Note
Production of ethanol from mesquite [Prosopis juliflora (SW)
D.C.] pods mash by Zymomonas mobilis in submerged
fermentation
Celiane Gomes Maia da Silva; Samara Alvachian Cardoso Andrade; Alexandre Ricardo Pereira Schuler; Evandro Leite de Souza ; Tânia Lúcia Montenegro Stamford *
UFRPE – Depto. de Ciências Domésticas, Av. Dom Manoel de Medeiros, s/n – 52171-900 – Recife, PE – Brasil.
UFPE – Depto. de Engenharia Química, Campus Universitário – 50670-901 – Recife, PE – Brasil.
UFPB – Depto. de Nutrição, Cidade Universitária, Campus I – 58059-900 – João Pessoa, PB – Brasil.
UFPE – Depto. de Nutrição. Av. Prof. Moraes Rego s/n – Cidade Universitária – 50670-901 – Recife, PE – Brasil.
*Corresponding author
ABSTRACT: Mesquite [Prosopis juliflora (SW) D.C.], a perennial tropical plant commonly found in Brazilian semi-arid region, is a viable raw material for fermentative processes because of its low cost and production of pods with high content of hydrolysable sugars which generate many compounds, including ethanol. This study aimed to evaluate the use of mesquite pods as substrate for ethanol production by Z. mobilis UFPEDA-205 in a submerged fermentation. The fermentation was assessed for rate of substrate yield to ethanol, rate of ethanol production and efficiency of fermentation. The very close theoretical (170 g L–1) and experimental (165 g L-1) maximum ethanol yields were achieved at 36 h of fermentation. The highest counts of Z. mobilis UFEPEDA-205 (both close to 6 Log cfu mL –1) were also noted at 36 h. Highest rates of substrate yield to ethanol (0.44 g ethanol g glucose–1), of ethanol production (4.69 g L–1 h–1) and of efficiency of fermentation (86.81%) were found after 30 h. These findings suggest mesquite pods as an interesting substrate for ethanol production using submerged fermentation by Z. mobilis.
Key words: renewable sources, bioconversion, fermentative parameters
Produção de etanol a partir do mosto de vagens de algaroba [Prosopis
juliflora (SW) D.C.] por Zymomonas mobilis em fermentação submersa
RESUMO: A algaroba [Prosopis juliflora (SW) D.C.] é uma planta tropical perene comumente encontrada no semi-árido brasileiro e apresenta-se como matéria-prima viável para o processo fermentativo por possuir baixo custo e para produzir vagens que contém um elevado teor de açúcares hidrolisáveis, os quais podem gerar diversos compostos, incluindo etanol. Avaliou-se o uso de vagens de algaroba como substrato para produção de etanol por Z. mobilis UFPEDA-205 por meio de fermentação submersa. O processo fermentativo foi avaliado por meio da mensuração da taxa de conversão de substrato em etanol, taxa de produção de etanol e eficiência de fermentação. Os valores muito próximos encontrados para o fornecimento máximo teórico (170 g L–1) experimental (165 g L–1) de etanol foram alcançados após 36 h de fermentação. O valor de contagem experimental de Z. mobilis UFEPEDA-205 (próximo a 6 Log cfu mL
–1) foi encontrado após 36 h de fermentação. As mais elevadas taxas de conversão de substrato para etanol (0,44 g ethanol g glucose –1), de produção de etanol (4,69 g L–1 h–1) e de eficiência de fermentação (86,81%) foram encontrados depois de 30 h. Conclui-se que as vagens de algaroba apresentam potencial como substrato emergente para produção de etanol por Z. mobilis por meio de fermentação submersa.
Palavras-chave: fontes renováveis, bioconversão, parâmetros fermentativos
Introduction
Fuels obtained from renewable resources have deserved a great deal of interest during the past decades
ma inly due to conc e rns about fos s i l fue l s depl e t ion.
R e s e a r c h e f f o r t s h a v e b e e n m u l t i p l i e d i n t h e l a s t
y e a r s a s a c o n s e q u e n c e o f c o n s t a n t i n c r e a s i n g c o s t s
a n d e n v i r o nme n t a l imp a c t d e r i v e d f r om t h e u s e o f
c r u d e - b a s e d f u e l s ( G r a y e t a l . , 2 0 0 6 ; P r a s a d e t a l . ,
2007).
Zymomonas mobilis is a Gram-negative, facultative
anaerobic that ferments glucose, fructose, and sucrose
as carbon sources (Viikari, 1998). These carbohydrates
a r e me t abol i z ed v i a the s ame biochemi c a l rout e , the
Entner-Doudoroff pathway (Paula et al., 2007). Z. mobilis
is a promising alternative to yeast in the synthesis of ethanol. In comparison with yeast, Z. mobilis has a higher
tolerance to ethanol and better kinetic characteristics
such as higher specific substrate uptake, higher ethanol
synthe s i s r a t e and hi ghe r subs t r a t e yi e ld to e thanol .Production of ethanol from P. juliflora pods mash 1 2 5
Sci. Agric. (Piracicaba, Braz.), v.68, n.1, p.124-127, January/February 2011
Moreover, it has advantages for the fermentation of glucose to ethanol that include a high yield of ethanol from
consumed glucose and a high specific rate of ethanol
production (Joachimsthal et al., 1998; Shene and Bravo,
2001; Tano and Buzato, 2003). The metabolic activity of
Z. mobilis depends on the strain and carbon source, while
many by-products can be produced during the ferment a t ion of suc ros e , such a s phenol , l a c t i c a c id, hi ghe r
a l c o h o l s , a c e t a l d e h y d e , m e t h a n o l a n d l e v a n
(Kalneniekis et al., 2000; Borsari et al., 2006).
Prosopis juliflora (SW) D.C., Leguminosae, a popular plant known as mesquite, is native to Central and
South America and has spread to North America. Mesq u i t e h a s g r e a t p o t e n t i a l f o r u s e a s a mu l t i p u r p o s e
tree in different parts of the world in comparison to
s e v e r a l n a t i v e a n d e x o t i c s p e c i e s ( K a i l a p p a n e t a l . ,
2000; Deans et al., 2003). Mesquite pods present a high
amo u n t o f c a r b o h y d r a t e s ( B a t i s t a e t a l . , 2 0 0 2 ) . P o d
production per tree can vary from a few kg to over
400 kg and is highly dependent on moisture availability to the plant (Riveros, 1992). In the northeast region
of Brazil, mesquite trees cover 150.000 ha (Tabosa et
al., 2000).
The aim of this study was to assess the use of mesquite pods as substrate for ethanol production by Z.
mobilis UFPEDA-205 in a submerged fermentation.
Material and Methods
Strain of Z. mobilis UFEPEDA–205 used in this study
was supplied by the Department of Antibiotics, Federal
University of Pernambuco, Recife, Brazil. Stock cultures
were kept in slopes Standard Swings and De Ley – SSDL
agar (glucose 20.0; yeast extract 5.0; agar 15 g L
–1
) (Swings
and De Ley, 1977) under refrigeration. For experimental assays, Z. mobilis were grown in 50 mL of SSDL broth
at 37ºC. After 48 h incubation, 5 mL of the culture was
added to flasks containing 95 mL of the same growth
media and allowed to grow at room temperature for 24
h under rotation (150 rpm).
Liquefied mash was prepared using healthy (with no
infection sign) mesquite pods. Mesquite pods used in this
study presented moisture 5.8; total sugars 56.5; reducing
sugars 4.6; total fiber 7.2; proteins 9.0; fat 2.1; and ashes
0.2 g 100 g
–1
(Silva et al., 2007). Pods were dried at 45ºC
for 18 h, followed for grounding in hammer mill with a
#4 screen to get the appropriate grind size. Hydrated
mash was prepared at 30 g 100 g
–1
of distilled water. To
prepare the mash, ground mesquite was slowly added
to distilled water in a constant agitation. After the addition of the proper ground amount, the mash was heated
to 50ºC, maintained at this temperature for 1 h and submitted to centrifugation (3000 rpm for 15 minutes). The
supernatant was vacuumed filtered using Whatman n. 1
and autoclaved at 121ºC for 15 min. After that, the mash
was cooled to room temperature and aliquots were aseptically dispensed in sterile Erlenmeyer flasks for fermentation.
The mash used for fermentations presented total sugars (sucrose) 16.1; reducing sugars 3.99; total fiber 3.99;
proteins 2.16; fat 0.63; tannins 0.09; and ashes 0.2 g 100 g
–1
.
It was reported a total soluble solids value of 18ºBrix
(Silva et al., 2007).
Submerged fermentation of mesquite hydrated mash
by Z. mobilis UFEPEDA–205 was analyzed. A 100-mL
aliquot of mesquite mash (added with 10 g L
–1
(NH4
(
2
SO4
and 2 g L
–1
KH2
PO4
) was aseptically distributed in sterile 250-mL Erlenmeyer flasks and inoculated with a 24
h-old culture (approximately 10
8
cfu mL
–1
). The flasks
were incubated at room temperature (28 ± 1ºC) under
static condition. During 72 h of fermentation, the mash
was analyzed for pH, glucose concentration, bacterial
count and ethanol concentration. The fermentation was
carried out in triplicate and the results were expressed
as average of the parallel assays.
Kit Glicose PAP – Liquiform (Labtest Diagnóstica,
Minas Gerais, Brazil) was used to measure the glucose
concentration (g L
–1
), while the pH value was found using a Micronal B474 digital pHmeter. The growth of Z.
mobilis was evaluated by the viable cell count procedure.
For this, at the pre-established periods a 100 μL aliquot
of the media was uniformly spread on sterile SSDL agar
Petri dishes and incubated at 37°C for 48 h. After the
incubation period the count of viable cell was carried
out and the results were expressed as Log of Colony
Forming Units per mL (Log cfu mL
–1
.(
Concentration of ethanol was determined using a gas
chromatograph (HP 5890, Hewlett-Packard, Palo Alto, CA)
fitted to a flame ionizer detector. A 2 μL-portion of the fermentation sample was injected onto a column (30 m; 0.25
mm i.d.; 0.25 lm, J&W Scientific, Folsom, CA). The chromatographic conditions were as follow: sample (without
dilution) injection volume 2 μL; hydrogen flow rate 5.0 mL
min
–1
; temperature program 120ºC (isotherm); injector temperature 100°C; detector temperature 120°C. The data were
processed using the Millennium Computer Program (Waters Chromatograph Division, Milford, MA, USA). Analyses were performed in triplicate and the results were expressed as average of the parallel assays.
The following parameters were used for assessing the
fermentative process: (i) Amount of consumed sugar: [S:
- (S
f
– S
0
)], where S: consumed sugar (g glucose L
–1
); S
f
:
sugar final concentration (g glucose L
–1
); S
0
: sugar initial
concentration (g glucose L
–1
); (ii) Amount of produced
ethanol: [P: (P
f
- P
i
)], where P: amount of produced ethanol (g L
–1
); P
f
: final ethanol concentration (g L
–1
); P
i
: initial ethanol concentration (g L
–1
); (iii) Rate of substrate
yield to ethanol: [Y
p/s
: P/S], where Y
p/s
: rate of substrate
yield to ethanol (g glucose g ethanol
–1
); (iv) Rate of ethanol production: [PR: P/t], where PR: rate of ethanol production (g L
–1
h
–1
); t: time of fermentation (h); (v) Efficiency of fermentation (n
p(%)
), based on the theoretical
yield according to the Gay-Lussac equation (51.1 g ethanol 100 g glucose
–1
): [n
p(%)
: Yp/s
.[100/51.1.
T h e d a t a w e r e a n a l y z e d b y A N O V A u s i n g t h e
Duncan test (p ≤ 0.05) and the software Statistica 6.0.126 Silva et al.
Sci. Agric. (Piracicaba, Braz.), v.68, n.1, p.124-127, January/February 2011
Results
During the fermentation the amount of glucose increased up to 24 h (Figure 1). After 30 h of fermentation
the glucose concentration dropped sharply and it was
absent in the media after 40 h of fermentation. Along
the 72 h fermentation period, the pH value of mesquite
pods mash did not vary (4.8 – 5.1). The maximum ethanol amount (165 g L
–1
) was achieved at 36 h of fermentation (Figure 1). The highest experimental count of Z.
mobilis (close to 6 Log cfu mL
–1
) was also noted at 36 h
(Figure 1). Highest rates of substrate yield to ethanol
(0.44 g ethanol g glucose
–1
) and efficiency of fermentation (86.81%) were found after 30 h (Table 1). Ethanol
productivities were 4.69 g L
–1
h
–1
and 4.56 g L
–1
h
–1
noted
after 30 h and 36 h of fermentation, respectively (Figure
1). At 72 h the values found for all assessing parameters
dropped sharply. 30 h was found as the shorter interval
time to obtain the highest ethanol yield from the fermentation of mesquite pods mash by Z. mobilis under static
condition.
Discussion
The amount of glucose dispersed in the growth medium was increasing up to 24 h of fermentation, although
i t wa s suppr e s s ed a f t e r 4 0 h. The inc r e a s ing g lucos e
amount found in mesquite mash up to 24 h was probably related to a continuous hydrolysis of sucrose dispersed in the medium. Silva et al. (2007) reported that
mesquite pods present high availability of sugars (56.5 g
100 g
–1
), particularly sucrose. High availability of sucrose
in the growth media increases the ethanol yield by Z.
mobilis (Favela Torres and Barati, 1988). On the other
hand, small yields of ethanol are found in media rich of
cellulose, inulin or starch since the bacterium is not able
to hydrolyze these polymers (Shene and Bravo, 2001).
Z. mobilis presents a prominent capacity of hydrolyzing the sucrose dispersed in the growth media and
r a p i d l y me t a b o l i z e s t h e r e s u l t i n g g l u c o s e a s c a r b o n
source for ethanol production by the Entner-Doudoroff
way (Swings and De Ley, 1977). The availability of glucose, fructose or sucrose in the growth media increases
the ethanol yield by Z. mobilis (Favela Torres and Barati,
1988). Extremely high levels of glucose in a basal medium could suppress the growth of Z. mobilis while causing no negative effect toward the ethanol yield (Morais
et al., 1993). The non-occurrence of changes at stabilization of pH in the mesquite mash during 72 h of fermentation could indicate the absence of contaminating bact e r i a i n h i g h n u m b e r d u r i n g t h e f e r m e n t a t i o n
(Narenddranath and Power, 2004).
The three times of fermentation (30, 36 and 72 h)
evaluated were chosen based on the higher and smaller
obtained ethanol yield. The findings suggested 30 h as
the shorter interval time to get the highest ethanol yield
from the fermentation of mesquite pods mash by Z.
mobilis under static condition, which could possibly reduce the overall time of the process and decrease the
cost for industrialization.
Aerobic cultures of Z. mobilis had higher ethanol
yield from glucose with maximum theoretical values of
0.51 g ethanol g glucose
–1
(Prasad et al., 2007). On the
other hand, small yields of ethanol from glucose (0.13
to 0.17 g ethanol g glucose
–1
) are found in anaerobic cultures (Viikari, 1998; Shene and Bravo, 2001). Tano and
Buzato (2003) reported a small ethanol production (29 g
L
–1
) from sugar cane juice by Z. mobilis ATCC 31821
with a small substrate yield into ethanol (0.42 g ethanol
g glucose
–1
) after 48 h under stirring (aerobic) cultivation.
According to the authors, this small yield was possibly
related to the amount of mineral compounds dispersed
in the sugar cane juice that could inhibit the fermentation by Z. mobilis. Regarding the results of previous studies the mesquite pods hydrated mash present a higher
efficiency for ethanol production in comparison to the
classical substrate sugar cane juice.
Table 1 – Assessing parameters of the fermentative process for ethanol production from mesquite pods hydrated mash
by Z. mobilis UFEPEDA-205 under static conditions for 72 h.
Average values followed for different letter at the lines differ (Duncan, p d” 0.05); Yp/s:
rate of substrate to ethanol conversion; PR:
ethanol productivity; N
p(%)
: efficiency of fermentation
Assessing parameters
Time of fermentation (h
–1
(
03 63 27
Y
p/s
(g g
–1
) 0.44 ± 0.02 a 0.35 ± 0.03 b 0.05 ± 0.02 c
PR (g L
–1
h
–1
) a .69 ± 0.02 4 b .56 ± 0.04 4 c 0.45 ± 0.01
Np(%)
(%) 86.81 ± 0.02 a 70.37 ± 0.01 b 11.27 ± 0.02 c
Figure 1 – Experimental values of the ethanol production and
microbial count in submerged fermentation of hydrat
mesquite pods mash by Z. mobilis UFEPEDA-205
under static condition.
-1
0
1
2
3
4
5
6
7
8
0
2 0
4 0
6 0
8 0
100
120
140
160
180
0 10 2 0 30 40 50 6 0 70 80
Ethanol production (g L ethanol production (g L–1) –1
) Microbial cou nt (Log cfu mL microbial coun t (Log cfu mL–1)
–1
) Glucose concentration (g L glucose concentration (g L–1)
–1
) pHProduction of ethanol from P. juliflora pods mash 1 2 7
Sci. Agric. (Piracicaba, Braz.), v.68, n.1, p.124-127, January/February 2011
Our findings suggest mesquite pods as an emerging
substrate for ethanol production using submerged fermentation by Z. mobilis. High levels of ethanol were produced from mesquite pods hydrated mash by submerged
fermentation using Z. mobilis under static condition.
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