for the low nutritive capacity of these products, Bauer-Staeband Bouvard l973! found that heating and drying had very marked negative effects on the availability of yeast-cell proteins to digestive enzymes,and Spicer l973! points out that excessiveheat processingcan reduce the availability of amino acids of fungal protein. Bressani l968! and Kihlberg l972! discuss the problem of processing of single-cell protein, they point out that treatments such as heating and drying can impair nutritive capacity by reduction of amino acid availability and/or loss of functional quality e.g. vater absorption capacity!. 8'hrlocherand Kendrick 973! foundthat detritivorousamphipods wouM not acceptdried fungal mycelium,though fresh rnyceliurnwas avidly eaten. After harvest of F74C, which utilized large and easily dissected shrimp, contentsof the foregut and hindgutwere examined. It wasobserved that the fibrous bran flakes were passingthrough the shrimp largely undigested,and that fungal material which had been dried onto the flakes was also passing through undigested productwas refrigerated rather than dried andspoilage did not take place!,a markedimprovement in performancein termsof maximalshrimp yield wasobserved Table ll, treatments 9 and 10!. Drying of the products is, of course, much more desirablethan cold storage in terms of developmentof an economicallydesirable product,and further experimentationmay demonstrate that othermethods of drying, or dryingof someother developmentalfungal feeds, do not detractfrom nutritive .a@a~I.P~. Desired:4 "- irqpnmd the rJitti.ti e .vaijceaf sirg1e-x «~P averagefinal freshweight per shrimp which were only 0.8 those of the maximalyield of untreated bran of F748. If we consider the question of relative quantities of nutritive value between the fermented and unfermented bran in this case, using conservative calculation of conversion of wheat bran to mycelial biomass, we find that unfermented bran contains 1.3 1.5 times as much nutritive material as fermented bran on a dry weight basis. 1n making these calculations,it is assumed that the portion of the wheat bran left unconvertedby the fungus was also unavailableto the shrimp see above,this section! and that the portion of the bran which was accessibleto the fungus was convertedat 40-50%efficiency. If these speculativecalculations are fair approximations,then the fungalproduct may have actually given higher feed-conversion efficiency than the unfermented product, based on weight of nutritive rnatter added. Experiments involving feeding of purely fungal materials no residual unfermentedrnatter! or with more efficiently converted substrates less residual unfermentedrnatter! would be necessaryto verify t,'lis conjecture. In order to partially correct the differences in quantity of nutritive matter amongfermented and unfermentedbran feed in F70C and F70D, we appliedfeeding treatments of 50% fungal material and 50% unfermented bran. We did so for the further reason that plant starch has been found to be a valuable component of artificial penaeid diets Andrews and Sick, 1973!, and wheat bran starch and other nutritive components, as discussed above, obviously serve well in this capacity. The result of this mixing of fermented and unfermented feeds in F7QCwas to improve the maximal yield of shrimp, using a Pestalotia product, from 0.7 that of unfermented bran to slightly better than equivalency Table l2!. If half the smaller end of the correction-factor range l.3! discussedin the above paragraph is applied to this yield, . ~ee6 off:r'rm~z >mern. r:-aaa«y ~! Zmvrhat. harL i eewc~Wee="~~v-'" .+he=Q niai ~ Experimental Marine Ration 20 in F7rrA. Feed efficiency was slightly lower in F7r!C than in F7%A for unfermented bran, indicating that the Pestalotia + unfermented bran feeding treatment performed on a par with the Purina product, on the basis of nutritive matter fed assuming that the ratio of shrimp yield from unfermented bran to. shrimp yield from the Purina product would have been the same in F74C as it was in F70 A!. The results of F740 demonstrate the marked effect which temperature {and salinity?! can have upon shrimp growth Zein-Eldin and Griffith, l969!: feed efficiency for wheat bran rose to twice its previous low value, and mortalities were, in general, higher than in F74A and F70C. Yield values for wheat bran and the Purina product were much lower than in previous experiments, and this was not enti-ely due to the lower stocking density, for average final fresh weights were also distinctly product both performed less well than wheat bran in terms of shrimp yield, th~ ugh the M th ' + unfermented bran product produced a yield which was only 4% less than the yieM from unfermented bran. Jn addition to the ambient temperature experiment and probably further obscured real differences between this ai d other feeding treatments. 'Jnder the conditions of this experiment, particle size in the range tested 0 - 60 mesh! had no evident effect on shrimp yield from dried Tr ichoderm a material. The shrimp yieMs of our F74 series of feeding tests are low and he feed efficiencies high when compared with some of the results of other workers who have investigated growth of penaeids on artificial diets Table l7!. They are, however, comparable to the findings of Caillouet et al. J975b!deveJoped in the same concrete tanks as usedin the present study. That they are a bit lower than Caillouet's findings is probably due to the fact that Caillouet used inorganic fertilizer in combination with his wheat bran feeding treatments, and we did not do so. There are too basic reasonsfor the Jow yields: I! among the penaeid shrimp, Penaeusduorarum, used in F74A, 8, and D, is one of the least attractive species in terms of growth potential under the conditions imposed by a mariculture faciJity Broom, 1973; Parker and Holcomb, 1914; compare with Forster and Beard, 1970!; ! the facilities and methodology used in outgrowth of shrimp at Turkey Point have consistently brought about Jowyields whencompared with other experimentalfacilities see Krantz and I976,'l ~~jg r~ iiltc fear thos@ r~~cnnc,pod.qt~q.pynqrj~eptyl AiffjpiLitjac~pg lnequivocally that fungal feeds can or should be faults discussed above, do not state ither, they suggest strongly that potential for used in rnariculture of shrimp. ermented feed exists, especiaJly in view of the development of a successful fungal O n a cD CO 00 aA g CV C0 Cl 0 0! 00 JJ CV Ch CV CV CV a Ch 00 cd CO cNw CVI a 0 CVI 09 CV 00 lJ g I I I I I I I I I i cd a a P4 09 a v! 47 dl Vl CO CO i' CO cPI a 0! 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W 0 ja cd C ccj 4 al Ql 0 aj aj C 4J ~ Ql Ql 4J 0 0 M 0 WQl jd al QD bd al 0 Ql W N crj 0 Qj c4! gu g 0 Ql 4Ql Cg rcj LJ cd Ql cj '4 '4 crd '0 aj Ia 0 0 4J V 0 0 al 'a ~8 0 4J 0 rd a gl O Ql ar W crd 0al d 0 c4 cd Ql 'aQj Ql cj cd 0 0 8 0 Ql cd 4J crj g A A 4 Crd g 0 U Ql al cd tll g 0 O WQl 40QI 4 4 W cd cd 4 0 W QD Qj 0 Qj il W a c'a w 0 0 aj cd Ql W 4J 0 Ql Ql 4 0 u/ crD 0 ~ rC4J Qj a 4J cd Qj dl W 0 W40 crj 0 W 4J W IIg Q! cj al W 0 0 cd d V 0 O V w Ql 4 cda 0 Ijc V 0 C3 9l fact that our findings are the result of a very small-scale research effort. 14. Culture screenin on deli nified b asse and straw ln our use of an agar plate method of screening, we attempted to circumvent the expense of instrument analysis and abbreviate the length of time necessary to determine ability of strains of fungi to convert test substrates into mycelial biomass. ....ee dVainbt'[email protected] 9b'rvn4-4hr~qnei~hxvdm-., ~Picwr.~nhicb! Tv'ri .!87< ~a~~ lllllllllllllllllliiiiIII f [L[[f J! ii'i'i""!i]I"I'I'I[[[[[ttii!I!PI!! !ttJTlg[[jJ[ [ JU[lIII~ffg[Nf[[fJ ji'IIIIIIJlf[[[lllllllllll [L[[f arrived at a more accurate estimate of rate of increase in mass of tested fungi on each substrate than with only measurement of colony radial growth rate. Rather, we took into account hyphal diameter and hyphal density and used these to arrive at an indicator of fungal biomassproduced. As Morrisonand Righelato I974! havefound, hyphal density and byphal diameter are important determinantsof the width of the peripheralgrowth zone it may be that refinement of methodologycould yield an estimate of specific growth rate usingmeasures of thesetwo charactersrather than atte,npting the moreexacting techniques of Trinci l97l!. There are objections which can be raised to the use of our methodology in this culture screen. Eslyn l969!, Nilsson 974!, Sharpand Eggins0970! demonstrated that assayof fungal ceHulolysismay yield different results whenagar-plate methods and other more direct methods are used. These studies, however, did not involve attempts to measure biomass. Another important fault lies in the fact that differencesamong test fungi in depthof penetrationinto the agar were not measured a few marked differences were noted!, and surface density and hyphal diameter undoubtedlydo not reflect submergedcharacters to the sameextent amongstrains of fungi. Whetheror not our methodachieved approximation of conversionability would be best determinedby conductingcorrelation analysisof fermentationresults protein production,-hitin production,available energy production! with our or alternative! 92 computed indicators. l5. Future directions Whenwe undertookthis project, it was the feeling of some of those already involved in the artificial detrital feed research, and some of those who were to sponsorour research,that the problemto be treatedwas a rathersimple one: just growup some fungi on aninexpensive substrate, submit it to the youngshrimp, and raise them to marketable size. After all, they were eating the same stuff in nature, weren't they, and growing just fine there? And fungi have been growing on lignoceilulosicmaterials in naturefor cons.All that wouldbe necessary would be to bring the processinto the laboratoryand accelerate it. '='dna'osone namurrnos: %gnrn8art t.-h.tfozrfgs~== '-=-;4haf. 4+ oeahlamaf 4evale~~ a successfulprocess for producingand utilizing naturalfood analogues is not as simple as it may seemon the surface. The seriesof stepsin a functioningprocess is diagrammedin Fig. l2. As Calarn L969a! points out, researchinto particularfer- mentationprocesses has demonstrated"how stubbornand complexa fermentation protec-::-:a+toe-=,"hnolTtovrrig~c~m- f j=.==='X~~tinaaaL-.-.~~of .-th. ~earl,. the ~even< studiesof L. V, Sickand his colleagues Sick and Baptist, l973; Sick et al., 1973!show that eveninducing satisfactory ingestion rates can be a very complexproblem. Regnaultet al. 0975!should be consultedregarding problems of form of feed materials. In order to successfullydevelop these steps in Fig. l2 so that they function as a workingseries, several factors or variablesmust be consideredfor eachstep. In manycases, the variablesconsidered under a given step will havesignificant interactingeffects on the functioningof that step,and to furthercomplicate matters,variables which must be considered under one step may have distinct effects QLI Jl4 I Jc9 t. --,. x iI:> ---.~ -' J --- >- !fr 4 tss r-on ~~Kn-'I-rrcoonIB> ~tea. af sabir ~ =-err:=~i. i n Car..ting~ ' A~- "QgnicR'itt anti ~~ ~-,oft tnguscapable of converting L2. As a further example,suppose we were to identify a 93 a treated lignocellulosicsubstrate under a given set of fermentationconditions such that the fermentedproduct contained a theoreticallyoptimaJ amino acid-reserve- carbohydrate-lipid-chitinbalance, examining as manyvariables under each of the stepsin Fig. l2 as possible.After havingdone all of this exhaustivework, our productmight well turn out to bea dismalfailure, returning us to ourstarting position,because of m ycelialunpalatability or even toxicity to our target aquaculturedorganism; Nikolei l96J!found that unpalatabiJityand/or toxicity appearedto bethe causesof failuresof hisgall-fly larvaeto growwell onsome of his fungalfeeds, and that this could be caused bystrain of fungus and/or fungal culturing conditions! Witha little imaginationand pessimism, one can conjure up several other discouragingscenarios. On the other hand, if theproblem is dealtwith intelligently, it shouldbe amenable to solution.We would propose that success not be attempted in leapsand bounds as it wasin the presentstudy successunder these conditions could only be the result of serendipity!;rather, careful, thoroughanalysis of someof the mostbasic questions should be the primaryeffort. Thethree major goals of early work wouldbe fungalstrain and substrateselection, determination of optimal delignificatian method, and determination of optimaltype of fermentation. Fungalstrain s!which wouid serve well in theprocess must be capable of both efficientconversion of the substrate used and satisfactory support of shrimpgrowth. Therefore,we feeJ that the problemof strainselection should be dealtwith by conductinga dual screen of asmany strains as possible, examining concomitantly the degreeof abilityof the fungifor substrateconversion and acceptability and nutritive capacityof the myceJiafor theshrimp. Those which ranked high in bothcategories would then be used in developmentof the fermentationand feed preparation processes.The thermophilicfungi makeprime initial candidatesfor this survey Barneset aJ.,l972; Cooney and Emerson, J964; Romanelli et al., J975!.Genetic engineering Esser, J974! and alteration of fermentationconditions could then be used as tools in improvement of the fermentation product in terms of conversion efficiency andnutritive capacity. A listing of culturesof potentially biodeteriogenic fungiavailable at threemajor culture collections is givenby Denizelet al. 970!. Substrate selection would be based on the criteria of Jow acquisition cost and relative facility of conversioninto fungal biomass.Substrates of low acquisitioncost wouldbe those agriculturalby-products of high contentof Lignifiedcellulose and thereby little valueas ruminantfeed. Theywould have to be readily availablein or near areas in which mariculture of shrimp has likelihood of commercial successin order that collection and transportationcosts be minimal. They would have to be amenableto simple, Jow-costdelignification processessuch as someof those which havebeen developed by the U. S. EnvironmentalProtection Agency Rogersand Spino,1973! and other investigators e.g. Bender et al., 1970;Han et al., 1973;Hartley et al., 1974;Heany et al., l973;the workof Bavor 974! mayalso be of interesthere! in order to permit developmentof a fermentationor ruminationprocess with high conver sion rates. Sugarcane bagasse is anexample of anagricultural by-product which may meet the substrate-selectioncriteria. It is grown in large quantities in tropicaJ climes wheretemperature conditions and land and labor costs are mostfavorable to penaeid shrimpmariculture operations. It is attractiveas a substratebecause it is available at centralizedLocations in the sugarprocessing pLants Callihan and Dunlap, I969!, and haslimited value as feed, fuel, paper,or constructionmaterial Srinivasanand Han, 1969!. Simpledelignification processes have been used on bagassewith successin increasingmarkedly its susceptibilityto bothbacterial and fungaL degradation from L5%to 70-80%! Callihan and Dunlap, 1971; Dunlap, 1969; Garcia-Martinez et al., 1974!. It must be noted here that Cruz et al. 967! claimed to have producedfungal- fermentedbagasse of 28%crude protein without delignification and without ~addi@ a ~nitroen source to thefermentation medium; their methods are cursorily reported, r0I- Q Ci ~ C CI z z ~Q~ O~r LLI ~ C c 5 XVX OO 2 r cl er 0C I LLI K 0~ LLI X ih Cn LLI ~0~ 0 K ILI LLI LLI 7 ZgE z o I-EO CO 0 r C 6 mz 0I-cC LL 0 ~O r LLI Lll g X r~ K I- 0 o~ 0 0! ~O 4Z g~v! Ij. % rI- 0 z «C 0 V LL ro 0 ~ % LLI Z g 2 I- g ~ K 8 LLI Z Ljj CO 0! 0LLL 4 Ol 0 Z Lil Z 8 LL. 0t g ~ +0 KO SZ 0 LL V! LLI 0 u. gD LLI 40 Cjj Ch 95 and their results are of questionableaccuracy.! See Mandelset al. J974!for a review of the susceptibility of cellulosic materials to fungal lysis. The choice of type of fermentation process which is to be used should be dictated by potential costs. A comparison of two basic types of fermentation processes, submer ged-culture and solid-state, indicate that the latter is best suited for artificial detrital feed production. By "solid-state" fermentationswe simply mean those which require only moistening, rather than submerging of the substrates to be fermented. Hesseltine J972! has recently discussedthe use of solid-state fermenta- tions, and lamentsthe fact that in the West,deep-tank, or submerged-culture, fermentation methods have been developedand used to nearly the exclusion of solid- state methods{a strongindication of this is the fact that solid-stateapparatus is not dealt with in the reviews of fermentation design given by Blakebrough l969!, Solomons J969!, or Steel andMiller l970! !. Hesseltinelists advantagesof solid-state fermentations; the most important and basic of these is the most obvious solid-state fermentationsrequire only small volumesof water. This removesthe necessityfor large vessels,and simplifies aerationmethodology. It eliminatesthe needfor supplies of large volumes of water and the problem of its disposal if inorganic nutrients are supplied in the proper concentration, washing of the product of the fermentation should not be necessary!~ It provides conditions which in themselves inhibit bacterial contamination {the fungi are favored by Jow moisture levels relative to the bacteria Hesseltine,l972; Gray and Williams, 197J!!. Harvestingof the product is clearly a very simple matter relative to the harvesting problems of submerged-culture fermentation; the finished product could conceivably be fed directly to the animals to be cultured. Each of these advantagesis a partial reason for the adaptability of solid-state fermentation to Jow-level technological operation this aspect is discussedby N. 3. Poole in a privately communicated manuscript which is now in preparation for 96 publication Poole and Smith, 1975;see also Irnrie, 1975!. %sBrook et al. 969! point out,solid-state fermentations are particularly well adapted to be carried out with simple apparatus at low cost, since this method of ferment,ition has been traditionally used in the production of fermented foods such as tempeh for over a thousand years Gray, 1970;Hesseltine, 1965!. What it amounts to is the production of a "vegetable cheese" term from Brook et al.! with emphasis on production of myceliurn. An example of a pilot-plant process similar to that which we envision for use in production of artificial detrital feeds is described for tempeh production by Steinkrauset al. 965! andfor productionof "mold bran" by Underkofleret al. 947!. r.i Iicity of tne% pI'qce0h0sperrrilt 4mm to De operatea jqa man5gec >y per'sons' 1~ ~ he s>rT al technical skill and training; this advantage has been recently recognized of mini agpslmis~X ~I 97/ L ~b~cu~c. the Jikelihood that successful maricuIture bv Tushy a wiJJ probably involve use of highly iabor-intensive plans, That the of crusta sic substrates which we have recommended could be adapted to lignocellu >ns of this kind is evident from the success achieved by Hesseltine et al. f erm enta' e productionof secondarymetabolites on forage materials including oat 968! in . g solid-state fungal fermentation, and from the preliminary results of straw! us iJ. 972! with solid-state fungal fermentation of paper and of N. 3. Poole Barnes et leagues personal communication! with solid-state fungal fermentation of and his c 3er y, 1972!- straw s~ uld seem that the potentiaJ for development of valuable information from ito artificial detrital feeds is strongly indicated. If enough support for research niJar to ours is forthcoming, and if thorough analysis of the set of basic projects . ivolved in developing a successful process of production and utilization of problems Is is carried out rather than hasty attempts at short-circuiting this these f e research!, then we feel that it is likely that effective prototypal fundamer etrital feeds will be developed in the near future. artif icial 97 16. Ran e of a licabilit As a final note, it should be pointed out that artificial detrital feeds of the kind discussedabove are not limited in their potentialapplicability to the husbandryof shrimp. Several other detritivorous or omnivorousanimals are aquaculturedand commercially valuable e.g. carp, mWlet, tilapia see B.'.rdachet al., 1972!. These presumably perhaps especially the mullets!have a digest:ve physiologygeared to the types of feeds microbial-detrital complexes!which would be produced by the methodswhich we propose. Feedsof this type might help changethe fact that the fl 1MITI j till I IV~r~k~"..~'.;imp-RSUNk4+N+>h~~'LVifi'X'i@=-'lJ+-. .L+~lkPLAl~W~W~& ..~mixmr ~ole.i iK sizeis that of feed Dassowand Steinberg, 1973!. Other aquaculturable anima/s might also be able to grow satisfactorily on cheaplyproduced fungal protein if it were a componentof their diets. W. D. Gray personalcommunication! relates that tropical aquariumfish have beenmaintained for severalmonths oi; a diet of fungal mycelium. 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