Cortes-Fernandez, J.

Gutierrez-Rojas, M.

Lonsane, B.K.

Roussos, Sevastianos

Serrano-Carreon, L.

Viniegra-Gonzalez, G.

Horizon / Pleins textes

MICOL. NEOTROP. APL. 6 : 15-26,1993. 
UTILIZATION OF SLAUGHTER-HOUSE 
BYPRODUCTS AS NITROGEN SOURCE FOR 
FILAMENTOUS FUNGI IN SUBMERGED 
FERMENTATION, WITH SIMULTANEOUS 
DEODORIZATION 
L. SERRANO-CARRE ON^, J. CORTÉS-FERNANDEZ~, M. GUTIÉRREZ-ROJASI, 
* S. ROUSSOS~, G.VINIEGRA-GONZÁLEZ~AND B. K. LONSANE3 
Autonomous Metropolitan University (UAM-I), Biotechnology Department, 
Iztapalapa, Apartado Postal 55-535, 09340 México, D. F., México. 
Institut Français de Recherche Scientifique pour le Développement en 
Coopération (ORSTOM), Centre de Montpellier, U. F. Biotechnologie PMC, 911 
Avenue Agopolis, BP 5045,34032 Montpellier, France. 
Central Food Technological Research Institute, Fermentation Technology and 
Bioengineering Discipline, Mysore 570013, India. 
UTILIZACION DE LOS SUBPRODUCTOS DEL RASTRO COMO 
FUENTE DE NITROGEN0 PARA EL CRECIMIENTO DE HONGOS 
FILAMENTOSOS EN CULTIVO SUMERGIDO, CON EFECTO 
DESODORANTE SIMULTANEO 
Accepted for publication February 17,1993 
The potential of slaughter-house byproducts (the meals of feathers, hairs, blood and 
meat scraps) as sole source of nitrogen in sustaining growth of Trichoderma har- 
zianum and Aspergillus niger in submerged fermentation was studied. The effect of 
different C/N ratios and the substitution of glucose with other polymeric cheaper 
carbon sources, such as cassava starch, showed that the efficacy of individual meals 
as sole source of nitrogen varies depending on the type of meal and microbial' 
species. The data established that these abundantly available slaughter-house 
QRSTQM Fonds Documentaire 
Cote ? y 
16 L. SERRANO-~ARFLEON ET AL. 
byproducts can be used as low cost nitrogen sources for gaining economic advan- 
tages in fungal submerged fermentations. Simultaneous complete deodorization of 
the meals was observed at the end of fermentation in most cases, which was not due 
to complete utilization of the meal present in the medium. This opens up an entirely 
new approach for microbial deodorization of these meals, which will make them 
more acceptable as components of animal feed, due to elimination of offensive 
odours, and may also provide higher returns than what is possible at present. 
Key words: Filamentous fungi, nitrogen source, fermentation, feathers, hairs, 
blood, meat scraps, Trichoderma harzianum, Aspergillus niger, delo- 
dorization. 
La producción extensiva de carne para consumo humano implica la obtención de un 
volumen importante de subproductos con alto contenido de proteínas. Dos factores 
limitan su utilización en la formulación de alimentos para ganado: 1) olor desa- 
gradable, y 2) desequilibrio en la composición de aminoácidos, lo cual disminuye su 
valor nutritivo. Se evaluaron cuatro de estos subproductos (harinas de: plumas, 
pelos, sangre y carne) como substrato para el crecimiento en medio líquido de dos 
hongos filamentosos: Trichoderma harzianum y Aspergillus niger. Se estudiaron 
diferentes valores de la relación C/N, utilizando como fuente de carbono glucosa o 
harina de yuca. Ambas especies fueron capaces de utilizar los diferentes subproduc- 
tos como fuente única de nitrógeno. Sin embargo, la eficiencia de cada una de las 
harinas como substrato depende del microorganismo utilizado. En todos los casos, se 
obtuvo una desodorización completa de los medios de cultivo después de 25 horas de 
fermentación, incluso antes del consumo completo de las harinas. Este trabajo abre 
nuevas perspectivas sobre la aplicación de un nuevo proceso para eliminar los malos 
olores de las harinas de los subproductos del rastro, así como para la producción de 
diferentes metabolitos de hongos filamentosos con alto valor agregado. 
Palabras clave: Hongos filamentosos, fuente de nitrógeno, fermentación, 
Trichoderma harzianum, Aspergillus niger, subproductos del 
rastro, harinas de plumas, pelos, sangre y carne. 
INTRODUCTION 
Increased world population and higher demands for animal products in the 
recent years have given impetus to animal husbandry, as well as animal 
product processing industries throughout the world. These developments, in 
, SLAUGHTER-HOUSE BYF'RODUCTS FOR FILAMENTOUS FUNGI 17 
turn, have culminated into generation of different kinds of animal byproducts 
in quantities. These byproducts, although extremely rich in organic matter, 
can cause environmental pollution if they are not properly treated before 
discharge into natural streams. This special treatment, however, involves 
high capital and operating expenses, without providing any financial returns 
to the industry in most cases (Lonsane and Krishnaiah, 1991). Consequently, 
the utilization of the byproducts, to obviate the need for waste treatment, is 
preferred (Lonsane and Ahmed, 1989). 
A number of industries have been established in Latin American countries, 
especially in Mexico, for processing slaughter-house byproducts, such as 
hairs, feathers, blood and meat scraps into meals (Ensiminger, 1978; Conrad 
and Campabadal, 1979). The only avenue available at  present for utilization 
of these meals is as a component of animal feed. However, they fetch very low 
prices due to different limitations, such as lower digestibility, poor protein 
quality, presence of toxic factors and highly offensive odours. It is of vital 
importance to these countries to find additional uses for these meals in order 
to get better returns to the industry and a more efficient utilization. 
The present study reports the potential of fo,ur different slaughter-house 
byproducts (the meals of hairs, feathers, blood and' meat scraps) as nitrogen 
source in fungal submerged fermentations and their simultaneous 
deodorization. 
MATERIALS AND METHODS 
Microorganisms 
Trichoderma harzianum Rifai, strain CCM F-470, was obtained from the 
Czechoslovak Collection of Microorganisms, Brno, Czechoslovakia (Roussos, 
1987); while Aspergillus niger van Tieghem, strain ORSTOM No. 10, was 
obtained from the ORSTOM strain collection, Montpellier, France 
(Raimbault, 1980). Both strains were maintained-and subcultured on potato 
dextrose agar at 4°C. For inoculum preparation, the spores from freshly 
grown slants were suspended in 0.01% solution of Tween 80 (10 ml/slant). 
The inoculum ratio was 3 x lo7 sporedg carbon source initially present in the 
culture medium. 
18 L. SERRANO-CARREON ET AL. 
Submerged fermentation 
The basal medium (pH 5.6) contained (gL distilled water): KH,PO, 1.3, 
Nà,HPO, 0.12, MgS0,.7H20 0.3, CaC1,.2H20 0.3, vitamin-mineral salt solu- 
tion 2 (Roussos, 1982). Glucose at 0.2 or 1.0% levels as carbon source and 
individual meals (moisture content 8- 10%; obtained from ('Energéticos para 
Alimentos Balanceados, S. A. de C. V.", Iztapaluca, Mexico), as sole nitrogen 
source and to give 1, 7, 10, 13, 16 and 19 ratios of C/N, were added prior to 
sterilization. 50 ml of the medium were sterilized in 250 ml capacity Erlen- 
meyer flasks at 115°C for 30 min. The inoculated flasks were incubated on 
rotary shaker (100 rpm) for 70 h, at 29" and 35°C for the experiments of T. 
harzianum and A. niger, respectively. In another set of experiments, cassava 
meal and wheat bran were used individually in place of glucose at  a C/N ratio 
of 10. The growth kinetics of the cultures in Biolaffite fermenter (1.5 L 
working capacity), with automatic controls, were also studied with glucose as 
carbon source at 0.2 and 1.0% levels and a C/N ratio of 10 in both cases. The 
fermentation medium was agitated at  200 rpm, while the air supply was at 
the rate of 60 L/h and the temperature was regulated at 29" and 35°C. 
Analytical aspects 
The samples were homogenized for 1 min in an Ultra-turrax (Janke & 
Kunkel, Germany) at 20 O00 rotationsper min before centrifugation at 6000 
rpm for 15 min. The supernatant-was used in estimation of reducing sugars 
by the DNS method (Miller, 1959), and starch as well as cellulose by the 
Anthrone method (Dubois et al., 1956), using glucose as standard sugar. 
The malodour of the meals at the end of a 70 h fermentation period was 
determined qualitatively by a panel of 5 researchers. The absence of the 
characteristic malodour of the meals was taken as a criterion of complete 
deodorization. The characteristic mycelial odour of the culture was, however, 
ignored while determining the deodorization. 
RESULTS AND DISCUSSION 
Utilization of meals as nitrogen source 
The growth of the microorganism in the medium is the most appropriate cri- 
terion of its ability to use the meal, because the basal medium employed was 
devoid of any oither nitrogen source. Direct biomass determination was not 
possible due to difficulties in separating the microbial cells from the meal. 
SLAUGHTER-HOUSE BYPRODUCTS FOR "TOUS ~ G I  19 
The mycelial cells ofAspergiZZus niger formed pellets and enveloped all the in- 
dividual particles of the meals and, consequently, the medium became almost 
clear by 48 h. However, no pellets were formed .by Trichoderma harzianum. 
Similarly, the estimation of protein as growth indicator was not possible due 
to the problems in distinguishing mycelial protein from substrate protein in 
the fermentation media. Therefore, the growth of the culture in the present 
studies was determined indirectly based on the consumption of the carbon 
substrate. The data indicated that both cultures were able to efficiently use 
each of the all four meals individually as sole source of nitrogen (CnV ratio 
of l), as the consumption of glucose was up to 94% in almost all the cases 
(Table 1); the sole exception being the consumption of 77.5% glucose by A. 
niger in the medium containing blood meal. However, the ability varies 
depending on the type of the meal and also on the microbial species. 
The meals of the slaughter-house byproducts can be used efficiently as sole 
source of nitrogen in submerged fermentations involving A. niger and T. 
harzianum. These results may also be applicable to other fungal cultures, 
yeasts and bacteria, as well as a solid state fermentation (SSF) system. 
These meals are available in abundance in Mexico and other Latin American 
countries, and are being used at  present as a component of animal feed, in 
spite of their limitations. 
Effect of CIN ratios on glucose consumption 
The consumption of glucose by A. niger increased with the increase in C/N 
ratios in the media containing meat and blood meals, while it was decreased 
' 
Table 1. Utilization of the meals as nitrogen source at a C M  ratio of 1 by 
Trichoderma harzianum and Aspergillus niger. 
% consumption of glucose at 70 h 
Nitrogen source 
T. harzianum A. niger 
Blood meal 
Feather meal 
Hair meal 
Meat meal 
94.1 
94.4 
100.0 
94.4 
77.5 
95.8 
100.0 
94.1 
. 
Fig. 1. Effec of C/N ratios on consumption of glucose by Aspergillus niger. Tempera- 
ture 35'43, ti e= 70 h. r $  
slightly between C/N ratios of 1-10 in media containing hair and feather 
meals (Fig 1). The total glucose consumption was up' to  90% for all 
the meals with C/N ratios up to 13, excepting the mediã 'containing blood and 
meat meals. On the other hand, the consumption of glucose by T. harzianum 
was up to 90% at the C/N ratios up to 13, excepting the hair and meat meals 
(Fig. 2). It increased with the increase in the C/N ratios up to  13 in blood 
.meal containing medium, and remained at 100% even when the ratio was 19. 
In all other media, the glucose consumption by both fungal cultures was 
found to decrease according to the increase in C/N ratios beyond 10. This is 
probably due to limitations of the nitrogen source. The final pH in case of T. 
harzianum was 5.2-5.4 in the media containing different meals, in contrast to 
. I  
& I  1 .  i. t .  
dic range (4.5-4.9) in case of A. niger. 
l 1 
SLAUGHTER-HOUSE BYPRODUCTS FOR FILAMENTOUS FUNGI 2 1 
n s 
8 0 -  
C 
O .- 
.CI 
6 0 -  
. 3 VI 
C 
O 
al 
v) 
O o 
1 2 0 -  
0 
o 4 0 -  
O .  
100- - I 
u 
- BloodMeal - FeatherMeal - HairMeal - MeatMeal 
I I I I I I 
Kinetic studies 
The kinetics o f  glucose consumption in a 1.5 L fermenter by A. niger and T. 
harzianum are shown in figures 3-4, respectively. The glucose consumption 
(%) was found to increase with time in both cultures. The lower consump- 
tion of glucose in the media containing 10 g initial glucoseL is probably due 
to  the limitation of nitrogen, because the medium contained only about 0.6 g 
meam (C/N ratio of lo). The data indicated that the consumption of glucose 
by both cultures was 100% with the use of blood meal as sole nitrogen source. 
However, the initial concentration of glucose needs to  be one-fifth for full 
utilization of glucose by A. nigèr in comparison to that possible with the use 
of T. harzianum. 
Efficacy with the use of cheaper carbon sources 
The efficacies of the meals to  serve as nitrogen sources with the use of 
cheaper carbon sources (C/N ratio of 10 with 2 g cassava meal or wheat 
22 L. SERRANO-CARREON ET AL. 
100 - BloodM. G2 
s - BloodM.Gl0 - 80 
c - FatherM.G2 
O 
n 
.- - FatherM.Gl0 .w 
60 J 
v) c 
O o 
a 
v) 
O o 
3 
40 
a 2o 
I I I 
O 20 40  60 8 0  
Time (h) -- 
Fig. 3. Kinetics of glucose consumption by Aspergillus niger in 1.5 L fermenter. 
bran/L) are shown in figures 5-6. The starch consumption by A. niger was 
higher than that by T. harzianum with the use of cassava meal as carbon 
source. In contrast, the consumption of starch + celluloses by T. harzianum 
was better than that by A. niger when wheat bran was used as sole carbon 
source, because this species is a strong cellulolytic fungus (Roussos and 
Raimbault, 1982; Roussos et al., 1991b, 1991~). 
Deodorization patterns 
Patterns of deodorization of the meals were interesting, as evaluated qualita- 
tively at  the end of fermentation period. Complete deodorization of four 
different meals, used in the present studies individually as sole source of 
nitrogen, was possible with T. harzianum in the media containing glucose, 
wheat bran or cassava meal as carbon substrate. Similar was the case with 
A. niger, excepting the C/N ratio of 1 in medium containing feather meal and 
the C/N ratios of 1 and 7 in hair meal containing media. The data indicated 
that the deodorization of the meals is not due to their complete utilization as 
SLAUGHTER-HOUSE BYPRODUCTS FOR FILAMENTOUS "GI 23 
100 
n 
* a? 80 
c 
O .- 
.c, 
60 
3 
v) 
CI 
O 
al 
v) 
O o 
o 4 0  
- 3 20 
'a 
O 
O 20 40 60 8 0  
Time (h) 
Fig. 4. Pattern of glucose consumption by Trichoderma harzianum against time in 
1.5 L fermenter. 
nitrogen source by the culture, because A. niger consumed about 75% of the 
total glucose at a C/N ratio of 1 in blood meal medium and still caused 
complete deodorization. In contrast, the odours were not eliminated by the 
culture in the media containing either feather or hairs meals in spite of 100% 
and 95% glucose consumption, respectively, at a C/N ratio of 1. 
The deodorization of these meals from slaughter-house byproducts by A. 
niger and T.' harzianum, or potentially any other microbial species, dor;s not 
appear to have been reported so far. This opens up an entirely new avenue 
for microbial deodorization of these meals in the fermentation process, where 
the meal also serves as sole and cheaper nitrogen source. The offensive odour 
of the meals is a characteristic which limits their extensive and gainful 
utilization. The acceptability of the deodorized meals by animal feed manu- 
factures and also by the animals will be much higher than what is existing 
now. Another possibility is to grow the desired microorganism on the meals 
24 L. SERRANO-CARREON ET AL.
Feat her 
u) 
Ca 
a, 
- 
2 
T 
I 
I 
Blood 
Hair I I 
t I .  
Meat 
5 O 10 20 30 40 50 60 70 SO 90 100 
S t a r c h  c o n s u m p t i o n  (%I 3 -  
T .- 
Blood 
1 l 
I I l 
... 
2 
Hair 
r 
I I 
O 10 20 30 40 50 60 70 SO *90 100 
S t a r c h  + cellulose c o n s u m p t i o n  (%) 
6 
, .  
Figs. 5-6.- 5:  Efficacy of the meals as sole nitrogen sources in cassava meal contain- 
ing medium for growth spergillus niger (0) and harzianum 
( I). 6: Consumption of 
wheat bran and 
and cellulose by the 
as carbon and 
SLAUGHTER-HOUSE BYPRODUCTS FOR FILAMENTOUS FUNGI 25 
using lower C/N ratios and the resulting biomass, along with the non-utilized 
portion of the meal, can then be used as a component of animal feed. This 
would have distinct advantages, such as improved protein quality due to 
microbial biomass, elimination of offensive odours and enhanced acceptabil- 
ity. The use of these meals as.organic nitrogen sources for mycelial growth 
also eliminates the need for the use of mineral nitrogen sources (Roussos et 
al., 1991a), which are known to cause problems in the nutrition of monogas- 
tric animals (Ensiminger, 1978). In general, this may lead to better returns 
than what is possible at present. 
CONRAD, J. H. and C. CAMPABADAL, 1979. Nutrient requirement ofswine. 10th 
Latin American Livestock Conference, National Academy of Sciences, New 
York, pp. 135-162. 
DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. A. REBERS and F. SMITH, 1956. 
Colorimetric method for determination of sugars and related substances. 
Analytical Chemistry 28: 350-356. 
ENSIMINGER, M. E., 1978. In: Producción porcina. John Wiley and Sons, New 
York, pp. 52-67. 
LONSANE, B. K. and M. M. KRISHNAIAH, 1991. Leaching of the product and 
further downstream processing. In: Doelle, H. W., D. E. Mitchell and C. E. 
Rolz (Eds.). Solid Substrate Cultivation. Elsevier Science Publishers, Essex, 
England (in press). 
LONSANE, B. K. and S .  Y. AHMED, 1989. Some neglected aspects ofwaste manage- 
ment: reduction, recycle, utilization and exchange. In: Souu. National Symp. 
on Impacts of Pollution in and from Food Industries and its Management, 
Association of Food Scientists and Technologist, Mysore, India, pp. 33-39. 
MILLER, G. L., 1959. Use of 3,5-dinitrosalicylic acid reagent for determination of 
reducing sugars. Analytical Chemistry 31 : 426-428. 
RAIMBAULT, M., 1980. Fermentation en milieu solide. Croissance de champignons 
filamenteux sur substrat amylacé. Thèse de Doctorat d'Etat. Sciences 
Naturelles (Université Paul Sabatier), Toulouse, France. 
ROUSSOS, S., 1982. Mise au point d'une methode pour l'étude des caractères 
morphologiques, biochimiques et nutritionnels des champignons imparfaits. 
Cah. ORSTOM. Sér# B i d .  45: 25-34. 
ROUSSOS, S., 1987. Croissance de Trichoderma harzianum par fermentation en 
milieu solide: physiologie, sporulation et production de cellulases. Etudes et 
Thèses (Université de Provence), ORSTOM, Paris. 
26 L. SERRANO-CARREON ET AL. 
ROUSSOS, S .  and M. RAIMBAULT, 1982. Hydrolyse de la cellulose par les moisis- 
sures. I. Screening des souches cellulolytiques. Ann. Microbiol. 133: 455-464. 
ROUSSOS, S., A. OLMOS, M. RAIMBAULT, G. SAUCEDO-CASTAÑEDA and B. K. 
LONSANE, 1991a. Strategies €or large scale inoculum development €or solid 
state fermentation system: conidiaspores of Trichoderma harzianum. 
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ROUSSOS, S., M. RAIMBAULT, G. SAUCEDO-CASTMEDA, G. VINIEGRA- 
GONZALEZ and B. K. LONSANE, 1991b. Kinetics and ratios of carboxy- 
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ROUSSOS, S., M. RAIMBAULT, G. VINIEGFL4-GON ALEZ, G. SAUCEDO- 
I 


Utilization of slaughter-house by products as nitrogen source for filamentous fungi in submerged fermentation, with simultaneous deodorization

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