influence of manufacturing variables on the characteristics and effectiveness of chitosan products....
TRANSCRIPT
Influence of Manufacturing Variables on the Characteristics and Effectiveness of Chitosan
Suspensions Products. 11. Coagulation of Activated Sludge
W. A. BOUGH, A. C. M. WU, T. E. CAMPBELL, M. R. HOLMES, and B. E. PERKINS, Marine Extension Service,
University of Georgia, Brunswick, Georgia 31520 and Department of Food Science and Department of Agriculture Economics,
University of Georgia Coll eg e of Agricu It ure Experiment Stat ions , Georgia Station, Experiment, Georgia 30212
Summary Chitosan samples manufactured under different conditions were compared for
effectiveness of coagulating an activated sludge suspension grown on vegetable canning wastes. Computer analysis of data from Buchner funnel filterability tests resulted in quadratic polynomial equations describing the response curves for volume of filtrate versus dosage, expressed as &liter chitosan1lOO g sludge suspended solids (SSS). The quotient of the filtrate volume and dosage at the inflection points of the equations obtained for 10 test samples and 1 commercial chitosan sample were compared to evaluate the response (effectiveness) per unit amount for each chitosan product. The product made by a standard procedure (deproteinated with 3% NaOH at 100°C for 1 hr, demineralized with IN HC1 at ambient temperature for 30 min, and deacetylated with 50% NaOH at 145- 150°C under NZ for 5 or 15 min) gave the best performance as a coagulating agent for this activated sludge system. Other products, including the commercial preparation, required higher dosages to achieve the same effectiveness. Products deacetylated in the presence of air rather than nitrogen decreased waste treatment effectiveness, which approximated the trends of reduced viscosity and molecular-weight distribution. The products containing min- erals were less effective than products from which minerals had been removed prior to deacetylation, but they were more effective than the enzyme treated sample and the commercial product. In general, although chitosan products obtained after 15 min deacetylation were more effective than those receiving 5 min deacetylation, effectiveness did not correlate linearly with viscosity and molecular-weight distri- bution trends. However, chitosan products deacetylated for 15 min did show that the higher-molecular-weight products (0.65- 1.1 x lo6) were more effective coagu- lating agents for activated sludge than the manufactured product having the lowest molecular weight (0.47 X lo6) and the commercial reference sample (0.56 X lo6). Thus, higher values for molecular weight were predictive of greater effectiveness for coagulation of activated sludge suspensions.
Biotechnology and Bioengineering, Vol. XX, Pp. 1945-1955( 1978) @ 1978 John Wiley & Sons, Inc. 0006-359217810020- 1945$01 .OO
1946 BOUGH ET AL.
INTRODUCTION
Previous studies have shown chitosan to be an effective treatment agent for coagulation of suspended solids (SS) in processing wastes from vegetable,' poultry,2 egg,3 seafood, and meat4 plants. Chitosan is also useful as a conditioning aid for dewatering activated sludge suspensions resulting from biological treatment of brewing and veg- etable canning wastes.4j5
The commercial chitosan products tested in the above studies were obtained through the National Sea Grant Program from Food, Chemical, and Research Laboratories, Inc. The manufacturing pro- cedure involved extraction of protein and mineral components from shrimp and crab hulls to obtain chitin. Chitin was then treated with 40% NaOH at 130- 150°C for 0.5- 1.0 hr to hydrolyze acetyl groups from the polymer. The resulting polymer, composed of /3-1,4-linked glucosamine residues, most of which have been deacetylated, is called chitosan.
If variants are introduced into the standard manufacturing proc- ess, chitosan products having different characteristics can be man- ufactured, as described in part I (hereafter referred to as I), of this study.6 The purpose of this part of the study is to investigate the performance of the 10 experimental chitosan products and the com- mercial preparation as coagulating agents for activated sludge. Of special interest are those chitosan products still containing mineral components from the shrimp hulls.
METHODS
Collection of Activated Sludge
Activated sludge was collected on a daily basis from the clarifier of a biological treatment plant operated by a local commercial veg- etable processor. The turbidity of the sludge was adjusted with a Hach 2100A turbidimeter to a reading of 1250 formazin turbidity units (FTU) before use. A neutral pH was required for the sludge. Suspended solids (SS) and filterability of the sludge were then determined for each batch before being used for the chitosan eval- uation. The SS concentration of the sludge after adjustment by turbidity measurement was about 7000 mdliter, and filterability of the control was approximately 110 ml in 30 sec.
Chitosan products were prepared using various manufacturing conditions as indicated in I. Variables in different manufacturing
PROPERTIES OF CHITOSAN PRODUCTS. I1 1947
processes involved: methods of deproteination (alkali o r enzy- matic); demineralization versus no demineralization; differing at- mospheres (air or Nz); and time (5 or 15 min) of deacetylation.
Buchner Funnel Test for Coagulated Sludge
The Buchner funnel filterability test was used to evaluate the various chitosan products for coagulation of activated ~ l u d g e . ~ Ac- tivated sludge suspensions (500 ml) were mixed at 20 rpm with a Phipps and Bird mechanical stirrer during the addition of the co- agulants tested, followed by an additional mixing of 2 min at 100 rpm and 3 min at 30 rpm. After mixing, the conditioned sludge was promptly filtered under vacuum (24 in. Hg) through Whatman No. 4 paper in a 11 1 mm i.d. Buchner funnel, and the amount of filtrate collected in 30 sec was measured. Six runs were conducted for each sample using six chitosan concentrations: 8, 12, 16, 20, 24, and 28 mg/liter. The Buchner funnel tests were repeated six times for six batches of activated sludge collected on six different days.
Analysis of Data
Data from six replications of Buchner funnel tests on activated sludge were analyzed using computerized multiple regression. For each chitosan product with six replicated runs, a polynomial equa- tion was developed for generalizing the curves of filterability (ml) versus chitosan dosage (g chitosad100 g sludge suspended solids (SSS)). These equations, describing the response curves for filter- ability of the sludge at different chitosan concentration levels, were used to represent the performances of different chitosan products.
An alternative equation (intercept equation) for each chitosan product was also developed, incorporating dummy variables to ad- just for differences in intercepts of response curves from different trials on the same product. Thus, one intercept equation can de- scribe each of the six individual runs of a certain chitosan product. Dummy variables were used only for the intercept terms (A',,), because it was found that the six response curves for each chitosan product tested on sludges collected on different days had similar shapes so that they could share the same coefficients for terms X , , where n = 1 or 2. For both generalized and intercept equations, several selections of equations with different polynomial degrees were developed.
Coefficients of determination ( R y were calculated to explain the percentage of variation in the predicted response curves. Standard
1948 BOUGH ET AL.
errors of the estimates and confidence levels were also calculated. Different selections of polynomial equations were compared, based on these statistical parameters, to choose the best-fitted ones.
The best-fitted polynomial equations (quadratic), developed for the response curves of different products, were used to calculate the inflection point of the curves by letting the first derivatives of the equations be zero. The values of the independent variables (concentrations of chitosan in g chitosanilO0 g SSS) and the mag- nitude of the dependent variable (volume of sludge filtrate) at the inflection point were taken as the points of optimum performance. Different chitosan products were compared based on these inflec- tion points. Since inflection points of different products occurred at different chitosan concentrations, both independent and dependent variables should be considered for product Comparison. However, when the ratio of the two variables were taken, the quotient became a single parameter representing the maximum effectiveness of the product. Thus, comparison was simplified using effectiveness quo- tients.
RESULTS AND DISCUSSION
For the sake of brevity, raw data values for X (g chitosanilO0 g SSS) and Y (ml filtrate) have been omitted. These raw data were incorporated into the generalized and intercept equations (of the forms illustrated in Table I) to yield the computed values listed in Tables I1 and 111. Coefficients for the multiple regression equations developed for only one chitosan product (111-A) are shown in Table I to illustrate the form of the equations and the statistical parameters used for selecting the best-fitted polynomial equations. The values at the inflection points for the volume of filtrate (Y) and concentra- tion of chitosan ( X ) , expressed as the ratio of chitosan to SS con- centrations, are shown in Table I1 for both the intercept equations and the generalized equations describing the different products. The coefficients of determination (R2) for the intercept and generalized regression equations (Table 11) show that the intercept equations represent the data better than the generalized equations, as was expected. The R 2 terms for the intercept equations show that 84- 95% of the Y values (volume of filtrate at the inflection point of the curve) were explained by the developed multiple regression equa- tions, which indicated that the response curves could be reliably estimated using multiple regression. Differences between the six replications could be adequately accounted for by adjusting the
PROPERTIES OF CHITOSAN PRODUCTS. I1 I949
TABLE I Multiple Regression Analyses on Polynomial Equations Developed for Predicting Filtrate Volumes Collected in the Buchner Funnel Filterability Tests on Activated Sludge in Response to Changes in Concentration for the Chitosan Product, 111-A
Regression Variable Description coefficient /3 value t value
I ) Generalized equationa
Y volume filtrate (ml) x o regression intercept x, X = (chitosan conc./SS)
XZ X 2
x 100
2) Intercept equationh
volume filtrate (ml) regression intercept (0, 1) dummy run 1 (0, 1) dummy run 2 (0, 1) dummy run 3 (0, 1) dummy run 4 (0, 1) dummy run 5 X = (chitosan conc./SS)
X 2 x 100
159.145955 853.902580 3.664667 3,66467
- 1097.805514 -2.810639 -2.81064
113.518547 69.157888 0.714689 8.82541 50.827098 0.525255 6.51360 56.508098 0.583964 7.24568 40.858770 0.422241 5.23235 35.684284 0.368767 4.56783
890.588887 2.762632 7.29295
-1171.997221 -2.168831 -5.71548
a Multiple R 2 = 0.5291; adjusted R 2 = 0.5005; standard error of estimate =
Multiple R = 0.890877; adjusted R = 0.863596; standard error of estimate =
25.848525.
13.507929.
regression intercept using dummy variables, as illustrated in Table I. Consequently, a good estimate of the inflection point of the curves was possible, and those points were taken as the optimum concen- trations for use of different chitosan products.
The values of X and Y at the inflection points indicated in Table I1 are also shown as bar graphs in Figures I and 2 . The A products shown in Figure 1 were all deacetylated for 5 min at 145°C. The B products in Figure 2 received 15 min hydrolysis at 145°C. In general, superior performance is indicated by greater height of the bar (vol- ume of filtrate, Y) associated with the lower optimum concentration of the chitosan sample in relation to suspended solids ( X ) .
The performance of different chitosan products was compared as shown in Table 111. The quotient of Y divided by X indicates the optimal response per unit amount of a chitosan product (ml/g chi-
TA
BL
E I1
Su
mm
ary
of P
aram
eter
s at
Inf
lect
ion
Poin
ts o
f th
e R
espo
nse
Cur
ves O
btai
ned
from
the
Slu
dge
Ass
ay o
n V
ario
us C
hito
san
Sam
ples
~~
Inte
rcep
t eq
uatio
n G
ener
aliz
ed e
quat
ion
Bat
ch
No.
Y
a ra
nge
Y" (a
vg)
X"
R 2 t
r Y"
X
a R
2 11
111-
A
282.
7-35
1.9
324.
9 0.
3799
0.
8909
32
5.2
0.38
89
0.52
91
111-
B 28
3.0-
345.
7 32
2.8
0.38
34
0.84
1 1
323.
1 0.
3924
0.
4768
IV-A
28
7.6-
338
.9
318.
6 0.
4737
0.
9308
31
8.4
0.47
29
0.69
28
IV-B
29
1.4-
343
.5
319.
0 0.
4171
0.
9041
31
9.5
0.42
38
0.62
97
V-A
27
5.3-
338.
5 30
9.9
0.41
92
0.86
39
310.
5 0.
4299
0.
4666
V
-B
275.
5-34
6.3
311.
6 0.
3872
0.
9006
31
2.2
0.40
05
0.49
40
VI-
A
289.
7- 3
43.3
32
1.0
0.46
00
0.94
96
323.
1 0.
4769
0.
7685
V
I-B
27
7.9-
334
.9
304.
8 0.
3927
0.
9191
30
5.5
0.40
49
0.64
74
W
0
C
Q z m
4
9 r
VII
-A
279.
5-33
5.0
307.
1 0.
4019
0.
8994
30
8.1
0.41
60
0.59
07
VII
-B
276.
9-33
3.9
300.
5 0.
4097
0.
9173
30
2.6
0.43
88
0.60
47
4-74
27
5.4-
322
.1
301.
5 0.
4239
0.
8388
30
2.5
0.43
79
0.61
04
100
g ss
s.
a Y
= v
olum
e of
filt
rate
at i
nfle
ctio
n po
int o
f cu
rve
(ml)
. X =
ratio
of c
hito
san
conc
entr
atio
n to
SS
at in
flec
tion
poin
t of
cur
ve in
g c
hito
sani
R =
coe
ffic
ient
of
dete
rmin
atio
n of
per
cent
var
iatio
n in
Y e
xpla
ined
by
a pa
rtic
ular
equ
atio
n.
TA
BL
E 1
11
Effe
ctiv
enes
sa o
f C
hito
san
Prod
ucts
Eva
luat
ed b
y B
uchn
er F
unne
l Fi
ltrat
ion
Tes
ts o
n th
e Fi
ltera
bilit
y of
the
Chi
tosa
n T
reat
ed S
ludg
e
111
I) G
roup
A (
deac
etyl
ated
for
5 m
in)
Run
1
926.
20 (
l)h
R
un 2
87
7.95
(I)
R
un 3
89
2.90
(1)
R
un 4
85
1.71
(1)
R
un 5
83
8.09
(I)
Ave
rage
87
7.37
(1)
2) G
roup
B (
deac
etyl
ated
for
I5
min
)
Run
1
901.
68 (
1)
Run
2
868.
14 (
1)
Run
3
876.
93 (
1)
Run
4
829.
92 (
1)
Run
5
836.
02 (
1)
I11
Ave
rage
86
2.54
(1)
Sam
ple
~~
VII
V
4-
74
VI
IV
% 78
0.46
(3)
78
8.21
(2)
72
6.73
(4)
71
5.67
(5)
70
9.67
(6)
=! E % E z m
83
3.42
(2)
80
7.58
(3)
75
9.82
(4)
74
6.37
(5)
70
2.52
(6)
7J
715.
54 (
6)
773.
84 (
2)
753.
73 (
3)
726.
01 (
4)
710.
77 (
5)
763.
59 (
2)
705.
69 (
3)
702.
02 (
4)
687.
% (
5)
645.
54 (
6)
738.
19 (
2)
723.
02 (
3)
703.
41 (
4)
696.
13 (
5)
655.
56 (
6)
0
-
-
-
-
-
777.
90 (
2)
741.
33 (
3)
723.
60 (
4)
711.
38 (
5)
685.
83 (
6)
3 9 z
V
VI
IV
VII
4-
74
844.
86 (
2)
788.
64 (
4)
799.
51 (
3)
720.
69 (
6)
726.
73 (
5)
726.
01 (
6)
797.
33 (
2)
785.
73 (
4)
788.
10 (
3)
793.
54 (
2)
755.
94 (
3)
741.
16 (
4)
728.
35 (
5)
702.
01 (
6)
894.
48 (
2)
852.
87 (
3)
823.
52 (
4)
814.
98 (
5)
759.
82 (
6)
0
U
C 0
+I v
726.
34 (
5)
e(
786.
63 (
2)
766.
01 (
3)
738.
52 (
4)
733.
92 (
5)
703.
41 (
6)
e(
-
~ -
~ _
_
823.
37 (
2)
789.
84 (
3)
778.
16 (
4)
756.
15 (
5)
723.
60 (
6)
a Ef
fect
iven
ess
is ex
pres
sed
as th
e qu
otie
nt o
f m
l filt
rate
/g c
hito
sad1
00 g
SSS
. N
umbe
rs r
epre
sent
the
rank
ing
of t
he q
uotie
nts
obta
ined
by
com
pari
son
with
in t
he r
uns.
1952 BOUGH ET AL.
3 i
31 - - E - g 3
3 0 > 31 I- U
YI
3c = Y
30
V
VI I
.40 42 . A 4
OPTIMUM DOSAGE x '
IV
1 .48
I
Fig. 1. Optimum chitosan dosage (&I00 g SSS) and maximum filterability for 5 min, determined by the Buchner funnel filtration test with activated sludge.
tosan/100 g SSS), and this quotient was calculated from the intercept equations for each replicate of each product. Since the data shown in Table 111 were obtained by polynomial regression analysis, they were compared only qualitatively by observation without further statistical analysis. The orders of magnitude for different products were generally consistent throughout the five replicate runs, as shown by the ranking system in Table 111. The sixth replicate runs for all products were eliminated in this analysis because these data were generally much lower than other runs, owing largely to the unusual conditions of the sludge collected that day. The order of effectiveness for A products was: 111-A, VII-A, V-A, 4-74, VI-A, and IV-A. The order of effectiveness for the B products was: 111-B, V-B, VI-B, IV-B, VII-B, and 4-74. The Y/X quotient at the inflection point may also be proportional to the effectiveness of a product used in a concentration lower than its optimal dosage, but it is not related to the effectiveness of a product at a concentration higher than its optimum.
These results show that procedure I11 produced superior products for coagulation of suspended solids in activated sludge. Figures 1 and 2 show that products 111-A and 111-B gave the highest filtration volumes with the lowest chitosan concentrations at the inflection points of their response curves. Although product VI-A gave a maximum filterability similar to 111-A, it required higher dosages of
PROPERTIES OF CHITOSAN PRODUCTS. I1 1953
chitosan to achieve the same effectiveness. The concentrations of chitosan products required for obtaining the maximum filterability in group B were generally lower than those in group A, as shown in Table 11, as well as in Figures 1 and 2.
The quotient of the filtration volume divided by the chitosan concentration (g chitosad100 g SSS) at the inflection point of the response curves reduces the data to a single number for each trial and product, as described by the intercept equations. Table I11 indicates that for those products which received 5 min deacetylation (A series), the basic manufacturing process (111-A) was the most effective with a YIX quotient of 877.37 mlig chitosan/100 g SSS. The quotient for product VII-A, 777.90, which was deproteinated with an enzymatic hydrolysis procedure, was lower than the quotient for product 111-A, which was extracted with dilute alkali. The quotients for products 111-A and VII-A were each different from product V- A (741.33 mlIg chitosanIlO0 g SSS), which was deacetylated for 5 min in an air atmosphere rather than under nitrogen gas, as was the case for the other products in the A series. Thus, the presence of air resulted in decreased waste treatment effectiveness as well as decreased viscosity and molecular-weight distribution as shown in I . Degradation due to the presence of air was also cited by Rigby in his patent on chitosan m a n ~ f a c t u r e . ~
- .;8 4'0 -.4'2 .4; ' . i 6 .41)
OPTIMUM DOSAGE x 100
Fig. 2. Optimum chitosan dosage (dl00 g SSS) and maximum filterability (mi) of different chitosan products deacetylated for 15 min, determined by the Buchner funnel filtration test with activated sludge.
1954 BOUGH ET AL.
Both procedures IV and VI yielded products containing minerals contributed by the shrimp hulls. It was of special interest to test their effectiveness as waste treatment agents. The chitosan manu- facturing process could be simplified to perform protein extraction and deacetylation of chitin only, if a chitosan-minerals product was feasible or desirable. The problem of logistics and supply of hydro- chloric acid has been reported to be a major limiting factor in developing a chitosan industry on Kodiak Island in Alaska, a major processing center of shrimp and crab in the United States.8 A chitosan-minerals product may be of special interest to this indus- try.
The mineral-containing products, IV-A and VI-A, which had been deacetylated for 5 min, were less effective than the demineralized products (111-A and VII-A) and any of the other products, including the commercial preparation, 4-74, as shown in Figure 1 and Table 111. On the other hand, when the deacetylation time was increased to 15 min (Fig. 2 and Table 11), chitosan-minerals product VI-B, which had been enzymatically deproteinated, the product IV-B, which had been deproteinated with alkali but not treated with acid, were both more effective than products VII-B and 4-74. Similarly, the mineral-containing product IV-B was less effective than the demineralized product 111-B. Yet, product VI-B was slightly more effective than its demineralized counterpart, product VII-B. The authors postulate that the presence of high concentrations of min- erals (mainly calcium salts) in chitosan products may reduce the chance of interaction among chitosan polymers and suspended sol- ids in the sludge. Thus, although the product without deminerali- zation had higher molecular-weight values owing to the elimination of the acid treatment as shown in I , it was not more effective than the demineralized product with slightly lower molecular weight. The effect of the presence of minerals overshadows the effect of greater molecular weight.
In general, chitosan products obtained after 15 min deacetylation were more effective than the corresponding products obtained after 5 min deacetylation, as judged by YIX values shown in Table 11. Exceptions to this statement were that there was no important difference in effectiveness between products 111-A and 111-B, and VII-A was only slightly more effective than VII-B. A clear trend for the chitosan products deacetylated for 15 min was that the order of effectiveness showed that the higher molecular-weight products, 111, V, VI, and IV (0.65- 1.1 x lo6) were superior coagulants to product VII and the commercial sample 4-74 (0.47-0.56 x lo6)
PROPERTIES OF CHITOSAN PRODUCTS. I1 I955
having lower molecular weights. Thus, the higher-molecular-weight values were predictive of greater effectiveness for coagulation of activated sludge suspensions. These results suggest that the value of the high-pressure liquid chromatography (HPLC) technique in product development studies is to develop the optimum product for a particular application where maximum effectiveness is desired.
The commercial batch (4-74) of chitosan was obtained through the National Sea Grant Program from Food, Chemical, and Research Laboratories, Inc., of Seattle, Washington. Rhozyme-62 concentrate was a gift from Rohm and Haas Company, of Philadelphia, Penn. The technical assistance of William B. Miller, Mrs. Sue Mc- Cullough, and Mrs. Sue Mahle is gratefully acknowledged. This work is a result of research sponsored (in part) by the Georgia Sea Grant Program, supported by NOAA Office of Sea Grant, Department of Commerce, under Grant No. 04-6-158-44115.
References 1 . W. A. Bough, J . Food Sci., 40, 297 (1975). 2. W. A. Bough, A. L. Shewfelt, and W. L. Salter, Poultry Sci., 54, 992 (1975). 3. W. A. Bough, Poultry Sci., 54, 1904 (1976). 4. W. A. Bough, Proc. Biochem.. 11(1), 13 (1976). 5. W. A. Bough, D. R. Landes, J . Miller, C. T. Young, and T. R. McWhorter,
Procerdings of Sixth Nutionul S . ~ ~ ~ i p o s i u m on Food Processing Wastes (U.S. En- vironmental Protection Agency, EPA-600/2-76-224, 1976), pp. 22-48.
6. W. A. Bough, W. L. Salter, A. C. M . Wu, and B. E. Perkins, Biotechnol. B iorng . . 20, 193 1 (1978) (preceding paper).
7. G. W. Rigby, U.S. Patent No. 2,040,879 (1936). 8. CRESA (Food, Chemical and Research Laboratories, Inc. and Engineering
Science of Alaska), Environmental Protection Agency Project No. 12130FJQ (Supt. of Documents, Washington, D.C., 1971).
Accepted for Publication April 3, 1978