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Biological Wastes 33 (1990) 161-168

Inhibi tory Effect of Citrus unshu P ee l o n A n a ero b i cD i g e s t i o n

E. Mizuki, T. Akao & T. Saruwatari

Fukushima Industrial Research Institute of Fukuoka Prefecture,Yame-shi, Fukuoka 834, Japan

(Received 3 October 1989; revised version received 18 January 1990;accepted 24 January 1990)

A B S T R A C T

A s ign i fi can t i nh ib i ti on o f methane f erm en ta t i on was observed i n anaerob ic '

d iges t i on o f Citrus unshu pe el a t loadings above 2 g / l i t re per day. This

inh ib i to ry e f f ec t was ma in l y due t o pee l o il , bu t i n par t t o o ther subs tances

presen t i n t he pee l . The l im i t i ng l oad o f pee l o i l t o anaerob ic d iges ti on was

abou t 65 p l / l i t re per day . A dd i t i on o f pee l o i l be low th i s l im i t l oad resu l t ed i n a

change i n t he microb ia l f l o ra and in gas p roduc t ion grea t er t han t ha t o f

d iges t i on w i th no pee l o i l . Citrus unshu pee l d id no t i nh ib i t me thane

, f ermen ta t i on a f t er rem ova l o f pee l o i l by s t ea m d i s t il l a ti on or aera t ion .

INTRODUCTION

Usually, anaerobic digestion converts solid wastes from fruit processing into

methane fuel. Previous workers, however, have reported that the citrus

wastes are toxic to the anaerobic digestion (McNary e t a l . , 1951; Wolford

e t a l . , 1952; Lane, 1980, 1983a, 1983b, 1984).

In Japan, C i t r u s u n s h u is the commercially most important citrus fruit and

a number of Japanese plants produce a large amount of canned and juice

products of u n s h u citrus. This creates serious problems in disposing of the

waste peel which is produced at more than 70000t/year. The primary

objective of this study was to assess the level of the toxicity of C . u n s h u peel tothe practical anaerobic digestion of the waste peel.

161

Biological Wastes 0269-7483/90/$03-50 ~ 1990 Elsevier Science Publishers Ltd, England.Printed in Great B r i t a i n



162 E. Mizuki, T. Akao, T. Saruwatari

M E T H O D S

M a t e r i a l s

W a s t e p e e ls o f C. unshu w e r e o b t a i n e d f r o m a c a n n i n g p l a n t a n d s t o re d a t

- 4 0 ° C p r i o r t o u s e . P e e l o i l o f C. unshu w a s o b t a i n e d f r o m a c o m m e r c i a l

j u i c i n g p l a n t . 1 9- L im o n e n e a n d c i tr u s p e c t i n w e r e p u r c h a s e d f r o m W a k o

P u r e C h e m i c a l s ( T o k y o ). O t h e r r e a g e n t s a n d s o l v e n t s w e r e c o m m e r c i a l l y

a v a i l a b l e a n a l y t i c a l r e a g e n t s o r l a b o r a t o r y - g r a d e m a t e r i a l s .

F e e d s t o c k s

W a s t e w h o l e p e el s ( fl a v ed o a n d a l b e d o ) w e r e c o m m i n u t e d f o r 1 0 m i n a n d

h o m o g e n i z e d fo r 2 0 m i n . T h e h o m o g e n a t e w a s t h e n s u p p l e m e n te d w i t h

( N H 2 ) 2 C O (1 07 .2 g / k g d r y w t o f p e el ) a n d N a 2 H P O 4 (5 6.0 g / k g d r y w t o f

p e el ), d i l u t e d w i t h d i s t i ll e d w a t e r t o 5 - 4 0 g d r y w t o f p e e l / li t r e a n d s t o r e d a t

4°C.

T h e p e c t i n m e d i u m u s e d a s a p e e l- f re e f e e d s t o c k c o n t a i n e d c i tr u s p e c t i n

( 2 0 g / l it r e ), ( N H z ) 2 C O ( 2 - 1 4 g /l it re ) a n d N a 2 H P O 4 ( 1 -1 2 g / li tr e ), a n d w a s

s t o r e d a t 4 ° C .

D i g e s t i o n s

D i g e s t i o n s w e r e c a r r i e d o u t i n 2 0 0 m l c o n i c a l f la sk s c o n t a i n i n g 2 0 0 m l o f

s e w a g e s l u d g e [ 2 7 . 5 % ( v / v ) s o l i d ] . T h e s l u d g e w a s i n c u b a t e d a t 3 7 ° C w i t h

i n t e r m i t t e n t s h a k i n g . A t 2 4 -h i n te r v a ls , a f te r t h e r e m o v a l o f 2 0 m l d i g e s t er

c o n t e n t s , 2 0 m l o f f e e d s t o c k c o n t a i n i n g 0 . 1 - 0 - 8 g ( d r y w t ) o f s o l i d s ( i . e .

0 - 5 -4 . 0 g d r y s o l i d s / li t r e o f s l u d g e p e r d a y ) w a s a d d e d t o t h e f la s k s to g i v e a

c o n t i n u o u s d i g e s t i o n o f 1 0 d a y s r e t e n t i o n t i m e . P ee l oi l a n d D - l i m o n e n e w e r e

a d d e d u s i n g m i c r o - s y ri n g e s w h e n d i g e s t io n s w e r e f e d w i t h f e ed s t o ck s . G a sy i e ld a n d p H o f d i g e st i n g s lu d g e w e r e d a il y m o n i t o r e d .

E s t i m a t i o n o f o i l c o n t e n t

P e el oi l c o n c e n t r a t i o n w a s d e t e r m i n e d b y t h e m e t h o d o f S c o t t a n d V e l d h u is

(1966).

G a s c h r o m a t o g ra p h y

P e e l o il a n d D - l i m o n e n e w e r e a n a l y s e d b y f l a m e - i o n i z a t i o n g a s c h r o m a -

t o g r a p h y u s i n g a Y a n a c o g a s c h r o m a t o g r a p h G - 8 0 w i t h a s t a i n l e s s s t e e l

c o l u m n ( 20 0 x 0-3 c m ) p a c k e d w i t h 8 0 - t o 1 0 0 -m e s h E G A U n i p o r t B .



Inhibition of digestion by citrus peel 163

M e t h a n e w a s d e t e r m i n e d b y g as c h r o m a t o g r a p h y ( Y a n a c o G - 80 ) w i t h a

s t a in l e s s s te e l c o l u m n ( 2 0 0 x 0 .3 c m ) p a c k e d w i t h a c t i v e c a r b o n (3 0- t o 6 0-

m e s h ) , u s i n g t h e r m a l c o n d u c t i v i t y d e t e c t i o n .

R e m o v a l o f p e e l o i l f r o m f e e d s t o c k s

P e e l o i l i n f e e d s t o c k s w a s r e m o v e d i n t w o w a y s , a e r a t i o n o r s t e a m

d i s t i l l a t i o n . I n t h e f o r m e r , f e e d s t o c k s o f h o m o g e n i z e d w h o l e p e e l w e r e

i n c u b a t e d i n a n o p e n - t o p p e d f e r m e n t e r a t 3 0 °C w i t h a ir s p a r g in g

(1 5 l i t r e s / l ' 5 l i tr e s f e e d s t o c k p e r r a in ) . I n t h e l a t te r , h o m o g e n i z e d p e e ls w e r e

e x p o s e d i n a s t e a m - j a c k e t e d p a n t o a s t e a m j e t f o r 4 0 m i n .

R E S U L T S

Inhib i tory e f fect of C. unshu p e e l o n m e t h a n e f e r m e n t a t i o n

P e e ls o f C . unshu w ere fed to d ige s te r s a t the load s o f 0 .5 , 1"0 , 2 .0 an d 4 .0 g d r y

w t o f p e e l (d .w . p .) p e r l i tr e o f s l u d g e p e r d a y ( li tr e p e r d a y ) . T h e c h a n g e i n g a s

y i e l d is s h o w n i n F i g . I ( A ) . F i g u r e I (B ) s h o w s t h e t i m e c o u r s e o f p H o f t h e

d i g e s t i n g m i x t u r e . T h e d i g e s t i o n s w i t h 0 . 5 a n d l ' 0 g w a s t e p e e l a n d 2 " 0 g

p e c t i n a l l a t t a i n e d a s t e a d y - s t a t e , w h i l e w i t h 2 " 0 a n d 4 . 0 g w a s t e p e e l g a sp r o d u c t i o n a n d t h e p H d e c r e a s e d w i t h t i m e a n d t h e f a l l i n p H t o b e l o w 6

a f t e r 1 0 - 1 5 d a y s w a s e v i d e n c e o f d i g e s t i o n f a il u r e.

Inhib i tory e f fect of C. unshu p e e l o i l o n m e t h a n e f e r m e n t a t i o n

G a s c h r o m a t o g r a p h y s h o w e d t h a t C. unshu p e e l o i l a n d c o m m e r c i a l l y

a v a i l a b l e D - l i m o n e n e c o n t a i n e d 8 9" 9 a n d 9 8 " 4% o f D - l i m o n e n e , r e s p e c ti v e l y .

T o d i g e s t i o n s f e d w i t h 2 g [ ( d .w . ) /l i tr e p e r d a y ] p e c t i n w e r e a d d e d 0 -9 0/~ 1 o f

p e e l o i l/ l it r e p e r d a y . T o a n o t h e r d i g e s t i o n f e d w i t h p e c t i n [ 2 g ( d .w . ) /l it re p e rd a y ] w a s a d d e d 8 2 -3 # l / li t re p e r d a y o f D - l i m o n e n e as a r e a g e n t t h a t w a s

e q u i v a l e n t t o 9 0 /~ 1 o f p e e l o il i n t e r m s o f D - l i m o n e n e c o n t e n t . D i g e s t i o n s

s u p p l e m e n t e d w i t h p e e l o i l a t 7 0 , 8 0 a n d 9 0/~ 1, a n d 8 2.3 ~1 o f D - l i m o n e n e a s a

reag en t f a i l ed a f te r 46 , 46 , 33 an d 43 d ays r e spec t ive ly (F ig . 2 ). A dd i t io n o f 0

o r 6 0/~ 1 o f p e e l o i l/ l i tr e p e r d a y t o d i g e s t i o n s s h o w e d n o i n h i b i t o r y e ff ec t o n

t h e m e t h a n e p r o d u c t i o n . T h e m a x i m u m a m o u n t o f pe el oi l p e r m is s ib l e fo r a

p r a c t i c a l m e t h a n e f e r m e n t a t i o n w a s a b o u t 6 5 # l / li t re p e r d a y ( F ig . 2).

L i g h t - m i c r o s c o p i c o b s e r v a t i o n s s h o w e d t h a t w h e n 6 0/1 1 o f p e e l o il /l it re

p e r d a y w a s a d d e d t o t h e s l u d g e , G r a m - n e g a t i v e r o d s ( c. 2 x 0 "3 /~ m ) p r e s e n ti n t h e p e e l o i l - fr e e s l u d g e d i s a p p e a r e d a n d G r a m - p o s i t i v e r o d s ( c. 3 x 0 "6 /~ m )

a p p e a r e d . F e w e r m i c r o - o r g a n i s m s w e r e o b s e r v e d i n t h e s l u d g e w i t h 7 0 /A o f

pee l o i l / l i t r e pe r day .



164 E. M izuk i, T. Ak ao, T. Saruwatari

i I i

20G

Ec_o

, ~ 1 0 CP

r ,0

A' ;o ' 2'o ' '0

Time (dQy)

8.0

p H 7 .0

6 .0

5.0

Bi i i

0 10 20 30

T i m e ( d a y )

Fig. 1. Mesophilic methane fermentation of C. unshu peel. (A) Time course of gas

product ion. (B) Time course o f pH of sludge. Loads are 0"5 g/litre per day (O) , 1"0 g/litre per

day (I-q), 2"0g/iitre per day (A), 4.0g/litre per day (0 ) and 2g (pectin)/litre per day (A).

T A B L E 1

Effect of Peel Oil on Methane Fermentationa

Lo ad o f Colour Volume Gas composition (%)c Residual

pee l o i l o f o f pee l o i l in

(Id/litre per day) sludge sludge (%)b CH 4 CO z sludge (l~l/litre)

0 Normal 45'5 48"4 51'6 0"0

60 Nor mal 37.5 48.5 51.5 47-7

70 Brown 48.0 26-8 73.2 394"2

° Measured 50 days after the start of fermentation.

b The per cent (v/v) of sludge after cent rifugati on for 10 min at 3000 rpm.

CH4 + CO2 = 100%.



Inhibition o [ digest ion by citrus peel 16 5

"2-

I~ 200

u

1:}o I 0 0

13.

m

A

0 1 ' 0 2 ' 0 3 ' 0 ~ 0 - - 0

T i m e ( d a y )

8 .0

7 .0

pH

6 ,0

5 .0

B

0 10 20 30 ~I0 50

Time (day )

F ig . 2 . I n h i b i t i o n o f m e t ha n e f e r m e n t a t i o n b y p e e l o i l. ( A ) T i m e c o u rs e o f g as p r o d u c t io n .

(B ) T i m e c o u r s e o f p H o f s lu d g e . L o a d s o f p e e l o i l a r e 0 / d / l i t r e p e r d a y ( © ) , 6 0 id / l i t re p er d ay

( O ) , 7 0 / d / l i t r e p e r d a y ( m ) , 8 0 # l / l i tr e p e r d a y ( [ ] ) , 9 0 iLl / l i tre per day (&) . Load of [ ) - l i m o n e n e

i s 82"3/A/l it re p er d ay (A ) . Loa d s o f p ec t in are 2 g / l i t re p er d ay in a l l cases .

T a b l e 1 s u m m a r i z e s t h e re s u lt s o b t a i n e d 5 0 d a y s a f t e r t h e o n s et o f

f e r m e n t a t i o n . I n t h e d i g e s t i o n f e d w i t h 6 0 # 1 o f p e e l o i l / l i t r e p e r d a y , t h e

v o l u m e o f s lu d g e (3 7" 5 % ) w a s l es s t h a n t h a t i n t h e d i g e s t i o n w i t h o u t p e e l o il .

G a s y i e l d w a s a b o u t 1 0% g r ea t er th a n t h a t o f t h e d i g e s t i o n w i t h o u t p e e l oi l.

A m o u n t o f r e s id u a l p e e l o i l w a s 4 7 .7 /~ l /l it re . T h e c o n c e n t r a t i o n o f m e t h a n e

i n t h e d i g e s t e r g a s e s w a s b e l o w 3 0 % , a n d p e e l o i l a c c u m u l a t e d u p t o

3 94 .2 / d / l i tr e , in t h e d ig e s t ion w i t h 70 / d o f p e e l o i l /l i tr e p e r d ay (Tab le 1).

An ae r ob ic d ige s t ion o f C. unshu p e e l a f t e r t h e r e mov a l o f p e e l o i l

F i g u r e 3 sh o w s t h e r e m o v a l o f p e e l o i l f r o m t h e sl u rr y o f C. unshu b y

a e ra t io n . A e r a t i o n f o r 1 2 h r e m o v e d 9 9 % o f th e p e e l o il . M o r e t h a n 9 9 % o f



166 E. Mizuki , T . Akao , T . Saruwatar i

1 0 0 - -- -- (3 -~

fE

I1)L

5O

I1)

F i g . 3 . R e m o v a l o f p e e l o i l f r o m s lu r ry o f C . ~'

unshu p e el. P ee l w a s c o m m i n u t e d f o r 1 0 m i n a n d

h o m o g e n i z e d f o r 2 0 r a i n . T h e s l u r r y o f p e e l w a s

t h e n i n c u b a t ed in a n o p e n - t o p p e d f e rm e n t er a t 0

30°C with a ir spa rging (15 l i tres /1 .5 l itres feed stock o 6 1'2p e r m i n) . T i m e ( h r )

t h e p e e l o il w a s a l s o r e m o v e d b y s t e a m d i s t il l a t io n f o r 4 0 m i n . N o i n h i b i t i o n

o f f e r m e n t a t i o n w a s o b s e r v e d o v e r 4 4 d a y s i n t h e d i g e s t i o n s f e d w i t h o i l-

r e m o v e d p e e l ( F i g . 4 ) .

D I S C U S S I O N

T h i s r e p o r t d o c u m e n t s t h e f i r s t o b s e r v a t i o n t h a t C . u n s h u p e e l c a u s e s

s i g n i fi c a n t i n h i b i t i o n o f a n a e r o b i c d i g e s ti o n . S e v e r a l i n v e s t i g a t o r s ( M c N a r y

e t a l . , 1 9 51 ; L a n e , 1 9 8 0) h a v e r e p o r t e d t h a t i n h i b i t i o n o f a n a e r o b i c d i g e s t i o n

i

Bv

o

L.,

~ ' 1 0 0+ 11'0 2tO 3ro ~ '0

T i m e ( d a y )

F i g . 4 . A n a e r o b i c d i g e s t i on o f C. unshu pee l a f ter rem ova l o f pee l o i l. Pee l oi l wa s rem ove d

from feeds to cks by aera t ion (O ) a s show n in F ig . 3 or by steam d i s t i ll a t ion ( /X) . T h e loads o f

organic substances were 2 .0 g / l itre p er day .



Inhibition o f digestion t5)' citrus peel 167

f e d w i t h c i t r u s w a s t e s w a s m a i n l y d u e t o p e e l o i l . I n o u r p r e s e n t s t u d y ,

C. unshu p e e l o il a ls o i n h i b i t e d t h e a n a e r o b i c d i g e s t i o n . T h e l i m i t o f p e e l oi l

loa d in g (c. 65 # l / l i tr e pe r day ) in th i s s tud y w as le s s tha n tha t (88-2/~l /li tr e pe r

d a y ) r e p o r t e d b y L a n e ( 1 9 8 4 ) .

I n a l l e x p e r i m e n t s , d i g e s t io n w a s s a id t o h a v e f a i le d w h e n t h e p H o f t h e

s ludg e fe l l to 6 .4 (Lane , 1980 , 1984). The d ig e s t ion w i th 70/~1 o f pee l o i l / l i t r e

pe r d ay , eq u iv a len t to the pee l o i l co n te n t o f 2 g (d .w .) o f pee l , f a i l ed in 46

d a y s a f t e r t h e s t a r t o f f e r m e n t a t i o n . S i n c e t h e d i g e s t i o n f e d w i t h 2 g o f p e el

( d .w . ) /l it re p e r d a y f a il e d i n 8 d a y s , it i s l i k e ly t h a t o t h e r s u b s t a n c e s p r e s e n t i n

C. unshu p e e l a l s o c a u s e d i n h i b i t i o n o f th e a n a e r o b i c p r o c e s s ( F ig s 1 a n d 2). A

d i g e s t i o n t o w h i c h w a s a d d e d 8 2 - 3 / A o - l i m o n e n e / l i t r e p e r d a y f a i l e d i n

4 3 d a y s . I n t e r e s t i n g l y , h o w e v e r , a d i g e s t i o n f e d w i t h 9 0 ~1 p e e l oi l c o n t a i n i n g

8 2 " 3 pl o f D - l i m o n e n e f a il e d i n 33 d a y s . T h i s s u g g e s t s t h a t t h e o t h e r

c o m p o n e n t s c o n t a i n e d i n p e el o il a ls o h a v e a n i n h i b i t o r y e f fe ct ( F ig . 2). T h e s e

o b s e r v a t i o n s a r e i n a g r e e m e n t w i t h e a r l i e r f i n d i n g s r e p o r t e d b y L a n e (1 98 0).

T h e a d d i t i o n o f 6 0 /A o f p e e l o i l/ l it r e p e r d a y t o t h e d i g e s t e r r e s u l t e d i n a

s u b s t a n t i a l c h a n g e i n t h e m i c r o b i a l f l o r a , f o l l o w e d b y a g a s p r o d u c t i o n

g r e a t e r t h a n t h a t i n t h e c o n t r o l ( w i t h o u t p e e l oi l) ( F ig . 2 (A ) ) . I n t h i s c a se , a

s m a l l a m o u n t o f r es i d u a l p e el o il w a s d e t e c t e d a t t h e e n d o f th e t e st p e r i o d

( T a b l e 1 ) . T h e s e r e s u l t s s u g g e s t t h a t m i c r o - o r g a n i s m s i n t h e s l u d g e

d e g r a d a t e d p e e l o i l t o s o m e e x t e n t , a n d t h e m i c r o b i a l f l o r a c h a n g e d t op r o d u c e g a s e s a t h i g h e r r a t e s a f t e r t h e a d d i t i o n o f p e e l o i l b e l o w t h e l i m i t

l o a d i n g .

T h e r e a r e s o m e r e p o r t s o n t h e r e m o v a l o f p e e l o il f r o m c i t r u s w a s te s b y

a e r a t i o n ( W o l f o rd et al., 1952 ; Lan e , 1980). In the p re sen t w ork , pee l o i l f rom

a s l u r r y o f C. unshu p e e l c o u l d b e s u c c e s s fu l l y r e m o v e d b y s t e a m d i s t i l l a t io n

o r a e r a t i o n ( F i g . 3 ) . D i g e s t i o n s f e d w i t h o i l - f r e e p e e l s l u r r y p r o v e d n o r m a l

( F i g . 4 ) . I t a p p e a r e d t h a t a n a e r o b i c d i g e s t i o n o f C. unshu p e e l w a s a b l e t o

p r o c e e d w h e n p e e l oil w a s r e m o v e d f r o m t h e p e el b y a p p r o p r i a t e m e t h o d s .

A p i l o t - s c a l e a n a e r o b i c d i g e s t i o n e x p e r i m e n t i s n o w i n p r o g r e s s t oe s t a b l i s h a p r a c t i c a l m e t h o d f o r a n a e r o b i c t r e a t m e n t o f C. unshu peel.

A C K N O W L E D G E M E N T

T h e a u t h o r s a c k n o w l e d g e D r S. M u r a o a n d D r M . O h b a f o r t h e ir r e vi ew o f

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