Production of poly(3-hydroxybutyric-

co-3-hydroxyvaleric acid) by fed-batch

culture of Alcaligenes eutrophus
with substrate control using
on-line glucose analyzer
Beom Soo Kim, Seung Chul Lee, Sang Yup Lee, Ho Nam Chang, Yong Keun Chang,
and Seong Ihl Woo
BioProcess Engineering Research Center and Department o f Chemical Engineering, Korea Advanced
Institute o f Science and Technology, Taejon, Korea

Alcaligenes eutrophus NCIMB 11599 was cultivated to produce poly(3-hydroxybutyric-co-3-hydroxyvaleric acid),

P(3HB-co-3HV), from glucose and propionic acid by a two-stage, fed-batch culture technique. The glucose
concentration of the culture broth was controlled at 10-20 g 1-1 usitlg an on-line glucose analyzer during the whole
culture period. Nitrogen limitation was applied at a cell concentration of 60- 70 g 1- l, when the glucose feed was
replaced by the mixture of glucose and propionic acid. The effect of the ratio of propionic acid to glucose (P/G ratio)
in the feed on the copolymer production was examined. The final copolymer concentrations of 117, 74, and 64 g l - 1,
polymercontentsof74%, 57%, and56.5%ofdrycellweight, andproductivitiesof2.55, 1.67, and1.64g1-1 h l
were obtained when the P/G ratios in the feed were 0.17, 0.35, and 0.52 (mol propionic acid mo1-1 glucose),
respectively, showing reduced production with increasing P/G ratio. However, the 3-hydroxyvaleric acid (3HV)
fraction in the copolymer increased with increasing PIG ratio, resulting in 14.3 mol% of 3HV at the PIG ratio of 0.52
(tool mol- 1). The concentration of propionic acid in the culture broth was maintained below 1.3 g l - 1 when the PIG ratio
in the feed was O.17 or O.35 (tool m ol - 1), but increased gradually when using the ratio of O.52 (mol mol - 1).

Keyworfls:Poly(3-hydroxybutyric-co-3-hydroxyvaleric
acid); P(3HB-co-3I-IV);Alcaligeneseutrophus; highcell density
culture; fed-batchculture;on-lineglucoseanalyzer;substrate concentrationcontrol

Introduction that of synthetic nondegradable plastics, it is important to

improve the fermentation and separation processes.
Polyhydroxyalkanoic acids (PHAs) are a class of intracellu-
A glucose-utilizing mutant strain of Alcaligenes eutro-
lar carbon and energy storage materials accumulated by
phus has been employed for the production of P(3HB-co-
many microorganisms under unfavorable growth condi-
3HV) by Imperial Chemical Industries (UK) using glucose
tions such as limitation by N, P, S, Mg, and/or O2.1,2 These
and propionic acid as substrates. The 3-hydroxyvaleric acid
polymers have recently attracted considerable research ef-
(3HV) content of P(3HB-co-3HV) can be regulated by the
fort because of their potential use as biodegradable ther-
variation of the ratio of propionic acid to glucose in the
moplastics. At present, the PHA copolymer of the greatest
feeding solution) Also, P(3HB-co-3HV) having a wide
industrial interest is poly(3-hydroxybutyric-co-3-hydroxy-
range of 3HV mole fractions (0-90 tool%) could be ob-
valeric acid) [P(3HB-co-3HV)], because it is more flexible
tained from A. eutrophus by using butyric and pentanoic
than the homopolymer, poly(3-hydroxybutyric acid) [P(3HB)]. 3
acid as substrates. 4
Since the cost of PHA production is high compared with
Fed-batch culture has been widely applied to achieve a
high cell density, which, in many cases, results in high pro-
ductivity and yield.5 With the development of on-line mon-
Address reprint requests to Dr. Ho Nam Chang at the Department of itoring systems, the substrate concentration in the culture
ChemicalEngineering,Korea AdvancedInstituteof Science and Tech-
nology,373-1 Kusung-dong,Yusung-gu,Taejon305-701,Korea broth can be directly monitored and controlled at the de-
Received25 August 1993;accepted 13 January1994 sired level. Suzuki et al. 6,7 developed a method to control

556 Enzyme Microb. Technol., 1994, vol. 16, July © 1994 Butterworth-Heinemann
 Production of P(3HB-co-3HV) by A. eutrophus: B. S. Kim et al.
methanol concentration for the production of P(3HB) by a
methylotroph. On-line glucose monitoring and control sys-
tems were also developed, s,9 Recently, we found that main-
taining glucose concentration at 10-20 g I - 1 was important
for efficient production of P(3HB) by A. eutrophus, and
obtained a high concentration of P(3HB) (121 g 1-1) at 50
h in a fed-batch culture with glucose concentration control
using an on-line glucose analyzer. I°
Despite significant effort, the final concentration of
P(3HB-co-3HV) obtained has been relatively low. Ramsay
et al. 11 produced 17 g 1- 1 of P(3HB-co-3HV) containing 5
tool% of 3 H V by constant feeding of propionic acid during
the polymer accumulation phase. Doi z also used a constant
feeding strategy to produce 6.4 g 1-1 of P(3HB-co-3HV)
with 59 m o l % of 3 H V using pentanoic acid as the substrate.
It has been reported that the growth ofA. eutrophus is
inhibited by as little as 0.1% (wt wt - 1) propionic acid. 3 Two
methods may overcome this problem. A strain that can
tolerate higher concentrations of propionic acid can be de-
veloped either by isolating a new organism or by improving
the existing strain by mutation. Second, the development of
a feeding strategy that allows the propionic acid concentra-
tion to be controlled at low level can be considered. Kim et
al. 12 developed a pH-stat feeding m e t h o d to maintain the
propionic acid concentration at a low level. However, the
final concentration of the product obtained was too low to
allow economical production of the copolymer.
In this study, we developed a new feeding strategy em-
ploying an on-line glucose analyzer for the production of
P(3HB-co-3HV) to a high concentration with high produc-
tivity by A. eutrophus. W e also examined the effect of the
molar ratio of propionic acid to glucose in the feeding so-
lution on the final concentration, molar fraction of 3HV,
and productivity of the copolymer.
Materials and methods
Organism and media
The strain used in this study wasA. eutrophusNCIMB 11599,13,14
which is a glucose-utilizing mutant ofA. eutrophusH16.
The medium for the seed culture was (per liter) l l: glucose, 10
g; (NH4)2SO4, 1 g; KH2PO4, 1.5 g; Na2HPO4"12H20, 9 g;
MgSO4-7H20, 0.2 g; trace element solution, 1 ml. The trace ele-
ment solution contained (per liter)IS: FeSO4"7H20, 10 g;
ZnSO4-7H20, 2.25 g; CuSO4"5H2O, i g; MnSOo-4-5H20, 0.5 g;
CaCI2"2H20, 2 g; Na2B407-10H20, 0.23 g; (NH4)6Mo7024, 0.1
g; 35% HCI 10 ml. The initial medium for the fed-batch culture
was (per liter)J6: glucose, 20 g; (NH4)2SO4, 4 g; KH2PO4, 13.3 g;
MgSO4.7H20, 1.2 g; citric acid, 1.7 g; trace element solution, 10
ml. Glucose and MgSO4.7H20 were autoclaved separately and
were added aseptically to the medium. The pH of the medium was
adjusted to 6.8 with NaOH.
Flask culture
Two-stage flask culture was carried out by the method of Doi et
al.4 to investigate the characteristics of P(3HB-CO-3HV) produc-
tion by A. eutrophus.
Fed-batch culture
The seed culture was prepared in a 250-ml flask on a reciprocal
shaker at 30°C for 1-2 days. One hundred milliliters of the seed
culture was used to inoculate the fermentor (2.51, Korea Fermen-
tot Company, Incheon, Korea) equipped with two six-bladed disk-
turbine impellers and three baffles. The initial volume of the cul-
ture was 800 ml. Temperature and pH were controlled at 34°C and
6.8, respectively. The pH was controlled with 2 Y HCI and 28%
NH4OH, which was replaced by 5 y NaOH/KOH during nitrogen
limitation. Dissolved oxygen concentration was measured with a
dissolved oxygen concentration meter (DKK Corporation, Tokyo,
Japan), and was maintained at above 20% of air saturation by
increasing the agitation speed and the aeration rate up to 1,000 rev
min - 1 and 1.5 1 min - 1, respectively.
Control of glucose feeding using on-line
glucose analyzer
Culture broth was continuously taken out and recycled by a peri-
staltic pump at the speed of ca. 100 ml rain- 1. The culture broth
was continuously filtered at ca. 0.1 ml min- 1 by a cross-flow fil-
tration unit composed of a ceramic membrane module (pore size
0.4 ~m, 6 m m x 80 mm, Ashai Kasei Co., Tokyo, Japan) manu-
factured in the authors' laboratory. Glucose concentration in the
culture broth was monitored every 10 min by automatically inject-
ing the filtered sample to the on-line glucose analyzer (model
2700, Yellow Springs Instruments, OH). The rest of the filtered
sample was discharged as waste. The concentrated glucose solu-
tion (700 g 1- l) was fed to control the glucose concentration at the
desired value using a peristaltic pump (Cole-Palmer Instrument
Co., IL) interfaced to a personal computer (IBM AT). This pump
was regulated using a PID control algorithm.
Effect of P/G ratio on P(3HB-co-3HV) production
The effect of P/G ratio in the feeding solution on P(3HB-co-3HV)
production was examined by carrying out three runs of fed-batch
culture. To allow polymer accumulation, nitrogen limitation was
applied when the cell concentration reached 60-70 g 1- 1. At the
same time, the glucose feed (700 g 1- 1) was replaced by the mix-
ture of propionic acid and glucose. The amounts of propionic acid
added to 1 1of glucose solution (700 g 1- 1) were 50, 100, and 150
g, which corresponded to P/G ratios of 0.17, 0.35, and 0.52 (mol
propionic acid mol- 1 glucose), respectively.
Analytical procedures
Cell growth was monitored during the fermentation by measuring
optical density at 650 nm with a spectrophotometer (Beckman
DU-65, Fullerton, CA). To measure dry cell weight for the deter-
mination of cell concentration, 2-5 ml culture broth was centri-
fuged, washed with distilled H20, and dried under vacuum at
60°C until no further decrease in weight occurred. The concentra-
tion of P(3HB-co-3HV) was determined by gas chromatography
(Varian 3300 Gas Chromatograph, CA) with n-butyric acid as an
internal standard.IV The concentration of propionic acid was de-
termined by high-pressure liquid chromatography on an Aminex
HPX-87H column (Bio-Rad Laboratories Ltd., CA) and a refrac-
tive index detector (L-3300, Hitachi, Japan), using 0.01 N H2SO4
as the mobile phase. The PHA content, P/X (%), was defined as
the percentage of the ratio of PHA concentration to dry cell
weight. Residual biomass was defined as total cell minus PHA.
Results and discussion
Production of P(3HB-co-3HV) by two-stage
flask culture
Initially, we investigated the characteristics of cell growth
and P(3HB-co-3HV) production by two-stage flask culture
ofA. eutrophus. The results of copolymer production from
Enzyme Microb. Technol., 1994, vol. 16, July 557
Papers
different carbon sources are shown in Table 1. The ho- 160 350
mopolymer of 3HB [P(3HB)] was produced from glucose as
a sole carbon source. The cell concentration and P(3HB) 140 -300
content obtained were 9.54 g l-1 and 78% of dry cell 120
weight, respectively. In contrast, a copolymer was produced -250
when propionic acid or pentanoic acid was added to glucose t- 100 "O
._o -200
with 1:1 (wt wt - 1) ratio. The mole fractions of 3HV units in ~ 80
the copolymers were 29.8 and 40.3% for propionic acid and S /

-150 oO
pentanoic acid, respectively. Both the cell concentration o
t-
60
and polymer content decreased considerably in the pres- O
-100 ~
o 40
ence of organic acid. This may be due to the toxicity of
organic acid in culture medium as previously described by 20 -50
Doietal. 4 Even though the use of pentanoic acid resulted in
a higher 3HV fraction, it was much more expensive than
, , , , , , ,
0
15 20 25 30 35 40 45 50
propionic acid, and was not further studied. Time (h)

Fed-batch culture with substrate feeding using on-line

glucose analyzer 20 100

We recently found that controlling the glucose concentra- 0

tion in the culture broth at 10-20 g 1- ] was important for g 16 80 ~
>
the production of P(3HB) to a high concentration with high -1-
productivity by A. eutrophus NCIMB 11599. l° A similar ,_~ 12 60
0
~
approach was taken for the production of P(3HB-co-3HV). ._
E
First, cells were grown to a concentration of 60-70 g 1- ] by
feeding glucose using an on-line glucose analyzer as de- '5 8 4o 9
scribed in Materials and methods. During the polymer ac-
c-
cumulation phase the feeding solution was changed to a O 4 20 v
mixture of glucose and propionic acid. The results obtained 5. X
0
from three runs of fed-batch culture with different P/G (1-

ratios are shown in Figures 1-3. ',0

The final concentrations of cell and PHA decreased
from 158 and 117 to 113 and 64 g 1- ] , respectively, as the
P/G ratio increased from 0.17 to 0.52 (mol m o l - ] ) . The
PHA content in the cell (P/X) and productivity also de-
creased from 74 to 56.5% of cell dry weight and from 2.55 to
1.64 g 1- ] h - 1, respectively. The 3HV fraction in the copol-
ymer continuously increased to 14.3 mol% with increasing
P/G ratio. Propionic acid concentration in the medium was
maintained below 1.3 g ! - ] when the P/G ratio was 0.17 or
0.35 (tool mol - ]), but increased gradually to 7.5 g 1- ] when
using the ratio of 0.52 (mol m o l - l ) . The lower polymer
content with the higher P/G ratio suggests that propionic
acid inhibited polymer synthesis. It was reported that the
optimum propionic acid concentration for 3HV incorpora-
tion was 0.5 g 1-].12
The yields of 3HV from propionic acid (YHv/PA) and
total PHA from two substrates (Yp/s) were calculated from
the data of the amounts of glucose and propionic acid fed to
15 20 25 30 35 40 45 50
Time (h)
Figure 1 Fed-batch culture ofA. eutrophuswith substrate feeding
using on-line glucose analyzer. The P/G ratio in the feed was 0.17
(mol propionic acid mol 1 glucose) during the polymer accumula-
tion phase. Ammonia feeding was stopped when the cell concentra-
tion reached 62 g I - 1 at 25.5 h. (A) ( x ) dry cell concentration (g I - 1);
( I ) PHA concentration (g I-1); (r~) residual biomass concentration (g
I - 1); ( _ ) glucose concentration (g I 1); (__) amount of glucose fed
(g). (B) ([]) P/X (%); (*) 3HV fraction (mol %); (A) propionic acid
concentration (g I-1); (--) amount of propionic acid fed (g)
the cultures. Both yields decreased with increasing propi-
onic acid feed rates. With the P/G ratios of 0.17, 0.35, and
0.52, the final YHV/PAof 0.344, 0.324, and 0.293 (g 3HV g - 1
propionic acid), and the YP/Sof 0.3, 0.221, and 0.2 (g PHA
g - [ substrates), respectively, were obtained.

Table 1 Production of P(3HB-co-3HV) by A. eutrophus from dif- Comparison of P(3HB-co-3HV) productivities

ferent carbon sources using two-stage flask culture
for different microorganisms and culture modes
PHA 3HV Table 2 summarizes the results from a number of studies on
Carbon Concentration Cell dry content fraction the production of P(3HB-co-3HV) by various microorgan-
source (g 1-1) weight (g 1-1) (%) (mol %) isms and culture methods. P(3HB-co-3HV) has been pro-
duced by fed-batch, one-stage or two-stage chemostat cul-
Glucose 20 20 9.54 78 0 ture techniques. The chemostat technique has been
Glucose 10
Propionic acid 10 2.6 33 29.8 frequently applied to investigate physiological phenomena
Glucose 10 and to find a condition that allows maximum productivity.
Pentanoic acid 10 2.2 46 40.3 For the production of PHA byA. eutrophus, however, the
two-stage chemostat has been known to be most useful

558 E n z y m e M i c r o b . T e c h n o l . , 1994, v o l . 16, J u l y

 Production of P(3HB-co-3HV) by A . e u t r o p h u s : B. S. Kim e t al.

140- -350 20 100

120- / -// P-'/ -300 -8

16- -80 ~o
--. 100- -250 ~ >1-
O
t-
O 80- 200 12 -60 ~
e-
¢/) v
~- 60" -150 o
o ~o 8- 40 P
t- 13.
O 40 rl00 (5 .o_
t-
0
.o 4- -20
20 50 o.
o Q..
13_

°10 15 20 25 30 35 40 45 5.0
0 0
55 11 15 20 25 3'0-3'5 4'0 45 5'0 55
Time (h) Time (h)
A
Figure 2 Fed-batch culture of A. eutrophus with substrate feeding using on-line glucose analyzer. The P/G ratio in the feed was 0.35 (mol
propionic acid m o l - 1 glucose) during the polymer accumulation phase. Ammonia feeding was stopped when the cell concentration reached 70
g 1-1 at 28.5 h. Symbols are as in Figure 1

120 300 20 100

/,,.' O
100 250 g 16 80
i /
>
~ 80 200 "~ T
03 O
C ,_~ 12 60
O ._o
t--
~ 60 150 ~ ._o
e- O "O O.
o
o -~ 8 40 o
t- 40 n
O
100 ~ ._o
o t-
O 4 -20
20 50 ~.
£
D_

10 15 2'0 2'5 3'0 3'5 4'0 45 0

15 20 25 3'0 3'5 4'0 45
Time (h) Time (h)

Figure 3 Fed-batch culture of A. eutrophus with substrate feeding using on-line glucose analyzer. The P/G ratio in the feed was 0.52 (mol
propionic acid mol - 1 glucose) during the polymer accumulation phase. Ammonia feeding was stopped when the cell concentration reached 60
g I - 1 at 26 h. Symbols are as in Figure 1

because maximum growth and PHA production cannot oc-
cur simultaneously. Some bacteria such as A. latus and A.
vinelandii UWD, on the other hand, are known to accumu-
late substantial quantities of PHA during their growth
phase.aajs In these cases the single-stage chemostat seems
to be suitable for PHA production, and productivity can be
improved considerably. However, continuous culture has
not often been carried out in practice because of the prob-
lems of contamination and culture instability. Thus, the
method that has been most favored in industry was the
fed-batch culture technique. As can be seen in Table 2, our
results obtained by the fed-batch culture of A. eutrophus
give the highest copolymer concentration and productivity
reported to date. The P(3HB-co-3HV) content in the cell
was relatively lower than that of P(3HB) homopolymer (75-
80%) due to the toxic effect of propionic acid. The 3HV
fraction has been known to depend on the carbon source
used and its concentration. With propionic acid as the sub-
strate, the 3HV fraction in the copolymer was relatively low.
Doi et al. 4 reported that the maximum 3HV fraction of ca.
40 mol% could be obtained using propionic acid as the sole
carbon source. Copolymers with a higher 3HV fraction
could be obtained by adding pentanoic acid. 4
In this paper we reported production of P(3HB-co-
3HV) to a high concentration with high productivity by
fed-batch culture ofA. eutrophus NCIMB 11599 with glu-
cose concentration control. It was found that the 3HV frac-
tion could be increased up to 14.3 mol% by varying the ratio
of propionic acid and glucose in the feeding medium during
the polymer accumulation phase. From a practical point of
view, no attempt was made to increase the molar fraction of
3HV units in the copolymer above 14.3 mol% because the
commercially available copolymer Biopol, having a similar
3HV mole fraction, has been shown to possess desirable
properties. Even though we were able to produce P(3HB-
co-3HV) with satisfactory results, further improvement

E n z y m e M i c r o b . T e c h n o l . , 1994, v o l . 16, J u l y 559

 O'1 Table 2 Summary of P(3HB-co-3HV) 3roduction by various microorganisms and culture methods
O~
O
Cell PHA PHA 3HV
m
Culture concentration concentration content fraction Productivity
N
-< Culture mode Organism Carbon source time (h) (g 1-1 ) (g 1-1 ) (%) (g 1-1 h - l )
(mol %) Reference
3
Fed-batch A. eutrophus Glucose + 46 158 117 74 4.3 2.55 This work
F; propionic acid
Fed-batch A, eutrophus Glucose + 44.5 129 74 57 12.2 1.67 This work
propionic acid
-4 Fed-batch A. eutrophus Glucose + 39 113 64 56.5 14.3 1.64 This work
o propionic acid
:3- Fed-batch A. eutrophus Glucose + 50 17 5 0.34 11
propionic acid
O
Fed-batch A. eutrophus Glucose + 48 9.8 6.4 65 59 0.133 2
pentanoic acid
(.o One-stage chemostat A. latus Sucrose + D = 0.15 h -1 2 43 18.5 0.3 11
t.o
propionic acid
< Two-stage chemostat A, latus Sucrose + 58 11 11
O propionic acid
One-stage chemostat A. latus Sucrose + 38 11
pentanoic acid
t._ Fed-batch Paracoccus Methanol + 120 9 2.34 26 60 0.0195 19
r-
,< denitrificans n-amyl alcohol
Fed-batch Alcaligenes sp. Glucose + 48 62 36 57 3 0.87 20
SH-69 yeast extract
Fed-batch Azotobacter Beet molasses + 19-22 59-71 8.5-23 18
vinelandii UWD pentanoic acid
 Production of P(3HB-co-3HV) by A. eutrophus: B. S. Kim et al.
s e e m s to b e possible if w e c a n c o n t r o l t h e p r o p i o n i c acid 8 Luli, G. W., Schlasner, S. M., Ordaz, D. E., Mason, M. and Strohl,
c o n c e n t r a t i o n in t h e m e d i u m . W e are c u r r e n t l y d e v e l o p i n g W. R. Biotechnol. Tech. 1987, 1, 225-230
9 Mizutani, S., Iijima, S., Morikawa, M., Shimizu, K., Matsubara, M.,
a p r o p i o n i c acid f e e d i n g strategy to test this possibility. Ogawa, Y., Izumi, R., Matsumoto, K. and Kobayashi, T.J. Ferment.
Teehnol. 1987, 65, 325-331
10 Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K. and
Woo, S. I. Biotechnol. Bioeng. 1994, 43, 892-898
Acknowledgements 11 Ramsay, B. A., Lomaliza, K., Chavaric, C., Dube, B., Bataille, P. and
T h e a u t h o r s are grateful to t h e K o r e a Science a n d E n g i - Ramsay, J. A. Appl. Environ. MicrobioL 1990, 56, 2093-2098
12 Kim, J. H., Kim, B. G. and Choi, C. Y. Biotechnol. Lett. 1992, 14,
n e e r i n g F o u n d a t i o n for financial s u p p o r t . 903-906
13 Bitar, A. and Underhill, S. BiotechnoL Lett. 1990, 12, 563-568
14 Lee, Y. W. and Yoo, Y. J. Kor. J. Appl. Microbiol. Biotechnol. 1991,
19, 186-192
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Enzyme Microb. Technol., 1994, vol. 16, July 561