\

HI

WW

1

105
988

 

”THS

EFFECTS OF CYANEDE ON TRICKLENG
FILTER MICROORGAMSMS

Thesis for the Degree of M. S.
MICHIGAN STATE COLLEGE

Donald George Daus
.1954

TH ES‘S

This is to certify that the

thesis entitled

THE EFI’ECTS or CYANIDE N TRlChLII-IG
FILTER l-ilCROOHGAItlSIfi

presented bg

Donald G. Dans

has been accepted towards fulfillment

of the requirements for
”a 6’.

MS; degree in W Engineering

 
    

 

Major professor

Date May 27; l9f§h

0—169

 

 

 

 

 

 

 

EFFECTS OF CYANIDE ON TRICKLJNG FILTER MICROORGANISMS

by
DONALD GEORGE DADS

tA THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the requirements
for the degree of

MASTER or semen IN CHEMICAL ENGINEERING

Department of Chemical Engineering

1954

 

 

 

rHESIS

 

 

 

 

ACKNOWLEDGEMENTS
The author wishes to express his sincere thanks
to Dr. 0. Fred Gurnham, under whose constant interest
and thoughtful suggestions, the experimental work and
the writing or this the sis were accomplished.

He is also indebted to Dr. LL. Mallmann for
suggestions on the media employed in the bacteriological
phases of this investigation. ,

Much assistance was given by Mr. Charles K. Steams,
graduate student. who worked on the same project.

Grateful acknowledgement is due various members
of the Bacteriology Department of Michigan State College,
especially Miss Lisa Neu, who supplied cultures used
in this investigation.

The writer deeply appreciates the grant by the
National Institutes of Health. since this financial
support made the investigation possible.

of” e r w
L t‘Lg (1:3

TABLE OF CONTENTS

Abstract........ .......................1
Introduction}...........................3
Survey of Previous Work.................#
Experimental Procedure;................l3

Fig. 1 Schematic Flow Diagram....14

Fig. 2 Cyanide Feed and .
Settling Tank................l5

Fig. 3 Overall View..............lS
Fig. 4 Trickling Filters.........15
Presentation and Analysis of Results...18

Graph 1 BOD of Trickling
Filter-Eff1uents.............19

Graph 2 Nitrates in Trickling
Filter EffluentBOe e e e e e e ee .0020

Graph 3 Nitrites in Trickling
Filter Effluents.............2l

Graph.4 Effect of Shock load of
Cyanide on Filter Effluent...22

Graph 5 Effect of Cyanide on
Nitrite Production in
EffluentsoeeeeeeeeeoefioeeeOeea

Graph 6 Effect of Cyanide on
Nitrite Production by
Psuedomonas aeggginosa.......25

Graph 7 Effect of Cyanide on
Nitrite Formation by
Unadapted 23. W. . . . . 26

/

Table 1 Toxicity Studies:
Glucose Broth................28

Table 2 Toxicity Studies:
_ Synthetic Broth..............29

Summary..................... ...........32
'Conclusions.............................34
Suggestions for Further Work............35
Bibliography............................37
Appendix I: Experimental Data..........40
Appendix 11: Analytical Methods......“..#8

ABSERACT

Sodium cyanide in concentrations of 0.3 ppm (as ON)
was applied to a pilot plant trickling filter. This was
later increased to 1 ppm. A second trickling filter,
operated in parallel, was used as a control.

The 0.3 ppm concentration of CN did not seriously
affect the BOD of the effluent, but a high nitrite
concentration appeared in the effluent. The nitrites
decreased after a week, falling below the control. Nitrates
were also below those found in the control filter effluent".

A CN concentration of 1 ppm retarded nitrite and
nitrate formation and increased the BOD of the effluent.

A shock load of 4 ppm CN also re suited in high nitrites.

Nitrite and nitrate formation in the filter effluent
is retarded by 40 ppm ON, especially in samples from
the control filter.

High cyanide concentrations (20 and 200 ppm) inhibited
reduction of nitrate to nitrite by Psuedomonas aemginogg
(both unadapted and previously adapted to 200 ppm cyanide);
2 ppm was slightly inhibitory, but 0.2 ppm had no apparent
effect. The high concentrations caused a slight initial
increase (followed by a decrease) in nitrites formed by
the adapted strain.

Laboratory toxicity studies using a glucose broth

-1-

-2-

and a synthetic broth indicated that choice of medium
is an important factor in demonstrating toxic effects.
Most organisms tested were tolerant to 20 and 200 ppm
ON on glucose broth. On the synthetic broth, consider-
ably less resistance was shown. Only one organisn grew
in contact with 200 ppm ON: Serratia marcescens; this
occurred only in the presence of 1 ppm methylene blue.

A cyanide tolerant strain of Streptomces M was
isolated from sewage.

The author believes that nitrite production is the
result of emergency use of nitrate as an oxidizing
agent by the exposed organisms. This action is also
sensitive to cyanide, but the inhibition is much slower.

INTRODUCTION

Since dissolved cyanides are generally toxic to humans
and to fish, it is in thepublic interests ‘to prevent
discharge of cyanides to natural streams. Gaseous
hydrogen cyanide, which is evolved on hydrolysis of cyanide
salts and other cyanide compounds, is also very dangerous.

Cyanide discharges arise from industrial wastes, such
as spent plating liquors and gas works effluents, which,
accidentally or deliberately, are dumped into streams or
into municipal sewers. The damaged treatment plants fail
to perform oxidation of the wastes and contain the cyanide
in the effluent. Cyanides inhibit the biological oxidation
of the sewage; thus the streams become overloaded with
organics, which are both a nuisance and a potential public
health hazard. These effects are probably more important
than the toxiCity of the cyanide itself.

In order to employ a biological cyanide disposal unit,
or to operate a sewage treatment unit in the presence of
cyanide contamination, the mechanisms involved must be
more clearly understood.

While many cyanide wastes contain the cyanides as,
hmayy metal complexes, only simple cyanides were studied

in this investigation.

SURVEY OF PREVIOUS‘IORK

The cyanide ion has two outstanding chemical
characteristics: it is easily oxidized and it forms very
stable metallic complexes. IMost of its biological activity
can be explained on one or the other of these bases.

Cyanide inhibition of cell respiration led Warburg
to discovery of the cytochrome systems. These cytochromes
have in common a heme (tetrapyrrole) nucleus and a heavy
metal ion as prosthetic group. Inhibition is due to the
formation of a complex between the cyanide and the
enzyme's metallic ion, thus reducing its effectiveness.
The toxicity of cyanides to mammals is due to inactivation
of the blood hemoglobin (21); Since the cytochromes are
frequently essential in hydrogen transport, repression
of these enzymes will 'seriously hamper cell metabolism.

Many other enzymes (1.9. the polyphenol oxidases)
contain heavy metals as active groups and are thus likely
to be cyanide-sensitive. The cytochrome oxidase system
(”indophenol oxidase") is also inhibited by cyanides (45)
(47»: ’ i '

-Not all enzymes are inhibited by cyanide. Urease is
activated (25); as is the plant proteolytic enzyme
(3-papain (24)% Some enzymes, as the dehydrogenases (47),
are neither inhibited nor stimulated by the cyanide ion.

.4.

~5v

Few general statements can be made concerning the
effects of cyanide on bacteria, as these are too diverse
physiologically to be covered in a few sentences. In
order to discuss these effects in an orderly fashion,
the bacteria will be considered in families.

Bacteria are generally classified in the class
Schizomycetes of the sub-phylum Fungi. Most bacteria, as
ordinarily conceived, belong to the order Eubacteriales.
There are eleven families in this order. Of these, the
following have representatives occurring in sewage and in
biological treatment units (6)(48)t Nitrobacteriaceae,
Psuedomonadaceae, Rhizobiaceae, Micrococcaceae, Achromo-
bacteriaceae, Enterobacteriaceae, Bacteriaceae and Bacillaceae.

The Nitrobacteriaceae are autotrophic. The chief
genera found in trickling filters are Nitrosomonas and
Nitrobacteg. Both have been reported to be inhibited by
cyanide in concentrations of 2.5 x 10‘5 (37) and 5 x 10'6
molar (31) respectively. These are equivalent to 70 and
140 ppm CE. Rae and Rao have demonstrated that Nitrosomqggg
fails to adapt to ammonia oxidation in the presence of
cyanide ion. These organisms are chieflyresponsible for
Iiitrite and nitrate formation on trickling filters and in
.5011. There are several reports of cyanide inhibition of
soil nitrification, including Lees (and Quastel (26) and
item and Clark (44). l

The Psuedomonadaceae are generally gram negative rods,

and are frequently found in soils and water. One of the

-5-

most commonly encountered is Psuedomonas aeruginosa.'Work
reported on this organism is conflicting: Quiroga and M0nte-
words (36) report that five strains of Pg. aeruginosa produce
HCN from amino acids. Mochtar (33) also noticed HCN pro-
duction. -

Barron and Friedeman (5). on the other hand, report
that this organism is completely inhibited bdeCN.

Acetobacter gylinum is reported by Cozic (13) to be
cyanide-tolerant, but six other species are inhibited by
4 x 10"3 molar KCN (10,200 ppm CN).

No reports on the effects of cyanide on Rhizobiaceae
were discovered in this survey:

Micrococcaceafi are gram positive spheres. Because
of their shape, they present less surface to their sur-
roundings and therefore would be more likely to be highly
tolerant of cyanide. cocci

Burnet (11) reports that gram positiveAare least
sensitive to cyanide; Streptococcus tolerates 0.5% CN
(5000 ppm).

Sarcina $333; is reported to be cyanide tolerant (18).

According to Braun (8), Staphyl ococci are more in-
fluenced by the cyanide than are the Streptocooci under
‘aerobic conditions. (This agrees with Burnet's data:
"Stagzlococcus amp" is inhibited completely by 0.04%
an.) ’

' The pathogenic cocci are reported by Sevas (39) to be

stimulated by cyanides at 10,200 to 51,000 ppm.concentrations.

-7-
0f the Achr0m0bacteriaceae, only data concerning
Alcaligenes fecalis are reported. Barron and Friedeman (5)
report inhibition; Mochtar (33) reports HCN production.

The Enterobacteriaceae arelhighly fermentative gram
negative rods‘,: which include such pathogens as Salmonella
typhosa and Shigella dysenteriae.

' Escherichia4 391;, the bacteriologist' a favorite, is
reported by Burnet to resist 0.1% (1000 ppm) 0N. However,
Aubel, Rosenberg and Szulmajster (4) report that cell
respiration is inhibited, except in the presence of pyruvate.
Stickland (56) reported that 0.0001 M CN (2800 ppm) inhi‘tit s
nitrate reduction. Dessy (14) reported that E. 29}; is
killed by 10% NaCN‘ in six hours.

Mochtar reported that Proteus produces HON.

Burnet reported that Shigella does tolerate cyanide,"
while Salmonella typhosa does not. Braun and Kurman (9)
reported that Shigella is less resistant under aerobic
conditions. ‘

Salmonella paratyphosa tolerates 0.01% but not 0.1%,
according to Bone. ‘

The Bacteriaceae are a 'miscellaneous" group; no general
trend was noted. .

Bacillaceae are the spore-forming bacteria. Nest
investigations have been centered on Bacillus subtilis.

As found by Boné (7). their spores survive five days
in 0.1% CH while the vegetative cells are killed. These
vegetative cells survive in 0.01% CN. Similar inhibition

-8- ‘
is found by Hartree (22), who found parallel results with
5,7~dichloro-8-hydroxyquinoline, also a heavy metal complexing

agent.

Cyanides are not particularly toxic to bacteria.
Southgate (41) reports more (total numbers) organisms in
a sewage containing cyanide than in normal sewage.

Burnet stated that all cyanide-tolerant organisms he
found possesed peroxidase (sfiheme-oontaining enzyme), except
Shigella. (Since hydrogen peroxide easily oxidizes cyanides
igflyitgg, this may imply a mechanism for detoxification.)

He believed that the major effect of the cyanide was to
lower the redox potential of the system.

Lbffler and Rigler (28) believed that ability to liberate
hydrogen sulfide from cystein is highly correlated with
cyanide resistance. 328 and HON both attack metal ions;
tolerance for one would render tolerance for the other more
likely. _

Molds have been reported resistant to cyanide. Dessy
(14) reported that while 5. 29;; is killed in six hours,
"molds” resist lOZINaCN for over one day. Tam and Clark (44)
reported that soil fungi and actinomycetes are little
(affected by calcium cyanide. Generally fungi are cyanide-
‘holerant. The chief interest seems to lie in demonstration
<3f ability to use cyanide as a sole nitrogen source.

Some work has been done on the toxicity of cyanide to
yeasts. Heisel (30) reported that Hg(CN-)2 is more toxic than
Ker which, in turn, is more toxic than NaCN. He stated that

-9-
long exposure to low cyanide concentrations causes a loss
of fat in yeast cells. This is not demonstrated in yeast
cells exposed to high concentrations of cyanide for a short
interval. I

Winzler (45) found that cyanides inhibit yeast res-
piration by inhibiting cytochrome oxidase and combining
with an (unspecified) enzyme system.

Some work has been done with green algae. Nitella and
Chlorella are among the genera reported to be cyanide-
tolerant (38)(15).

Some HON is produced by higher plants, but its function
is unknown. ' "

The toxicity of cyanide to mammals and fish is well
recognized. I

An organism has been isolated from the effluent from
a trickling filter adapted to thiocyanate containing wastes
by Happold and Key (20). They named it "Bacterium thig-
exaggxidags". but it is not well characterized. Meyerson
and Skupenko (32) reported finding thiocyanate in streams
containing cyanide. (This may be a clue to the mechanism
of cyanide destruction or detoxification.)

The effects of cyanide on sewage and sewage treatment
may be considered as the summation of the effects on the
single organisms.p However, the populations involved in
the disposal process are not well characterized.

ludzack, Moore,.Krieger and Ruchhoft (29) have cone

ducted a study on the effects of cyanide in sewage samples

-10..
in the biological oxygen demand (BOD) test. They concluded
that cyanide causes a lag in, or inhibition of, metabolism.
but does not sterilize the sewage. Five percent inhibition
was caused by 0.3 ppm CN. They further conclude that the
chief disposal mechanism in natural streams is volatilization.

There are two general methods 'of aerobic biological
disposal: activated sludge process and the trickling filter
method. Both employ a zoogleal mass of bacteria which oxidize
the material; in the activated sludge process, the mass is
not attached to any support.

The earliest reported work on the activated sludge
process was that of Wooldridge and Standfast: in 1937 (48).
They found that 10“]! ON (200 ppm) or the vapor from solid
KCN inhibited the bacterial actioh.

In 1946, Nolte and Bandt (34) set up a miniature plant
using a modification known as thefllagdeburg process and
‘butyrates or g-cresol as the organic substrate. The protozoa
were shocked by 5 ppm KCN but later recovered. At 62 ppm,
after adaptation, the effluent was free from cyanide and
butyrate. If normal activated sludge is supplied in the re-
cycle, 330 ppm KCN will not interfere with oxidation of the
butyrate. (Evidently multiplication of the organisms is more
Sensitive than their metabolism.) Similar results were
noted when m—cre sol was .used as the substrate.

In 1948, Lockett and Griffiths (29) noted that 1 ppm
HON partially inhibited the oxidation process. HON was blown
Off. in the aeration tank.

-11-

Coburn, in 1949, (12) found that 5 ppm caused a partial
inhibition of oxidation by the activated sludge, but 20 ppm
caused complete inhibition. This latter inhibition was over-
come on removal of the cyanide.

There is even less data on the trickling filter process.

Pettet and Thomas, in 1948, (35) noted that less than
1 ppm HCN had no effect on the BOD of the effluent. (This
disagrees with.lndzack'2§.§;.) Increasing the cyanide to
2 ppm had little effect on BOD but nitrate formation was
retarded. An increase to 4 ppm resulted in an increased
BOD in the effluent, as also did 10 ppm. After the filter
was in contact with these concentrations for a time, the
abnormalities dissappeared and the cyanides were more-or-
less completely destroyed.

when the cyanide was increased to 30 ppm, nitrification
was completely destroyed. About two months were required
for adaptation. Then, total nitrogen in the effluent was
greater than in the control.

The'Water Pollution Research Board (Great Britain)
published some work on cyanide toxicity in their 1951
annual report (1). They stated that 1 ppm had little effect
on the son of the effluent but that the permanganate oxygen
demand had increased. Nitrification was initially inhibited;

In 1952. this group reported (2) that 50-10025 of the
cyanide fed to the filter could be included in the ammonia,
Initrites and nitrates in the effluent. an adaptation, ‘

cyanide in copper, zlnc and cadmium complexes were also

-12-

almost completely destroyed. This was not the case for
the iron and nickel complexes. .

These studies do not agree closely on details, but
owing to the possibility of different flora on the filters
and different materials in the sewage, this is not unexpected.

To generalize, the BOD value is less sensitive to
cyanide than chemical oxidation values. Nitrate formation
is inhibited. The cyanides are eventually destroyed on the

filter, if continuous exposure is provided.

EXPERIMENTAL.PROCEDURE

Two pilot-plant trickling filters, two feet in diameter
and with a rock depth of six feet were set up in parallel,
complete with separate settling tanks ( 70 gallons capacity),
recirculation pumps and constant-head tanks. These units
are located in a special building on the grounds of the
East lensing municipal sewage disposal plant. Each filter
has a capacity of 600 gallons per day, when operated at a
recycle ratio of six to one. Figure 1 is a schematic flow
diagram of the system. Figures 2 to 4 are photographs of
these units; Figure 3 is an overall view of the pilot-plant.

The sewage, free from industrial wastes, is obtained
. from the primary settling basin in the East lensing plant.
Control of flow rates is obtained by using orifices installed
below constant-head tanks. A fan and ducts are installed
to remove contaminated air from the room.

After the filters had matured, one "was exposed to a
continuous feed of 0.3 ppm (as GR) sodium cyanide. The feed
unit was essentially a supply bottle inverted over a .funnel,
with a stopcock to regulate flow rates. (See Figure 2.)
Cyanide concentrations in the sewage were varied by changing
the concentration of cyanide in the feed solution. It was
necessary to use distilled water in these solutions to avoid
precipitation of the cyanides.

After four weeks, the cyanide was increased to 1 ppm.

-13-

t . callu'ia‘si.v..l.lmis..>i' It. .’-. ‘00" :‘l:l").’:t '1'.... .(i‘ i...n'.--'.u'.!.i D'.’ b. II‘VI":I'

.
nv‘wll Il.l\r.||l. 051' ¥’.IIIDI 1": .r i

weaned o»

 

h and.“
wean-em

 

 

 

 

 

 

 

 

 

 

 

 

 

nngsm nopaah.llthu
daemonalodadmho

sewnmac . .

JE-

 

 

 

 

 

 

 

 

 

 

 

EI} [Pl

EdmwéHn. 30AM

OHdeamum

H chewak

 

 

 

 

“fir"‘iV'T-‘r

 

doom pndpmdoo -

 

 

 

_ . hopawm

 

 

Honpdoo

 

Figure 2
CIANIDE REED AND

SETTLING TANK

Figure 3
OVERALL VIEW

Figure 4

TRICKLING FILTERS

 

-16-

Sampleswere taken daily: at the bases of the filters
("effluent"); at the recirculation pump intake ("influent")
and at the constant-head tank proportioning the raw sewage.
(To retard clogging of orifices, a.gcragn is inserted above
this tank.) Daily duplicate determinations of biological
oxygen 1 demand. ' . (BOD) and single determinations of
oxygen consumed by dichromate (DOC) by Shaw‘s method (40),
nitrates (phenoldisulfonic acid-method) (3); and nitrites
were performed. Nitrates and nitrites were determined I
colorimetricadly, employing a Coleman Model 9 Nephlo—Colorimeter.
(See Appendix II for calibration curves.)

In addition, formaldehyde determinations by chromo-'
tropic acid (10) and by condensation with phenylhydrazine
(43) and cyanide determinations by Prussian blue formation,
by picric acid reduction, and by ferric thiocyanate formation
were also made. Calibration curves for these analyses are
included in Appendix II.

At the end of the pilot-plant investigation, the control
filter was subjected to a shock load of 4 ppm cyanide by
adding the necessary amount of cyanide solution directly to
the settling tank. Nitrite production was followed in this
‘test.

Toxicity studies, using pure cultures of organisms
reported from sewage and trickling filters (6)(48), were made
in the laboratory, both on a synthetic medium and on glucosl
‘broth. The organisms were obtained from various members of

the Bacteriology Department. Organisms employed were:

-17-
Aerobacter.aerogene§, Alcaligcggg fecalis, Qhromobactegygg
amethystinum, Escherichia 321i, Proteus vu1garis, Psueso-
.mgngg aeruging§§,l§§. fluorescens, Salmonella typhosa,

Serratia marcescens, "Staphylococcus auregs ” ( Micrococcus

pyrogenes var. agreus), and ;1§treptococcg§ liggifaciegs.
Isolation and identification of cyanide-tolerant

 

organisms from sewage and trickling filter effluent was
attempted. The procedures outlined in Bergey's manual (6)
were followed. -

The effect of cyanide on nitrite production in the
filter effluent under aerobic conditions was also in-
vestigated in the laboratory. Fifty-milliliter portions
of the effluents from each filter were exposed to 40 ppm ON
in sterile BOO-ml. Erlémeyer flasks loosely stoppered with
cotton. ‘fianples were withdrawn aseptically; nitrites were
determined colorimetrically as" in. routine analysis.

The temperature was 22thC. I

Similar experiments were performed on two strains
of fig. aeruginosa, one not previously exposed to cyanide,
the other adapted to 200 ppm GN. Diluted 48 hr. cultures
were added to 100 ml. of a sterile medium consisting of
physiological saline plus 0.1%.glucose and 0.1%.potassbmm
nitrate. These organisms were added aseptically to the
flasks together with various concentrations of cyanide
(0.2, 2, 20 and 200 ppm). Nitrite concentrations after
various time intervals were determined. A.temperature
of 20°C was maintained during these tests in an air

incubator.

PRESENTATION AND ANAEYSIS OF RESULTS

- ‘ 9 g a. .

Graph 1 shows the effect of low concentrations of
cyanide on the BOD of the trickling filter effluent. Cyanide
in a concentration of 0.3 ppm was not particularly detrimental
to the effluent quality, but addition of 1 ppm ON resulted
in a marked tendency for higher BOD values. The mean BOD
of the control filter effluent was 14 ppm oxygen, with a
standard deviation of 1.6 ppm.

A similar plot of dichromate oxygen consumed showed a
scatter which.was difficult to interpret. The effluent from
the control filter had a mean of 43 ppm oxygen, with a
standard deviation of 29 ppm. A correlation between BOD and
DOC is included in Appendix II.

Graph 2 shows that 0.3 ppn.cyanide partially inhibited
nitrate formation, and that 1 ppm caused even more marked
inhibition. Neither concentration inhibited nitrate formation
completely. The mean nitrate concentration in effluent
from the control filter was 3.1 ppm (as N). The standard
deviation was 1.6 ppm. . —

Graph 3 shows the concentration of nitrites in the
filter effluents. Immediately after addition of 0.3 ppm
ON, very high.nitrites were noted. After a week, the
nitrite concentration decreased below that of the control.
After the cyanide was increased to 1 ppm, the low nitrite

an 18-

 

 

 

-19-.

 

 

 

. ‘. WV.

.

noudfiu

3:3“ H9330 x a

.. . . ..
. IO . . o
. . . .1. .
. . . . .. .

 

«3% EB Gaga Mo

#5

.33

.....

.....

 

 

Ir 7:
tillers-4 w_1 mar-mm -.
- .2 on .m- o. m - on .3 2 a. o- m . .
4. it erPIP FLL’VLL» ? r r >)L+LFL E ralblrirb ILIL rb FPbLbb'P *hv p — p Pllbl
1 i #4 11<Iu 414144ld4i<44qllt a M14 1a a 11 1 .4 Is 1144411 iqdi -.14.441 ”441 H
. a .wr . fim.
. x I .
.0.
IO... '7 '''''''' t I III III" I. III. III! nllI-lrllallplv II-
. I. 11. .ill. 0.le :IW?
.- . .n. . x .
o ..o . a o O 1%.
I. ... .K.. ‘1 ..h
. . I , .
Heap-8:53 r _ a... .. . .__
,_ 4". ‘‘‘‘‘ lilo" ll‘ Inuit-1111...!!!- ell! Lyle IIIIIIIOIII'I.&|‘.IIII“'-II
n... ¥ K . . uni...
.._ Q .. H _ . ..,. . . .. .
aw . bmb+n . .06... E. J6 . ... .1 fl...
iv . w...’oele.l.ll O.l.0'l 5!. II A... III .w‘M‘IT’pQIJ ‘.IJI i'.'..‘h. 1+6 .I‘Cl..'.. t Ilv III-IO! tL‘l'.’~+-¢'F.?1-.IIJS

 

.6\.

I

mfifiotfistbveom ‘ V ‘ ‘ ,

( mfifixo fmdd. ) _‘1fPWW(Ii

 

 

 

 

 

 

litrate Nitrogen. (m)

.\.O~

5.0-4

6.0“

:"
o

S“
o

2.01.

 

X0

.-20- _
mass as. means "rttm' mm

0.3 ppm. -~-—-~~<-~:~'~»-

' 1

x Control Filter
0.31pm“ Filter

--'.._1 m, Cyanidsé-s-v—
0 .

 

 

 

 

K x7e,

 

_._ -J

 

-‘_~'_ - -- _--_.

 

 

 

 

 

 

‘49“

Q8-

06‘

(ppm)
5b
\N

3‘
*3

litrit‘e . Nitrogen
9

.o.'/.

 

 

' names 3me

.,. .

 

  
  
 
 
 
  
   

 

 

 

enmxmmf—

i..

l

. ... I.

...
. ..

1

x- .ocami filter
:0 Ignited, filter

.‘-W.
I

.....

(x

sclel-

 

 

 

1 Harsh

 

h.

 

_.—.—

 

 

-22-
concentrations persisted. The steady decrease in nitrites

on the control is not explained. Nitrites on the control
averaged 0.16 ppm (as N), the standard deviation was 0.12

PPm-

Graph.4 shows the effects of a shock load of 4 ppm ON
on a filter not previously exposed to cyanides. An initial
peak of 0.07 ppm Noe—N was observed an hour after the cyanide
was applied. In three hours this decreased to the current
average value for the fiiter (0.03 ppm NOE-N). A second
delayed rise to 40.28 ppm N was noted on the sebond day,
which returned to normal in about two weeks.

The effect of 40 ppm cyanide on nitrite formation
in filter effluent (under aerobic conditiions in the .
laboratory) is shown on Graph 5. Cyanide initially stimulated
nitrite formation in the effluent from the filter exposed
to 0.3 Ppm ON, but inhibited it in the previously unexposed
effluent. Both controls behaved similarly. Nitrate
formation occurred in the controls; relatively little in
the cyanide containing samples.

Graphs 6 and 7 show the effects of various cyanide
concentrations ( 0 to 200 ppm ) on nitrate reduction by a
pure culture of Psuedomonas aeruginosa. Cyanide at 0.2
ppm had no effect ( or was possibly slightly stimulative).
Cyanide at 2 ppm was slightly inhibitory; at 20 ppm the
inhibition was marked. A.cyanide concentration of 200 ppm
had a slight stimulation on the strain adapted to 200 ppm,
but had a decisive inhibition of the unadapted strahr.

 

 

P

1(IIICIIV(IGI (.1

 

 

 

 

 

_
.........

 

J
I

l
. . . -e
. . .
.
. . . . e
w
, , . . . . . , .
‘ I
’ cm .
or . , _-
. t . _
, t i

........

 

 

 

l
(

 

CRAP? '4'
.1

~shamanism:

\
r
t

ppm. mimics

Vile l..|!I.I’l|'ll’Ll‘-l. I (In, (L

t

l
.----_- .i-iirwsci_.r

e . o I o . .
"w”-enfi‘... - .-
. . -

Cpé
.._

1
I
i
s
)
1

n .

 

i
w_ A
Ti? it- I-
*

H!(ill.( Ill" 9‘.) .IIv

. 8|!» ll)¥)ol. -

 

 

.- .. ._. w ---..__.r..

 

(

a

1
. . a I .
,-._.;;___ii._i...ii--

“ .

‘(-l'|u

(Lil

.....

 

- -TIJ

 

 

 

 

......

......

 

 

 

 

.....

 

-o—4t—.o

\ .o |
t we IL
a _
. 1L
.

.. . Fl.

7 . . I
7

 

 

 

 

 

 

xvlilt. .

 

 

 

 

 

 

m --.. .wni- - -..
. i _ 0
new: newonpaaWeSH-Zm

 

 

 

, . . . .
I
.4— “.a_-.- H-..“ 7-._.... it.

 

a..—
I

 

V.

W

-
Y

_.__1. J

l
l

 

 

..........

r
V

..nays.a£tert_ehbok

I J7

 

I .
I"

i.

/

 

 

 

3
.0 ..

a“

|
l
. , I,
fiL’I aw?) -
, '~ t_'

view

i

TUB-“3 «enema-Fa

3.33":

I'll}. : 4 1' (I)

 

 

 

 

 

 

 

 

 

 

 

 

w.
n... n- he
I I. .
is Q?
V
. a?
O
._ m ..
. ecu-none in.» 9..- .mns need». ween-nun- W .-
. . ~33» deco-Hugs .- .. aw .
. . . ova-Eh"- .HE o..- dam Saga Hoke-Bo e m u Hex
- ; . 8:: Hes-see c . .e . .
4.. . .w... no?
. w .. T. I_.\
w m _
U ‘ .d‘
\ _. ‘ mean-.5— 55:- efidoH-n- 9-
, . 638an ES:- 8 .530 B eon—.m-

m E48

 

as

 

 

 

 

 

 

3...
«6.:
8 am.- e. .e

g mam-mammals- Baa-3

33..- 8 33». a. means
a. mans .. w

 

 

 

 

 

.2 q.

. see

3

(add) sonnets put: its

. 2.6

 

the

 

426- ,

 

 

 

 

dogwadooéu head «ham
1 R . w o
n ._.
3 . n a a ..
“a . .33
_ H
. H mm
. 1v
. u
n
.. com a «a _ I .
. 3. ON H 0 . . . .1."
.. I , CON In . + . . #de 1 .
. . 8 N00 I? _ . m
. . . .. - . )
. m
h . ..
n .96
_
. .
. .
‘
x
.
__ .35
_

 

ENHHH no ”H5590 mo mgg

 

. g amonnpmm gas .3 282.5%
A _ 5 l3

 

 

 

-27-
with respect to nitrate reduction. Evidently adaptation to
cyanide did not involve a significant change in nitrogen
reduction.mechanism.

Tables 1 and 2 show the results of toxicity studies.
.All organisms tested were more resistant to cyanide on
glucose broth than on the synthetic broth. Pigment for-
mation was retarded in several instances.

Enterobacteriaceae and close relativesng. aerogeneg,
g, ggli, Proteus vulgaris, and Serratia marcescens were very
tolerant; fortunately Salmonella typhosg was the most
sensitive.

The most cyanide-sensitive organisms were Alcaligene§_
(an Achromdbacteriacea) and Qhromobacterium amethystinum
(a Rhizobiacea), especially on the synthetic medium.

'gg; asruginosa and Pg. fluorescens were moderately
tolerant of cyanides.

Of the gram positive cocci, Staphylococcus aureus and
Streptococcus liguifaciens were both tolerant; Staphylococcus
‘was more sensitive. This agrees with Braun's findings.

High concentrations of cyanide (200 ppm) were very
toxic in synthetic broth. Only Serratia grew after 24 days;
then. only in the presence of 1 ppm methylene blue in the
mediuni

Some of the organisms (i.e. Proteus) were revived on
synthetic broth after contact with cyanide by addition of

1 ppm methylene blue to the subculture.
All tolerant organisms were catalase positive after

massage pacemfim Ofipmflnmpomnsno *

agopm +
spzohw on t «mom

.1. .I. .I. + mnofioemadw: mfiooooo anonpm
+ A+v .. I muonfiw mfioooooahnmdpm
++ ++ ++ ++ mnmomoonms mapspnom
.I. + i... + @8393 Samsoaaem
.I. .I. .1. + mnoomonofifiw mgosododwm
.1. .I. _.«I. + mmonwwamm monoBOdofimm

manage.» 3690.5
.300 .mEoZonomH

+ .+ e... + .mm gfinopogosonno
+ + + A...“ fiasco.“ modowfiso:
.1. .I. .I. .I. monomonod nepogonod

um flu N. 4. "when
mu ammo

figafigfig

H u made

am 23.30

-29-

gable g

Togicitz Studies on Sznthetig Broth

2Q ppm QT
Organism Days: 1 2 3 1+ 5 10 ll
Aerobacter aerogenes (+)H~ .- .. .- .- ..
Chromobacterium sp. .. .. .. .. (+) H- H-
Proteus vulgaris ' .. .. .. .. .- + -H-
Psuedomonas aeruginosa .. (+) +1 4-!- ++ ++ -H-
Psuedomonas fluorescens — .. .. (+) + -H- -H-
Serratia marcescens .. .. + H -H- -H- -H-

0 pp CH

Aerobacte : ae rogene s

Chromobacterium sp. k+)(+) .. .. .. .. .-
Psuedomonas aeruginosa. - — .. .. .. - ..
Psuedomonas fluorescens - .- .. a... .. .- ..
Serratia marcescens — - .- .. .. .. .-

( Organisms grown for two suceseive transfers on

Wnthetic broth before innoculation.)

Composition of Synthetic 'Ioth:

Sucrose 30 gm.
KZHPOu. 1 gm.
MgSOu 0.32 gm.
03.01 0.08 gm.
(NHLSZSOQ, 1 gm.
Water 1 liter

-30

growth in the presence of cyanide. The cyanide did not
noticably affect the level of growth attained but affected
only the period of lag.

A cyanide-tolerant organism was isolated from raw
sewage containing 200 ppm ON, and was identified as
Streptomyceg w. Table 8 in Appendix I describes its
characteristics. It is very cyanide-tolerant, surviving
three succesive transfers on a synthetic medium containing
cyanide as sole source of nitrogen. This Strep, gébgg is
also catalase positive, but does not release hydrogen sulfide
on Kligler's Iron Agar.

The high.nitrite concentrations can have two likely
origins: oxidation of ammonia or reduction of nitrate. Since
the chief ammonia oxidizer (flitroscmonas) is reported (37)
both to be completely inhibited by cyanide and to be unable
to adapt to tolerate cyanide and since the Psuedomonas
experiments show that low cyanide concentrations do not
inhibit nitrate reduction, reduction of nitrate is the
more likely. major source of nitrites in the effluents from
filters exposed to low concentrations of cyanide. Nitrates
may be used as emergency oxidizing agents, if the cyanide
attack on the cytochromes impaired the use of oxygen as the
oxidizing agent. This nitrate reduction can stop at nitrite
or procedeall the way to ammonia or nitrogen. The secondary
decrease in nitrite may represent either a gradual inJHbition
of this mechanism or development of a more efficient reduction

system. Since nitrate concentrations were not need during

'3?‘
this secondary repression, this second possibility should
not be ignored. In any case, there is insufficient nitrate
to oxidize a significant fraction of the sewage.

’ The‘gguedomonas experiments show that cyanide does
inhibit nitrate reduction, if the cyanide level exceeds 2
ppm. At lower concentrations of cyanide,.gradua1 inhibition
may take place.

The shock effects may be similarly explained. Initial
stimulation of nitrites, and rapid repression may represent
the desperation attempt of the trickling filter organisms to
offset an impaired cytochrome system and then gradual
inhibition of the nitrate reductase system. _As the cyanides
are washed out, the reductase system may recover more rapidly
(or the attack is more readily reversed) than the cytochrome
system. Thus, a second increase in nitrites is not unlikely;
The extzeme duration of this second period of increased
nitrites indicates that # ppm OH is very toxic to the filter.

Contact of the microorganisms with somewhat higher
cyanide concentrations than previously encountered resulted
in an initial increase in nitrites often followed by a
decrease. This was demonstrated on the filter itself (both
under continuous exposure and under a shock load), batchwise
contact with the filter effluents and in nitrate reduction
by Psuedomonas gggggggggg. ~All are involved with.nitrate
reduction: The extreme duration of nitrite persistence
on the shocked filter eliminates cyanide itself as the
possible precursor of the nitrites:

SUMMARY

Sodium cyanide in concentrations of marginal toxicity
(0.3 and 1 ppm CN) was .applied to a pilot-plant trickling
filter.- At the- end of these runs, the control filter was
subjected to a shock load of 4 ppm CN.

BOD of the effluent was not affected by 0.3 ppm CN,
but an initial increase and later decrease in nitrites
and an initial decrease in nitrates were noted.

A concentration of 1 ppm cyanide retarded nitrites
and nitrates in the effluent and also caused increased BOD
values. The shock load also produced the high.nitrites and
‘low nitrates. '

Cyanide in a concentration of 40 ppm retarded nitrite
and nitrate formation in the effluent from the filters.

‘gsuedomonas aeruginosa reduced nitrate to nitrite in
the presence of 0.2 ppm CN, without inhibition. Cyanide at
2 ppm is slightly inhibitory; 20 and 200 ppm are decidedly
inhibitory.

Laboratory toxicity studies were attempted on organisms
reported from trickling filters or sewage, using both
glucose broth and a synthetic brothe Most organisms were
tolerant of 20 and 200 ppm ON on glucose broth. On synthetic
broth, 200 ppm was very toxic: Serratig marcescens was the
only organism to grow, and this only in the presence of

-33-
1 ppm methylene blue‘. Cyanide affected the length of the

lag period, but did not appreciably alter the level of
growth attained.

The author believes that the nitrites originate from
emergency reduction of nitrate: this oxidative alternative

may also be inhibited by cyanide, but at a much slower rate.

CONCIIJSIONS

(1) Cyanide in a concentration of 0.3 ppm interferes with
the nitrogen metabolism of a trickling filter operating on
municipal sewage, but is not appreciably detrimental to the
BOD of the filter effluent.

- (2) Cyanide in a concentration of 1 ppm is markedly
detrimental to the quality (BOD) of the effluent. Nitrate
formation is partially inhibited.

(3) Cyanide at 40 ppm concentrations inhibits nitri-
fication in the filter effluents.

(4) Shock loads of cyanide applied to an unadapted filter
result in increased nitrites in the effluent.

(5) Cyanide toxicity to microorganisms is a function
of the medium supporting tbeee organisms.

(6) Cyanide toxicity involves increased lag periods,
rather than decreased population levels attained. Cyanide
exerts a bacteriostatic action.

' (7) Most of the sewage organisms tested are found to
tolerate 20 and even 200 ppm ON on glucose brofih.

(8) Cyanide in concentrations over 2 ppm retards the use
of nitrates as auxiliary oxidizing agents by fig. aeggginosg.
A! lower concentrations, it is only slightly inhibitory.

(9) The increased nitrites probably have their origin

in reduction of nitrates.

~34?

SUGGESTIONS FOR FURTHER.WORK

(1) Investigation of conductimetric titration with
nickel and "versene" as a possible analytical method for
cyanide. h

(2) Investigation of the effects of methylene blue on
a trickling filter inhibited by a shock load of cyanide.

For some organisms, methylene blue can act as a substitute
for the cytochrome system.‘

(3) Investigation of formaldehyde as a possible de-
toxifier for cyanides. Reaction kinetics might be determined
.by following the cyanohydrin condensation reaction in a
conductivity cell.

(4) Isolation of the denitrifying organisms from the
trickling filters. The general organism present, Zooglea
ramigera, is reported by Bergey's (6) not to be a denitrifier.

(5) Attempted adaptation ofANitrobacte; and Nitrosomonas
to low concentrations of cyanide. Metabolic activity may be
followed as nitrate or nitrite.

(6) Investigation as to the correlation between ammonia
production and nitrite depression. )

(7) Systematic investigation of the significant components
in media for the demonstration of cyanide toxicity. This
might yield some insight into the mechanisms involved.

(8) Investigation of possible intermediates in cyanide

' -35~

-36-
destruction. For reproducible results, the use of pure
cultures of well-known organisms is suggested.

(a) Formats might be measured quantitatively by
reduction with acid and magnesium, with colorimetric
determination of the formaldehyde formed.

(b) Formic dehydrogenase might be demonstrated
in the organisms by decolorizing methylene blue in a
Thurnburg vacuum tube.

(c) Thiocyanate may be determined colorimetrically
as the iron complex.

(d) Nitrite may be determined by the usual method
employed F in this work.

BIBLIOGRAPHY
(1) Anon., Dept. Sci. Ind. Res., Rept. Water Pollution Res.
Board, 1951, 36-40.

.(2) Anon., Dept. Sci. Ind. Res., Rept. Water Pollution Res.
Board. 1952’ Bl'h‘e -

(3) Anon., "Standard Methods for the Examination of Water and
Sggge", American Public Health Assn., New York, 9th Ed".,
1 .

(4) Aubel, E., Rosenberg A., and Szulmajster, J., Biochem.
(31; Biophys. Acts. 5 228-5; (1950): Ann. Rev. Biochem. g9 17
1951'. - '

(5) Barron, E.S.G., and Friedeman, T.R., J.Biol. Chem. 121
593-610 (1941); CA :55 3570.

(6) Breed, R.S., Hurray, E.G.D., and Hitchens, A.P., "Bergey's
Manual of Determinative Bacteriology," Williams and_Wilkens -
00., Baltimore, 6th Ed.,'1948.

(7) Boné, G., Compt. rend. ‘scc. biol. lag 96-8 (1936);
as 29 5608. '

(3)33§aun, H., Schweiz. Z. Allgem. Path. u. Bakt. g 309-18
9 9 ‘

(9) Braun, H. and Kurman, V. Schweiz. Z. Allgem. Path. u.
Bakt. _1_§_ 403-14 (1951); cs (6 3610(1952).

(10) Bricker, C.E. and Johnson, H.R., Anal. Chem. ll #00 (1945).
(11) Burnet, F.M., J. Path. Bact. 29 21-38 (1927); on g; 1474.
(12) Coburn, 5.1:... Sewage Works J. g; 522-4 (1949).

(13) eozie, 11., Rev. gen, botan. 52}, 212-4 (1936); CA g; 4897.
(14) Dessy, G., Sperimentale 1g 87-96 (19210;. CAEfliO}.

(15) Emerson, 12., J. Gen. Physiol. 19. 969 (1927.); ca _2__1_ 2719.

(16) Finkel'shte'ln, D.N., Zhur. Anal. Khim. 2 188-95 ((1948);
CA g 8708.-

-37-

-38-

(17) Friel, F.S., and Wiest, C.J., Waterworks and Sewerage 92
97 (1945).

(18) Gerard, R.W., Biol. Bull. _6_Q_ 227 (1931).
(19) Grant, w.M., Anal. Chem. _2_g 267 (1948).

(20) Happold, F.C. and Key, 1., Biochem. J. 3; 1323 (1937);
01 31 8886.

(21) Hartree, E.F., Bull. soc. chim. biol. 2; 377-80 (1950);
CA 4.6 9470.

(22) Harrop, 8.1., and Barron, E.S.G., J. Exp. Med. 4_8_ 207-33
(1928); on g 3691. -

E23) Hudlitz, F. and Flasher, 3.12. Anal. Chem. _13_6_ 185-8
1952); ca 4_6_ 9470. ,

(24) Irving 0.71., Fruton, J.S., and Bergman, J., J. Biol. Chem.
139 568-82 (1941); CA 35 7983.

(25) Jacoby, 11., Biochem. Z. 181 194-206 (1927); CA g; 2000.

(26) Less, H. and Quastel, J.H., Biochem. J. 49 803-15 (1947);
CA 4_1_ 2516.

(27) Lockett, W. and Griffiths, J., Sewage Works J. _2_(_)_ 357 (1948).

(28) L'dffler, E. and Rigler, R., Klin. Wochsc'her. 9. 712-3 (1929).,
as 3 8609; CA 5212 4570.

(29) Ludzack, F.J., Moore, W.A., Krieger, H.C., and Ruchhoft, C.C.,
Sewage and Ind. Wastes 21 1298-1307 (1951); CA 46 3189.,

(30) Meisel, M.N., Zentr. Bakt. Parasitenk, II, Abt. _8_8_ 449—59
(1933); CA 28 1074.

(31) Meyerhof, D.I., Arch. ges. Physiol. 165 229 (1916).

(32) Meyerson, C.A. and Skupenko, P., Wastes Engineering _2_5
17 (1954).

(33) Mochtor, A. and Van Veen, A.G., Geeneesk Tijdschr,
Nederland-Indie _8_1 874-9 (1941).

(34) Nolte, E. and Bandt, H.J., Beitr. Wasser-Abwasser u.
Fischereichem. Magdeburg 1946 9-14; CA 42 336 (1949).

(35) Pettet, A.E.J. and Thomas, H.N., Inst. Sewage Purif. J.

(36) Quiroga, S. and Monteverde, J., Rev. Sud-American
Endocrinol., Immunol. y Quimioterap. 22 565-73, 612-24 (1940);
BA _15 9006 (1941); CA 35 1440 (1941).

-39-
(37) Rao, 0.0. and Rao, W.V.S., J. Indian Chem. Soc. lg
681-90 (1939); CA L4 4510.

(38) Ross, E., Proc. eoc. exp. biol. med. :2 09 (1934)
BA_2 15583 (1935)-

(39) Sevag, 11.9. Naturwissenschaften _2_:_1_ 466-7 (1943)
BA _11 1741 (1943). _

(40) Shaw, J.A., Anal. Chem. 23, 1764 (1951).

(41) Southgate, B.A., J. Soc. Chem. Ind. (london) fig lT-4T
1933); BA 9 7517 (1935).

(42) Stickland, L.H., Biochem. J. g5 1543 (1931); CA 26 1636.

43) Tanenbaum M.E. and Bricker, C.E., Anal. Chem. 22,3ER-7
1951); 01 45 4633.

(44) Tam, R.K., and Clark, H.E., Soil Sci. 51 359-65 (1944(:

.(45),w1nz1er, R.J., J. Cell. Comp. Physiol. 21 229-53 (1943):
CA 38 137.

(46) Wooldridge, W.R. and Standfast, 11.13., Nature 110 665 (1932):
CA g1 3272.

(47) Wooldridge, W.R. and Standfast, A.B., Biochem. J. g1
183-92 (1933); CA g1 3763.

(48) Wooldridge, 17.11. and Standfast, A.B., Biochem. J. 39
1542-53 (1936 ; CA 31 1134.

BA; Biological Abstracts
CA; Chemical Abstracts

APPENDIX I
EXPERIMENTAL.DATA

Table 3 Trickling Filter Effluents..............41
Table 4 Trickling Filter Effluent After Shock...43

Table 5 Batchwise Nitrite Production
in Filter EffluentSOOOeeeeeeeeeoeeeeeeeonOOM

Table 6 Effects of Cyanide on Nitrite Formation
by ‘Unadapted Psuedomonas aeruginosa........45

 

Table 7 Effects 0f Cyanide on Nitrite Formation
by Previously Adapted Pg. aeruginosa.......46

Table 8 Characteristics of Cyanide Tolerant
Organism Isolated From Sewage...............47

-40-

(x:

H.” )IDI‘A I?“ Clea.

.,..

fit

 

 

   

3w
(mmm

J..._..2.__.L_2_..l_.2...3._..2._.

m0

2::(ppm O)

Nitrates
:th)

Nitrites
(mmm

 

Temperature

- g 00.)
.1.

mm

mmm
-e.e

-41--

m
0
wmmOOMOOHmllmlldom [mo lom
H mmN HH H H Hm? N3 HHS:
nn new he
NmmmwammlemlIwwdolNlmNNNNH
: N d H NNH m HHH
lwmlmmll omtmOIm IIIIIII
N Nm 30m mg m" |"
IomldmmlmmthtllmllmmlIluog.
Hm N mN m 0 mm m

00:30
0....
NHMNN

omoo
Ndmé

Nmmm
C C O .
Hooo

WWW

II
000

0.3 ppm GN

mommHmmmdomomm ONNNmONm
eeoeeoeeeeoeoe'
HNOHHHHHNNHHOO r-lr-(t-lmr-(r-‘IHO

ooocommm‘mmmmmmdmmmmmfio
mmHmdNHH ##NNHHHHdmméHH

m dwmmm demnm H
HHOWOimHHOOOOO OONOOOfiH

66HHN666666666 66666690
mmmmmmw

MMNMHHSH 8000000H80H38
66666666 66666666666600

2
3

A.

Amfl l

lmmfl

ECW“

.q‘

I‘l‘k .

-42-

~|.A 14V

 

0.a N.0N 00.H
0.0a N.me 00.0
I. I I. I MoH I.
n.0a 0.0 u .1 m.m m.m
0.0m an x u N.m 0.0
0.0a m.HH Na m.m m.m
0.6m mm on 0N 0.N a.m
NN mm. a 1 m.a m.m
mm s a x 0.H m.m
0m ma 1 1 0.H ~.m
mm 3 mm :0 t ..
mm mm .60 mm 0.0 0.0
on m.~a .1 I 0.0 0.0
.nw..1wn .nw. .iq: .nw..1qa
Ho 8000 Huang“ .Hniamflwu
not 009 aoesseaz

seesaw

ummomxa. N

nonaao Hosesoo a

 

 

mNH.o oOHpmH>00 nonwsmpm
mmH.o mafia

mm0.0 u 1 0H .

I. I m. MH “(UH

no.0 00.0 ms ma

00.0 00.0 mH 6H

no.0 NN.0 WH MH

no.0 $0.0 0H 0H

“0.0 00.0 #H m

m0.0 00.0 NH m

50.0 00.0 0H m

00.0 no.0 0a 0

@0e0 0He0 NH W Hwhgd. 20 Egg H

rdW.;41. 14%. r.
CH E08: ~00 V
mopHasz maspmuomsoa open

Table 4

Trickling Filter Effluent After Shock Load of 44p0m annide

Elapsed time Nitrites
(minutes) (P9111 N)
-40 0.03
-20 0.036
0 0.033
10 0.058
20 0.062
40 0.066
60 0.066
80 0.051
100 0.040
200 0.030
220 0.028
(days) (ppm M
1 0.24
2 0.28
3 0.17
6 0.19
7 . ‘ 0.08
8 0.17
9 0.06
10 0.16
11 0.07
13 ' 0.06
14 0.044
15 0.036

-43-

- :wlmnu

0003000 Sam 0: «N

Houpqoo “H

 

 

._ w}: d m m HH NdH
mmpmanz 0.H 0.H 0.H 0.H 0
H0:.o m.H mm.o m.N w :H H NdH
mw:.o :0.H mm.o m.a N.0 MH H mNH
000.0 00.0 00.0 00.0 0.0 0H N 00H
000.0 H0m.0 H0.0 00.0 0.0 0H m 00
000.0 000.0 00.0 00.0 0.0 0 m 00
om.o 00H.o Nm.o mH.o m.N m N am
00H.0 00H.0 0H.0 0H.0 0.H 0 m 00
mHN.o NHN.o NN.o NN.o N.o m.H H m
00.0 00.0 00.0 00.0 00.0 0.0 0.0 H
00.0 mo.o wo.o wo.o 0 «H0 0H0 o
00Hq0mo 89m m.o o» dwmomxm hHmSOHbmnm um
g m.: N.o m.m NH NdH
00000000 0.H 0.H 0.H 0.H 0
Hm.0 00.H 00.0 0.0 0 0H N 00H
Aw 000.0 00.0 00.0 H.H N.0 0H H 0NH
.4 000.0 H0.0 00.0 00.0 0.0 .«H 0 00H
. 00H.0 00.0 00.0 00.0 0.0 0H 0 00
HmH.o mdm.o 0N.o 0m.o 0.: m N on
dH.o wma.o mH.o 0N.o m.N m N Hm
miH.o HOH.o mH.o mH.o m.H 3 N 0N
00H.o mmH.o mH.o 0H.o N.o m.a a m
0H.0 00.0 0H.0 00.0 00.0 0.0 0.0 H
NH.o NH.o NH.o NH.o 0 Adv AHV 0
1mm 89: CA 693 A125 A233 «2005 «0.533
0 :JW. N H dubosmm
000A madaob oEdHob mafia
mouwnpaz_0opownpoo mopwppfiz H000900000>m H0009 oamfidm dommdam

oqummo on anamomkm msoa>mum oz fid

 

mmnmsammmngmpHfim 0H 000000000 opHup z -umeno¢00

w OHQGB

 

'45-

 

 

Hliects of (halide on Kit-rite Formation

 

' Huada ted Psuedomonas aemginosa.

   

Time elapsed Cyanide Concent rations

 

0. .. A: “g

(hours) 0 punL 0.2; Dog _2 ppm 20 ppm 200 pop;
Nit-rites (ppm No :
0 0.0 0.0 0.0 0.0 0.0
0.5 0.088 0.016 0.014 0.022 0.036
1.5 0.006 -0.0 0.014 0.022 0.032
2.5 0.0 0.020 0.008 0.008 0.042
4.0 0.022 0.027 0.025 0.029 0.037
6.0 0.007 0.012 0.029 0.016 0.046
7-5 0.012 0.023 0.027 0.017 0.046
9.0 0.029: 0.030 0.034 0.027 0.10
11.5 0.09 0.823 0.017 0.014 0.028
12.0 0.125 0.25 0.022 0.014 0.012
12.5 0.25 0.42 0.034 0.022 0.042
13.0 0.28 0.41 0.022 0.008 0.010
13.5 0.43 0.56 0.056 0.012 0.025
14.0 0.48 0.61 0.115 0.022 0.034
14.5 0.54 0.74 0.056 0.018 0.034
15.0 0.74 0.89 0.070 0.018 00036
15.5 0.90 1.10 0.067 0.022 0.044
16.0 0.92 1.18 0.089 0.082 0.031
16.5 1.05 1.34 0.095 0.034 0.036
17.0 1.20 1.55 0.11 0.022 0.046
2205 0.24 0.022 0.019
23.5 0.34 0.020 0.020
24.5 ‘ 0.45 0.018 0.015
25.5 0.52 0.034 0.036

Innoculum: 7.44 x 107 cells

Temperature : 20°C

' Medium: Physiological saline with 0.125 K1403 and
0.1;9 glucose

-45-

Table 2

Effects of Cyanide on Eitrite Production Ex

Previqugly-Adanted Psuedomonas aeruginosa

 

 

Time yanide Concentrations
Elapsed 9 ppm. 0,; ppm 2_ppm 20 ppm
(hours) Nitrites (pmeN)
O 0.0 0.0 0.0 0.0 0.0
0.5 0.016 0.012 0.020 0.075 0.0 6
1.5 0.020 0.014 0.010 0.050 0.086
2.5 0.020 0.016 0.020 0.014 0.080
4.0 0.022 0.018 0.018 0.023 0.062
6.0 0.022 0.012 0.016 0.013 0.050
7.5 0.015 0.015 0.014 0.023 0.053
9.0 0.022 0.024 0.028 0.029 0.068
11.5 0.042 0.010 0.024 0.018 0.064
12.0 0.006 0.0 0.014 0.010 0.052
12.5 0.036 0.030 0.030 0.021 0.070
13.0 0.010 0.007 0.008 0.008 0.054
13.5 0.027 0.033 0.016 0.012 0.056
14.0 0.055 0.080 0.020 0.016 0.059
14.5 0.10 0.082 0.023 0.028 0.073
15.0 0.16 0.20 0.029 0.018 0.038
15.5 0.29 0.30 0.060 0.025 0.050
16.0 0.37 0.39 0.062 0.025 0.026
16.5 0.45 0.61 0.080 0.042 0.059
17.0 0.60 0.79 0.10 0.030 0.048
22.5 0.022 0.033
23.5 0.025 0.031
24.5 0.70 0.040 0.029
25.5 0.050 0.035

Temperature: 20°C

Innocuium: 6.75 x 107 cells

Characteristics of Czanide-Tolerant Organism Isolated From Sewage

Morphological

Mycelium: Conidia formed in chains
I'iuch branching, small elongated cells

Colonies: White on.Nutrient agar, potato plug. calcflum
malate agar, Kligler's iron agar, starch agar.

Flakzey white on surface ”of nutrient broth

Physioloaical
Aerobic
Reduces nitrates to nitrites
Peptonizes litmus milk
Actively proteolytic, liquifies gelatin

Catalase positive

Survives three sucessive transfers on synthetic

medium (agar), with 200 ppm GN as the sole nitrogen
source.

Isolated on synthetic agar in the presence of 200 ppm CN.

Characteristics fit best those of Streptomyces a1§g§,wBergey's

(6) page 934.

. —.. . .c-Mm.
‘! l' ' s

APPENDIX II

ANALYTICAL METHODS

General Discussion............49
Correlation of BOD and DOC....50

' Calibration Curves: '
Nitrites................254
Nitrates.................55
Cyanide as Thiocyanate...56

Cyanide:
c Picric Acid ReductionS?

Cyanide as Prussian Blue.58
Formaldehyde 'Freeuooo.o059
Total Formaldehyde.......60

-48-

‘ W'.'.l. .-
q' '.
...
g.

 

 

 

 

 

 

ANALYTICAL METHODS

Routine analyses of BOD, nitrites and nitrates
were perfprmed according to the procedures outlined
in Standard Methods (3). The nitrite procedure was
slightly modified to give greater accuracy at low
concentrations: the sample is diluted to 20 m1. instead
of 50 ml. before colorimetric comparison.

Shaw's method for DOC was chosen because of the
relative speed and simplicity of the procedure. The
analytical results showed too much scatter to be useful.
.A graph showing the correlation between BOD and DOC
is included. The correlation is poor. There is no
significant difference between the results from the
treated and the control filters. This can probably be
attributed to the poor precision of the determination.

It isalso difficult to obtain a suitable analytical
procedure for cyanide.

There are two common general procedures for cyanide
determination: titration and colorimetry.

Titrimetric procedures include (1) titration with
silver nitrate and various indicatoro(liebig method),
and (2) titration with divalent nickel.

Colorimetric procedures include (1) formation of the

.49-

 

 

mCQMZM u

C. .2430 .Z k U)

-51-
iron thiocyanate complex, (2) reduction of picrate to
igggpurpurate, and (3) formation of Prussian blue. A
fourth method is available: formation of a blue color
with pyridine~pyrazalone reagent, but the reagents are
so unstable as to render the method useless for occasional
determinations. . a
Thiocyanate complexes, using the procedure recommended
by Standard Methods, are formed along with an appreciable
quantity of colloidal sulfur. Filtration of the reaction
mixture is difficult and can be a source of appreciable i
error. To increase the method's accuracy in low cyanide
concentrations, the procedure outlined in Standard Methods
was modified: final dilution to 25 ml. instead of 50 ml.
Picric acid reduction can be employed where a
distilled sample, free of volatile, readily oxidized
organic compounds is available. The procedure used
was that of Finkel'stein (16). Since almost any reducing
agent interfere, the method is unsuited for direct
use on sewage samples. If numerous determinations are
desired, the initial .distillation generAlly recommended
should be avoided, if possible}‘
Prussian blue, formed after evaporation of the sample
and resuspended for colorimetric comparison is usable
for colorimetric analysis at concentrations of 10 ppm CN
or higher. Extremely large (over 100 ml.) samples are
required for lower concentrations. Variation in particle

size of the Prussian blue reduces the accuracy of this

-52-
of this method. For this investigation, the procedure
of Friel and Fleet (17) was employed, diluting the
sample to 5 ml. before comparison.
Titration with silver 2. nitrate is not feasible in
the presence of chlorides, commonly present in sewage.
Simple acidification and distillation is not adequate,
unless the chlorides are previously removed. Removal can “i
be accomplished bit is tedious. ;
Titration of cyanide with.nickei, using dimethyl-

glyoxime as an indicator was unsucessful, due to the

‘firhfl I _

sluggishness of the nickel-glyoxime reaction. Further
diffuculty was encountered in that cyanide areduced the
sensitivity of the dimethylglyoxime, possibly by forming
uncolored condensation products.

Addition of excess nickel and back titration with
"versene" (diaminoethylene-tetra—acetic'acid) is feasible
if a suitable indicator can be found. Mureoxide (ammonium
purpurate) is suitable (23), but is relatively expensive
for routine analysis.

Titration of the cyanide directly by. nickel in the
presence of excess ammonia was attempted. There are
several disadvantages: the end point is weak and the fumes
are irritating to the analyst. The cyanide complex is
more stable than the nickel ammine. Ammonia might be
used as an external indicator, but this also is cumbersome.

Conductimetric titration with nickel or with.nictel

and versene may besuitable.

-53-

Cyanohydrin formation was also considered as a
means for estimation of cyanide. Excess formaldehyde
, is added; the excess free formaldehyde is determined
by the method of Tanenbaum and Brisker (43). Under
conditions for formaldehyde determination, the cyano-
hydrin seemed to be quantitatively decomposed. This
was almost instantaneous.

Calibration curves for all the colorimetric methods

mentioned were prepared. These are included in this Q

“9' I LA. '
‘ -

appendix. A second formaldehyde determination is '
included: formation of a colored condensation product

with chromotropic acid (10);

.1. .0'. 0000 '0. 0... .0" 10 0.01.010 ‘00. . . .010 '00 ‘0 0'1 0'10 10 9 " 01 .150 0.1 009' 0401' 0.0 0

 

.'1 00 0.0 0'01 0..010 '.0. .. 0 .10 o 1.0 .0100 0 10 '10 '04 9.0.10. 0'0 0 . .0 00 '0'! '00 .10 '00.. 00 1
.... 001 . .00. 0. Y 0. 00010 011 0 0' 00 0.0 0 . . .00. 010.0 0.1. 0. Y 00.0 00 HHH. 0
.0 0 0 0 0 009 '0'0 000 00' 0 0 0 00010 1.0 0 '0 0100' 0
1.0 010 O 0 O 0 O 04 '10.

00 0 o '0' ' 0 0 0.0
00 '1? 0 ' v.0 0 Y
01 '10 I. 00. 0 O 0 0' 000 0'
0 0 00 0 . 00 . 0.0 0 0
' 0 0'4 0'0 0 0 0 e 0 0 00 ' 0 l
011 000 '1 '0? '00 '0 01' 0'0 01H!“ 0 010
' .0 04 000. '0 .0 0 10" 0.0 0 00 0. 'L 0110.

0001 000 u 00 .04 0.. 4.00 00.0 . 4 0. ..0 . 0. '.1 01 . .1 . 0 0 0'0 0 0 ..1 .0 '0.0 000. ... . . .111 '0"
f'» 001" 0 "0 00 0 0 .019. 0 01.0 0" 4' 0 0"0 0'0 010
0" "‘1‘ 0 00'0 00 £1.10 0 0‘ ' .' 0 .

0.' . n . 0 10 1. 0
01 0 00 1 0000 00 '00 00001 0 0 1"0 '0 010.10 00 ' 'o 0 010.' 0 0

0 01.00 00 '0 0.00 '0 0.10101 0.0 100 'HIO 00 0010 01000 0'. 010100 0.9.00 0010 00100 0. 0 00

e r '00 '01010 010 0 01"10 000 0 0 0 .0100 0. .10 0'00 0.00 0 o "r 0 '0 ' A 0
0. '0'0 1.0 00.0 r1 010 1 00010 0.0 001'... 00.n0, 00110 01... 01.0 0100 0 . " ' '0 0 1' -101
o-.. 00 '10 0.00." 0'00 . 0 . 0'01. 0 V0 0' 0011. 1'10 .0... .000 .100 7'0 0'0 0'10 0 0 ' '00 00 '4 t
0' '1010 0.?40 >100 '000 0.. 0.01'10 '10 00 001010 '10" '0 .1000 110. e... '00 0' 0 .0 '0 0100 .0010 1' '00 0.
0.0 01 00 .01 ... 0.0 0014 '0 r10 .901? 00100 0.00. 0...0. .1.. .' 00. .0.. ...0 '.0 "' .0.0. .' 0 v. .1010
01’ Y 01.0 a 01110. 0.0.10.0 0100 .00 'V1 01. 0 0 .0. 0.110. ..0. 00 1.1.0 100 0 ' I
0" »"0 .010 10100 0100 '010 0 0101 0 0'10 .0.0' .1. '1' 0 ..'. 010'0. 00 .' 0 'c'. .0 $0
+10 00 0 0 0 01000 0 . 0100 .'0 01'0 .0100 .0? 100001910.0 .0. .100 ..- 0. 01. 01010 0 f 0' 0
' 0 00 .0 ... 00. 101.1 .00 a .10. 00 .1 .0' 00'0 0 01". .010 '11 00
01' 0 0910' 0. . .0. .00. .4 '0'0 v0.0. 01. .0? 00'0 0.000 . 00.10 1 0 0 . 01010 01
000 4 0010 0010. 1 0 010 .1105 '0 .0 "0 0" 0'0 .0 0'0. ..0'0. 1'0 00.1.0. 0 0 0 .w10 1'0 '00 '10
0'0 0 0'.00. 0 .00 0'01. 00 . 0'0. ' .L. 000. '01 0 00110 10.0 . 0 .00? .c0.
'0 .10 «1010 0 0100 04' 0.10 0.00 00 0.00 0.00 .00 010.010 0 .o 00 0 0 .000. 0.10 10010
' .0' 0 . 00 00 0.00 .01.. 000.1 " v 0.' 000 ' 011 . 0'0 '1' 00 .10 0100 .0 0 0010 00 00.0
4 010.1 000 0'01. 0.0 0 ..0' a 0' .000 00 0 0101010 0000. 0'00 0". 0'0 0' 00.0 . 0" ‘."0. 004 ' '00.'
'0 0 '01 0.0.1 .0. 000 10. 00 0 '00 0010 00 .0 01' .0101. 0.10 '4. 0 0 000. 4110. ' 0..' n '10 0 .000...
0 0190 014' .0 4" u .0' 100 "0 00100 ' 00011 10.1 '0 .0 v' 0.0 01* ' 0 .01010 '0 0010 0'00 00 11'
'01. 0 r 0 .001.1 1100 0.1'.. .0 '. 000101 0.00. .0..0 '00. .0. 0'0. 10 ..'. 0.' 1'0 00 10 1.. I 0 '1' 1'0 0 0 00 0 ' . u '10..
1101' '10 0 '1000 00'. T0010 .' .0..0 10. 004.0 .. 1.0.. 00.0 100 s 0. .. '0'? .0?0 .0 0 00 ..0 0.1.10 0 .0 ' 0 '01? 0010 010 0.100 '0 10
0 ' 0'0. 0 '0 9'0 0000 0 014." 10?.1 091.1 000 .0'1 0v.0 010.. 0000 00 00'. 100' 10100 .010 '. 1 00 0'0. ' 0010 0
0 '01. 010 00 '00 '00 .701 '10 .0 v 0 0' 0 0 '10. .0. '10 0'0. 001' 1'0 p'vl 00 10 0 0'0 0' 0 0
.0 v .1 001? 0 . 0..0' .r1.9.0.00 .9' 0 0 '000 0'01 101 010.0 0' 1' 000' '000 01'. 7'} 0 0100 000
." or" 0'0. '0' 0000 0'00 '00 001.0101 0.10. 0 .0 O0 0" 0110 .0 0.01.0
010 '0 0 11V ,_0 0 0 0'0 '0 0000 uo1' ..00 10' "00 00 400 0' 0 '10... 0 0101' 0000 0'00

'0 "00 00' '0 '.0 0.1.0 001010 0 10. 0 0 00 0 0 0 010 1 00.01. '0 . '0.

I . ' 0 . . 0. 0 0 0 .0. 1101' 001' 0" 0.0 0 0 . 0 9.. '01010 1.0 001.0 0 0"0 . 010.0 ' . r. 0 0 '0
0014 .0190 0401' 0'00 .1 10.0.0 00. 000 0.0 0.0 I 100 01004 '010 0 0 0.400011 1 0010 0.00 v1.1 0'0. 0. 10.0. 101 10 0101' 00 o '

V01. 1'0 P0010 .0 .07. . "0' 0000 .0 010. 0.0... t ' 00 .0. '10 0 0001' 0'00 '0. "'0 000.. 0.00 0 . '00 '00 00
0f 0.0110 0.0. 0+ 0 .0'1' 01. 000 1 .V00 '01.' 1'00 010 . 0 . 0000 01 .1 '10 1'10 010 00 01101 0 .1
0.0 '0 1 01. 90 0100 0.001. 00 00 001' ' .0 0 u '1 '0 0000 0 010 01'. 0101' 00 '100
.0110 0 01-0 00 0 00.0 V 10.. .v. 000. 0.10 0 010 000 0 .0 0 '01. 00. 1'0 0.. .00 . '
00.40 0 '0. . 0 00.1.0 01'00 0 0 0010 10101010 0 '0 0 . 1.00 0.0 00 0 01". 0 .1.. 0100 .0'0 0 1 .0.
. 0... 1.00. ' 7.0 0.. ..' 00.0 0110 1010' 00 0" 0. «'00 ..01 0 .010 . 0 00 0. .0 00 .0. . 010 0010' 1
. .
..'0 0 0000 010' 0 0 0,0 00 090 O'.L 10 00'. ' 01 00100 . '410 0.00 010 '001 ...1 0.010 001' 0 1 00 0 0 0.0100 .0.'.
0 0010. 1' 0... 0 1'0 0 0.. 01000 '0 1 '01. .e'. 01 00... . o 0 ..0 ... 0'19 01. 001 0 101 .10. 010 ..0 4.0.0

. 110 . . 04 .o.. '00 0.1. ”H .00. .0.V ..0 0 .. 1'00 1. 0.0. 0 011. 01 0.0 01' 00.. ... .

. '04 1'10 .. 000 0 .0100 010 4 010. 1 000. .'f10 0 .0 0 -1000 0".0 0 '0-0 '0 0 .0 0' .0 0 0 00
1. . 01020 '.'00 0' 1100. 0010.. '0 4441. >0. 1.0' .01. 11 01.010 0'0 010 0 .0.0 00 011.. 0. .1 .

'000 0101. 01010 '0 001' 0140' 0 0 0. 000 0.00 0.0.0 .' 1.". 00' 0 '01' J .0 0 001'
00'. 0.00 0100.0 .0 00. .00. 0. '01' "0 .1090 0 041 . 0' 'o..0 0. 0f'10 '.0 .00 .0 0 10 '0 . 0. ' '0 . .
. . _ .

. . 0 .. . .. .<. .. . .n. 0 v. .... . .1. .00. 0.. 0 0... . . 00.01 0 0 1 0'. 00 ' 0 00.0 0 0
000' 0". 010 o 1 .11. 00.01? .0. 0'00. 010.0 0.000 0000 . 10-. '01.. ..._ .._. .. .0 .0.. r ' .0 '0 01 0 00 '000
01' 0 0. 0 000410 1' 700 .0. 000. 00. 0.010 0. 101 000 .' II. '.0 0 r. '1'. 00. '0 00 10 001'0 V.

. v10 0 0 0 0'1. ' 00 . .0 0.-.. 000.9 00010 .5010 00 01‘ 0. .00 .0. .110 .1' 9'.01 .0100 .0' 0.0.10. 0... 100 0000 0010 011 0' 7'0 0'0 '10
0 0 f0 00 00.? 04. 0 0 . 101010 00.010 0..- 00 010 .'00 0.. 01". 00.0 00.. ..10 '0 1 I 0

11' '0 '0 0000 0.. 0'0 ..0 0.9 . .000 r 000 0. 01" Q 7'19 010). ‘00 '0. '10 0010' 1' 010100 0

'0 010. 0». 00 I '00 '00! 0100 0 D 00 000 00,010 000.0 0. .0000 0.... 00. 0 '0 '0 0100 '0
0100.. 000. 100' '00' 00' 010' 0" ' 00" '40 '000 00 1'0 0.0 101000 00. 000. 010 10 00 0.0 . t

1 001'. .0.' .010. 04.01. 00.010 00" 01. '0 000.0 .000 0.0'0 010 0010 0.'. 0.00 100. ..0.0 V. .. 0 . .'0 <00 0.0 10'

0110 00 010 0.0. .00.. "0 0.0 010'0 '01. 0'».0 0.1.. 0." 00 00.10 0 0 010 0.100 ..01.. 1.1.0 14 4100.0 01.0 .0.
. 00 0.11 0100 .07 00... 0.1141 .. 110 o 0 0.1. 0 0.11 10.0 .100 .1 00. 0. .0 1. .010 0 .0 .0'0 411'. .0Iv 0. 001

000 01010 '01- 1010 0.!0 .'0 000' 000 >0 0 V0.. 01100 00.0 .1000 '00' 0 '10 0 '10 000 .0 0 ... 10.0 v .10 '0 0'0 0
.00 010.00 11. 0 ..0 010.1 . 0 .11... .01.. 0.0 .00. --.0 1 0+0 0' 1. 0 000 ... 1 .0 0 0.0' 0, a... 9001' 00.. '0. . 0 0 0
.1.. 0. 10 001010. .1.. 1.100 . 0'0 r0 '..0 .o. 01 000 000 0 .00 ' 00|0 17 0101 0'11k. .4... 0v0 0.. '10 0 0 ' r00
00.0 .0 00 0 01.' 000' ' 0.0.0 01 '00 10 0 0 00 .V '0 010 00 .... 001'. 0'.0 '1 01.. 00.00 00010 0 0.0 0 010 '0
1. 0 .0101 01" '10 .0. .000 .000 .00 '000 00 0 a. 1 10 0 .. 0.0 -o .000.-. 1. 010 0010 01'. 010100 00.10 " P10 00
0' 1 '~ 00. 00'. 1' 00' 00. '1 0100. 0.00 .0 90101' ' ' 0 01001 0 00 '0 u. 00 .0 01 0'0 0.0 0-0 010.00 000010 0 *'1l
"0 01010.10 0'00 000 '10.. 10. 01 . '0' 010 ' "' 0 ’1'4 010 00 1'0 11" 0.0. 01. v 0 010 101019! 0' 000' 0 0 0 010 0 .0"
011 .o .010? a. 10-00 00. .1... 0000 0.0 100 .0 .000 .0 ' '10 .V'. . 0.0 . P. .0 0 1 01000 00' '10 0 0 0 .0 0 0 '10.

.0.. r .01' V10.0 '0. ' 0010.01 '100 .0 00 10 01k 0 k '0. 0 010 0101' 0.. ...0.0 .001 1' '0. 700' 0 000.0
.110 1'0. 0000 001010 .0... 000' 00 '1.0 .0? .0100 000V 10. '01 .11 .00 .0'0 .0. 11. .1... 0000 .11 .10...- 01 '101' v '1. . '1. .010 01...

0 I0 '0010 '.0 0. 0'0 0. .0.. 010. '00 0.100 010 0 0'0 ..0 ...0 1...0 0.0 . 0.0 00 0
0" 0. 00 01" 0100 .0. 0.010. r0 10.0 01. 0 . ..10 0... 0.0 0 '000. 000 . .. 0 . .0. '0 . 00 .0' 001010 .0 001"
'1010 '00 1'0 0'00 01.00 0 00 . v v .1. 00 '0 0.0 ' 0100 00 0 0. .0100 0 It 0 V00 0100

0'. 0.0 0. 010 '0. .10. .. ..0 01100 00' 0. r .10 00 0 . .1.». 0.1 .00. .11. o .11 c. .. 0101'- 0' 1 0.. 0 '0 10, .010 '. 0".
00 01.. '010 00010 .1. ..0 0 ' 0'10 0010.0 0.00 0010' 1..- '00 00. 01 00- .1011 0'-.. 0 .1 1. 0 00.1.0 0 000
0' ' 0 0'01 0.0 0 0011 .1 I '00 1| «'0 0... 1.1 .10 1000 0.0.0 .10.? 0.11 . .1" 0.110 11700 1'0 0 ' r11 0 0, 1010 00'0
100 10'. 1000 0 0.. 00 0.00 01... 101.0 0 00.1 10. -0100 010 L410 1001' 100 .0001'. 00010 0010 0. '0 0 'l'& 010
."0 11.0 001001 04'00 11.0.0011. .1.0H110... .... 1'0 0'0. 00. 10. 0.. 100.. o'00 010? 10.. 00 10.00 .000401'0... 01 '0 '0' 0000 0.0 0 0
11.1 0 101 .110 0' 101.1 0'.. ..'0 00 '0.' 0' 0 v.. 0.0 .1 ...0 110.01 0.10 000 00. 0. .v'!.. .'00 0" 0% 01'. 0.1 '0 ' .0' 01'. 0001.
.01' 0.... 0110 01.0.0 1 1 '0'10 00 . 0 ..11 0. . 1'. 0 .1 1 a 00 ' . 1. .. «cur . u 0 0.00 . .
011' 0 .v'v. .. . .0.1' 1 0 0' 0 0'7 100.0. 0 1. '10' 0. ...0.0 .1. .0..010.00 .010. 1. .01 '01.. 0 010010 '0 0100 "'1 0 .00.. 000 010

010 '0'..01'1. .0.101.1. 0.1. .0... 0101.0 0.10 '0'7 '000 1. 10 '0.0 .0 0 .'.. .010 '1000 00"0... .00 .00. 010.0 '01'10 1.0 1. 0 0 1. '0'. 010 '00
'0 ..'0.0 .001. 0-00 .. 11010 i .. .011 010.0 0.... .. . ,1.. . 001001 0.1.. 0010 '01.. 001. 091. 0... ...0 .0 1 .11. 01.10 0". .1. 0'0. 0. 0000 0010 '00. 00100 00.0 ..0 .
0 0. ... .0 .1 1101' -00 e . ..'0 .0. 0.- 1... 00 0 '.. 110. ...010 01. .oo 11'. 0. ..0. .1.. .0 .0. 0.. . 0.010 1 01.1. 0'. .0.' 00 0 .1. .000
0'10 0. ..1 0010 . 0 .. . v 1 01100 11' 10110 0 . 0100f ..' -.. .0 ' 0. '00 0000 '010 0000 '. . 000.0 .00.. .000 110 Y... 1 0 00100 0 0'. 00 000 r.» ' 0'0.
U.u.'h H1H0'HI .101 - 1 .' 1v.1 ..1v1 . . ”1'1. 0. 101010 1.0- ..11 11.00.1010 .100. 0 . 0 - 11110 .10 0 0 01010 '00 00. .01' .00 0 o 101
- .011 .1 - .10- -.11 .1-. .110 11.. .10. -110 ..0.1 .11 ..00 -10. 0..00 0 000 1'
. u . ..00 0 00 00. 4 '0 ..0. 1
11 1.1. 1 111 . . . .1. 1 - . - 1 0 0 . 0.. '0 '14 'L. 9.0 0 V 0 0'00
.1. . 1 '

1 v 1111 v .1 '1 .. 1 0.- . 1..1 00.. 1. 00. . 0.'.. . .0
110 0' 11 0 O. 01.0 0. I r 0.0. 0 110 00 1 '00 1'00. 00 .000 0 ' ' 0110 10I 000' '10. 0 0 0 '.'1'0
.1 . 11 1..1 .-.1 . . 1... . .70. .1. . ... 1. . .1.. 01-. u. . ..- . '1 10.. ... .1.0 -.0 _

-1 0 '00 . . 11.-. ... .0 .. 00.0 0 . . . 11000.0 1. v .

.1.- '11 11 -.10 :0. 1- 11.1 . .0 0.. .... 01.... .. .... r. 0.. ...+... 0000 0 0.0 41? 00 .00 '00.
1 1 .0 1 010.. 0. 1 111 . 1 01 10 0 v1 . .1. 1 u . 0 . .1 .

11' H1. 11. 101.1 . 011. ..011.0 ... 1 .00.-. ... 0.0 00.0 ..00 00'0 I 0 0'0 00 O .0

.1 .0 1...1 ... 10 1 .. .0 1. .-1 - . 0.. .0.0 .01 - '1'01010 . 10.0 «.1 000 .0.. u '0 000.4 ". .‘0'1' 0 0

10 .00. .1.' 01 n .0 0 ..'1. .
..L. '4 00'. .0. .0.
0 '7'0 00 I '01 0
0 '0... .0 00" 0,0 0.0..
00, '9 '6 ..00 '00.
O 0 0. 'VICI. '*‘
.
00'0 . #001. .0 '
O
. . o.
0.. 0 '0 v 0.0 .90 .0 .«0.0
01010 400.0 1' ’0 _
0,00 0 00" ' 10'0 .. 10.9
0 _ 9
0 0.0 0
0 .7'" 400 0 0|. 0
0
a 0
.1... . I..- 0. .40.. L00 .0 0 .01. ..
010 I
0. .... ..0 .1. 10
0.. 0
0 o 0 0.
. 0 . .
.r. .v..- . .0. 01 . 0.... .. 9. 1.
0 00 I .
01' 010 J .0 41 '000 '00 0.4.00 .00 0*
I; e '0 '0‘ .0
0o 0 f I .0 , 0 1.000 0101 0.19 .'0. 0000..0b.
I
. 0 0’
0 10. 0 V . 0 0 .0- I
I 0100 z 0010 a 00 V00 00 '0 0 . v ..
0' '
' 0 0. 0. .1.
0 0 01f

0 Ole" [I ,0 0.010 e. .0100 I0 .09 019'. ‘1’. ‘0 V0.0. V94

 

.1. x: 1.. . e . .1..
II. 0 0 ‘0'? ..t.0.0>o 00100 .0010 0'90 0'00
0‘1
'0. 0010 .0 0
/.10 -00 .
.1. 4/
In 1 I1: I 10'. .ro'nl.0.00.10'1 ‘7‘. .6

-,,—-.’--. .A. » .— —-...-

1.0111l0' 1

inll '11

0100'." .

-5
.

..101'

 

 

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

  

 

     
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

w; . 1411 14 1| 1|. 1‘11 11 1 H1 1 0. I 1 1 I 11 1 1 11 A ..1 11l 1 .1 .0
. m . . . 0 . o - . m , p , . A
_ , _ p 0 , .. . . ,
. _ . . . . , . . . . . . .
.w1 0 1 ,. .1 *1.01.. ....f... ... 11.10 '01 0' 0, ..0... + li.101,.0 1. .01 0. . 1 .
0 . . 1 . 0 .- . -. . _ c . . . _
. , .. . .N . -* . ., . , . - .
. o + . . . . 1. r 1 . . . , .. ..1. . _
.0'0 L1 1'41 . . ,. I141 111 10 11... .
. ' _ . 0 .0 _ . . . o . . 1.». 1.. w. 1
, . W . . .. - .. . . , . .. . - .. . -. m
u . - ¢. . . . . o - 1,, 0.. _
. . . . . . . . w ,. 0 .. - , .A. . *
. 01 . v 1... . -1 W.. I 0. 0 *1 0r .1. .11. . 10- I. .. . ...1 .111 .010110'01'11 .,.11"1IO . . 1,1
. #fi . . .. . , v. .. a. 0 .1, . . w
, . . ... L. H M ;._. . .1.-$33,“... .
_ , ,- . . .. . .. . . . u . , w . . .
11.1. 11 ...fi.. 11. .14 11-11011. 1v. .11'11010..,111110.111T1.1.1-141,1.101.1..1 {$.11 v ,1 4 .1 11 111‘
. . .. o 1 o. . . . . . . . . .4 .n 0 ~ .
c _ . ,. ., . 1 'L . . . .. ..*. ... q ., . . H
. H .. - _ . v . . 0 . . .. .0.. w . . . . .
. . . . . ,. o , . ... . 0 .. . . .
- . -- , 1-.....- ,1 -..--.Liff- . -1.-1..--..1 -- 1:. - f 2.1-. .-.-.1 ...--I ._
., .w ,. ..,. -,, w .-..- ,q —...,..* .y- ... , . m
. . , , o. . .. . . v... . 0 .... . . -. ... .0’
. a M .. c, . . _ . - .0, . .. .. o. , .,.
0|..' 1 4 1 1 1 1 111 H {.1 1,111 114 11., L
. . . , ..... -. . . .. . .. . .... . . . v ,
_ ,, H . .. ..h. , .. .., —. ... ..-........,, .,.--... _ . . A“
. h . v .4. .. . . .. 1 ... . . . “,. “-..... ...-#1 .«.,. M. a , .-
,. -. _ . . 0 . . ....0 ... . ..0...,‘ 0.1.1.0. . .
v I . 11.. '1+10.111. -.11 1110fi110. 0w1'0 ".1'1 .-.11' .1011W1'0..40.l1 .f- 0.4100010+.0'.'010..Lh1"'.w01'0.'+l1 ”x. 1“
. . M . .. . .- .. .. . ... 0. . . . .. . - ..11 -.- 0 1
, . .LW. 1 . - , ,. _- -.~ . 0 1 . ... , A ,...0.“. ... . n
.. . . . .. . . .. ... -.. .0.. .1. _—, . . -,1.. . . ...
. . 00 h . . , .. ... h . . , 0 p .. . a
.1. . 1. 1. 17.0 1'. . 4 11 111. w A .1 W 0 01 1 w 1 1 L. 1 '1011. 0111—
. . .. . .. . . -, . ,v 4. . . 1. + . . .—
,. 1 . ... .0 .. .0. 0 1 . 4. .. . , h .w. .- . -. .-
. .. , u h, 0, . . ..H .- . . U . .... .. .. 0 . ~
, . . . . . .. . . . ... .- .. . .. .. . . .
_ . 1-11W I 9 a o . 01 0.1 00000 0,.1' 11 0 . Av. .10.«111'- 0 01 1 11.1100I11 .001."1§ 101.f0 '11 9'.+ .Mv'.01 | .
. . . ' . .., . . .,. 1 . . m .. w ... a e . ‘ . . . . .H. ...
. , . . . ... . . . ._ . ,_. .. . .. y .. .- ..
. . ....... . a 0. a ., . .v .. . *0 o. 0 . v H 0 0 . 0
. . ., _ . . .. .. . . _
“1111 . H . 0 0 '1 P .r 1“ .r 1101». 11 .
1 .. 4 4 114 '1 _
Q . .... .. . ... . . . ... ~ . . , o 1 . .— . , . .
. .- . . 0.. . a .0 .. 0. w .0 0 .o 0 ...0. _ . . . 0 .H . . . 1 . 1 .,
a h u . . , . m . .. . * . 0 . V . . 1. , o . . .
-. . - . . 1 .. .0 .- . a . . . . . o. -
4 . 5.- 101 . «1 .1 . -11-.111.+ 1.1.00 -..Av1111.1011.010. 110. . 111.. 0' 111101111. .10..0-4-10.h.$100.111011+ 0 ,0.-.1Y 1- r . 1 0.1 711. . 0n... 1
. ‘ . . . . . u . . .o . ... . .. .. o. 0 . . - .
- , . , . _ _ . ..... . , . M - M _,4 ,
. - .. fl a , . ~ . ,. “ , ... . . ,. . . . . . - .
0 1 11110 1111.. .,'0 '10 11'041101'1'0101'110101110i1 0 0 1' 1'1 1 a 10 11L'1ll0010111 r .1r11 1111 1.1 1.
0 . . .... a- ... g . . ., . . n ,. . ...u .0 . .. ._ . 4 ..
. d , . .. 1| , . ., . . .. . . .0 -, ,0
.. . - . . . . . .0 . a ...,. o w o ~ .0 .M
0 1.0 . 0111 .w-1101 1 '1 '1 . 1.10.011 V.101 .11. - .1111 '01 V01 10: 11' .... T'01 .0.00.1.01 110100 111001 0,. 0
# . ,. ..... q. 1,.1M . . .. o 0 . . “,1- . ..
~ . . . w _. .. .. . . . ... o .
, ,. H u. ..‘1. ~ . . _ 1 W . r. .
_ ,, . . 0... . .p .. -. .-.
0.1 ”1+ 0 r w W11 1.11 H r 1”
. r , . . .. . .fl . . . .. .
p . _ , - . ., - . . a . .
. . p . a ..fi. 0 ~ A 0 , + a 0
. .11---. . .2111- «...-1- f- - ----,1. ...-I-.-- :F. ,
m . . .. .. _, . . _ ..fi
. J . .. u. . - w , .
V . . , n - . . f . , H .. , .
1' 11.,p111'1 ,1.'_111H.."..':. 0111' .101. 0" 01...- 1- 1010101411 110101.1H1'11011 1.1 . .-..111'4f10111'11. 1h
, 0 . . u .. . .
. _ , ,
. e . _. .~ . h w
. . . . .. .. 1 .
. . . 1 -, ,0--1-1..0|1.1..~.1 ....111+-l. .- 01 , .1 . 1 o 1 .u
. n ,. .. o . s w n
. - ,. , . , . . . _ ..
.- . . . . 1 .
. M . . ._ u , . .
111.011'.1|+'1H . m1 '1" 1+ .... A 7.0".."LT'1'1'111
. _ . , _ ,. , . .
. . . . . r . ~ .
0 - . 0 _ .
1 . 11. .0. . 0..s 0 1 .1. ,0- 01 o. .0001)- 0 A 01.11 I 10 1|
_ . .. . , _, . . - .
. . .. . . . 0 . . .
¢ . . - w . . . .. 0
. .. . . , . , . - Q1 ,.
' 0 1+1 01' 0 101+. . 01010101.! 101. '0 '11 0'11 1 W'i'illu? "" .01 .010 0 .1100 0' '01..”"011'1 '00 . 11' 1110101 '1
- . .. . . A .
. . g , . . . .. 4
0 . — . .r W I . 0
o , ,,. H . .. u 1., .
_ 1 0 .. .Q '1 9 . 1 1. 0 I. 0 1110 . 0 . .1 1 1 .+ .0 0 1. . I10 01 1 0 ' 1|“ 1.
o 0 0 . . . a ..
N . . , v - . - .. . . . o
. ~11 . ... . 0,,.. 0 . .. .-
. . . . - .0 0
1 <1 0, 1 J11 111+ 1,051 . 011.11
— 0 . 0 ,
. .. w ., n. ,. __ . , m w,. ..
u L . H . . . . ._ u. .
.. 01 "101 111..-.0101" 011 . 1. 'I. '1110 . .11, 101‘ .0 . . .1 . s
M . . _. 0 ,_ . .. A , u ' .
, . . . H . I,- . . t w
. .. . . . .. . . .
, . . .. , . . . - u . g . < , o
«11" 1 1 1L. '1. 0- k Y0 110 01" 01 ' 1.101011110113110. 10 + 0 111011 1. 1 01 1;. V . 1 '1'! 4 .1101 . 0 111111001 .1111. 1' 01 101' .11 1
. . v . . . ... A . . .
v 0 . . . . . , 0 .
H _ 1. . . H. . W . .y .0 k . m p. _ a.
...0 .1411.... I 0 ..I*.' 1 0 1v ..-- .1. d 1.11..'0| 11I '0 0101.1 10 1 01 1111 “.100 ' 0 0 h 10 1 , '0 1 . H
. ._ . . . . . .. . _ . . .
. w . . ... . . a . . . , .. . .
. . . — . ~ H U . , . * ., - . ,. 0
. . . + . . ,
111'1 - . . . .q . 1.... . ...... 11..” . w ...-y 1 '01'0"0.L.."".10"'0L
. , . , . . . .. 4 _ . . .H . 1 H ._ u .-
. H _ . , . , ._, -.. a ..H M - . . _ H u . n ,. . . . _ F . ..
.--.- ,1..- ,. 71.1... ....-...._ ...-g. ::.. ...-.. -- , -., ... f .H ... , _
. _ . . , . . . , . . -. L . .
, . . . . . .. . . A _ . o . . ,
. . . . . . * . . . a . 1 h , , . . . w , .
0 . . . . u . . F .-. . , v p
1%, 1 1 1 01111 ,W -11 111 111M111l 111 110' 111.41'011'1'10'11 vl.1'0 1.4, 1 0'1 ..1 0.1 1 F ~ v , H . . _ .
0 fi . . 1 . . . . 1' 1M1 1-001001 Av 707.0 1 0100010 101111 1"!1.‘01011f010|01.f110|1010101'l 110 I 10.00 i . , , . H1 . . n
. . . , 1 _ . . 011' 1011011111 .0100.1. 011.- .0' .
, . , . . , - . _ ., - _ , ,H , . . . . , 1 w .. , . _ i -. - . - 1 .
_. ..., . , . v . _- . . _. w . , , . . , , . . . , .
. . . 1 1 . -. ..-...1 1 .11 1.0.11. 1 . . 1 . . 4 . . . .. . .
a . , «1 .11.. . v. 1 n a , v . _ .
. . . . - H n , . .A, . L . , .. . _

 

 

 

 

o. -A‘+;A . .

.9’. ‘0

vd I|T. -

 

 

ncanzn QBNOM: 8. 20. UJm U

 

 

 

 

 

 

 

.I I < 4 . I . . v
. . o . n . .
.. , F . V . 4
F . . . F F . F V F . . F .
. . a . V .. .. A F
F. . V I I ..... F II. I II I F F I I n o I I w . I .L . II n I F I I
. a b V 4 .
. . _ .fi _ V I F . . F . . . . ... F V F 4
V < n F . V . - . F
v , . . . . F v < . .
_ 1 QQIII ‘II -9 IIIOILIIYIIII'I Iifi‘lfifiinlfifiitfi4I. r )1 I’...VIIIIII~|"||I‘I]|I IIIfiIII firI fl I L ..ltfi).fi1..l'! Ii? I’IIOI I IVIIIIIIfiIIIF. I‘IIII’IA’ I III, I... III— IOIIII 'I If I‘d. 0 III
. . . . . .. F . . . a. _ . . . . . .
. h F F . . .V V . F F . . . . . . . .
—
V I V . . IF . . F . . _ F .
. F , ...r . n V . V a _ . . . F . . _
. I I . . I a I I II. V I I II I III V . I . V I .4 _ I I V. .II I I. I I » II V I I I w I I l I I
. H . . . . _ H V _ .V . a I , . w
. F . F . F . . . . _ 4 . . . . . . .
. n F V . O r . . v V .
F II III4_. IV I |I.II IVIIIIII .fiYIII lfiVVIIIIIIIII Fr 'II rilanfiI.Ifi|LloIIlILrII IlIItlQIltfil VI'IIfiIOIIIOI IIATII VIIIQILI ..Vfin‘fiIYICI L 1'01 I‘IIV?IIIIfiI|rQ I vInIIIOIOIo IlfiIIo II‘IIIIFFV‘I IlfiIIIIJIwIII I I fin IOIIII It I {I II? I I F II
. V . .V .V . V. V. . V _ .V . _ . . , . _
. _ F V . V . . _ V _ h V u . . . . . V. V
. . a I > . . . . . . F . . . m m .. . F F . <
F. . VF . . . _ . . . . u . . a F F . F
.q I I I . . II .I I . IV. ..... .F I . .¢.I F . III: . .F In «I A. . I 4:. IWLIIT. III. III. I I I IVII I» .. _ F .F I . . . 4
V . .. ._ . V . 4 V I . . . _ 4 . A
.. — . .o F V . F . Q .’ I . _ . _ V. .
.. WI _ . . F .. F . I F ..... . F I I . F F F _. ”0
~ . V o .F . V v . . . . . .
. lnIIfiIIIVIlIIIIII II 4. I0 Al IIIIIIUIIIIII ILIIII flII IJIIII IIII V I I I I I L IIIII Ilinlillj . OnoleIIII IIIrfiIII
F . F . . . , . . . 4. . .F .. .V ... . F 40
_ . . . . V . I 4 . a VI .
_ F V 5 . F . . I .04. . . I . .VH I a F I .
. . _ . u . . . n I v ...n - . a F V. I. .n .
. . - -- V .II. II II. IIIIT II.-. . ...... I; II . 31.. I..I.II r. 4.V IIIIf.-IJ.I.II IIIIIw .-III.I-IFI -Ir IIV+-I.LI.I-.I I.I . .I-.- V F II-I.V I I V .
. . . IF H a. .. V F $ I . . . 7. F
‘ . . I . V. I .V . F. . .F. . I . . . ... . .
F . F .. . V ..F. . IV I A . . . . . .
. a V V o n n . o I.

VIVVy‘.
:“7

l
-l
1 I
- I
‘ I
V l
'I
I

T
“3

.I. IJIIID IIIIIIYIOIIII fIfitII'II.‘_ IfiIIII I .Irnl'p $71-..- 1
F a . HID V .. _. .. I . .
. . V . . . V . u‘ I . F. .
_ u . . . V . FV V V . 4 V I . .... .
_ c I o I . I I I‘II a VI I I I QIv I II Y ‘ VI t Ii v I IIVOltl II .II II .Lf- I. L .I II] D I n n c I I I 1 I
. .I _. n .F . . .
y . V V . . V V .

4
l
T
F—F+-o¢-o 9 ¢ ~FFF FF
0
I
F
I . V
2
l
I
b
0
fi
. -
6
I
F.
l
A
|
l
4
I
l
I

 

 

.t .V Ql 1| III ifin} ‘ifiz

 

 

         
  

L--—. -4 .F.—. 0-F.-
I I
.

V I V 5 n - . I
V. .-V-... -.V.—...,” .. ..I ...-
--

.

.

. F-¢F-_F-F-F..
V II I

 

 

 

 

     

 

 

 

 

 

 

 

0 I
V d . f
I . .F . I .F o . F F '3 .
I . . I. IIIVwVII ”III -I I ..L -.F I-I .... IF F 1-7.- .I I II.” II,.I..h . I I - I I. .FIVII. T’.
. . . . F .. V. V. . I h V I n I _
F 4 I I. w . * . F VI .. F F . o. .
F H . . I . . F . F F v. .. . I .V . .. F I... ._ .
. I II I II VIII I5 III II TI". {It tIfiIIfiIIflIIII‘IIIIIffiIIIOJOI' IIIIIfilIIIIt IIIfiIIIIII UfilIIffiIII‘ITfiVfiLIIV 'VIIIJVIIIIIPIIIIIIIIIIVILI V IOI'I IOII 1.1.90 ilfiIIIIIVI AV. .IIIIVII ..VV VIII‘IIIIIIAVIII'IO III V+J I.
. . F . . . . ... I. . .. F ‘ I.F . . I
u I .a I r . . F I F . V. » . . .0. 9 F . .
. o .o I I I. I o h ~ 0 . ,9 F ,
. V . . V .— 4 . . . V. . _
F . I I I I If I”! .IIIm V1I||I In A” I I fl II”. «I In .4 r I ”I n I. III._ IA VIIIIIIV. F.I. .» (I 4 . I II In».V .. ... I
» . . V. .H ...... ., .V ..V . H “ +J .. ..m V . u w g F H
V n . V VII“. .... H .... n 4 gr . .VV q .
V I ’I‘r.‘ I? II. II kw {IL N ‘fi‘. IL it :41 O‘ I 'LflIJ j. ifi‘lfilfi lkln III I) IIIIIfi lolIfink.I|Inf.l vfi00|IOII .Ivfilwl IIIIIII' ...
V .V.. .fV...a .\.I. . - V ..V. . . W .V _ . .V V
4 ~ I V. I. ‘ w .5 F 4 9 . V... _ F . . V . w .. V
. . . V . I I . . o . _ . F
I IIIIII. FIII.II I IIII I IV I IIV L III I I IOIIII. I F 4 ..I II
. 8 . . .. .. V . % FI . p . F 4 * . . ... F > . .. A u
I . . . V. 0 . _ .
O 5 . . m . V . 1. , w M V . .H V V m . O V
a I”: I - III| 5|! IIfiIliI'IIIIL :‘ILI‘II‘II'I IAFIIIOI 1 VIII?» It'll II IIIIII'IIQI+.I1|yrIIIOIIIIL~IIII|IOfiIIIIfiI.h. HII \YlfiI «III .I00 I IJII I fl IMIIILTI IN .T I I n
p h _. . o . V V. a . .5 V . . M H n a . 0
. . .V F . t .. V V I n . .V . . _ w . F I I
p o . I. v ‘ a J
V # III. I . II," .. III III- III, I V .I I II . #V I *2 ..z.. r -I-.. I” . . II F .L. I I . .. I
I V . . . 9 . . .
_ . . F .n V .V I F V . V . H _ . H V
. . . . . . _ . .
. . V n
‘l I‘ll/‘It'- Ll. Yr? . *4 M IV
. . .V .

I
1

I

I

I

O
L.;
Q

I

9

l

I

 

 

 

 

 

 

 

 

 

 

 

 

 

. .V .
.V III! II. I IOV II II ..I
I v . . u+ V : . o .- W
_ h . H . Fr u... . . n .V F V .
LI I! II -- .1 “HI . IIIIIIIVIII II».-. I .IIIstIIIIIIFiII- IV-.--,VI-.-+I_II_;.II..I. .
I V . . . ....V.
VV . . .. B . . h F H F . V .
. _ . F .4 . V. F . F .
F I _. IV II a I IVV .I'II III AT? II _9VI II II I IIFI I I .. .III I’IIQYOYIIAI
_ _, F ” 1. . m. _ .7 F . .
o F I. . . . n . . I
. V . . . F F V. .
“VIII! I'Iltil. .I k I... If flii #II r “r VII I
. V V . c
F . . . . V V n _. I .
. . . . . F _
F . F . H , . . . F
.r F _ n.IJ.V .V Il~.. .IIIII m 0' .I I III. fl I Ifi I I5 I];
. . _ V . . . _ _ .
. . . F .V
F 4 . o .. . I . I F
u" I . VI ' II! o I o.|' IVIPV l .InIIQnIIl. I rI! V IIIIIIfiIO VW 0‘ {fl V I1 6 IIhIIIO ‘LvIVfilfiII .'IIIII filtII Il.‘ L I‘ll I
. . . c . I4 .
V _ F V. m v V. ; V.V V.
. . ,V . F . V .. $8? F .b
. . I V s
n F F F _. ,.V
_ v . F V . . .
..VIIIIII IIIlt|II|nIIIII LVIIIIPIII IF blII
M n _ a .f A .....V “I V I
_. F o, . I. .. n V
F b 0 V. 09 III? I w v
F I . .
. F _
V. . .
I I III II I IIIIIo YIInIIOIIIIIIIToIfiI I I IIIIII. III I
V . II 4 . .II
—
_ _ . F F
H I - ... V V H -. w _
V F . F W V
a

.-.-...

"'"W

.-y—A._.

 

 

 

 

 

 

 

 

 

 

    

  

 

 

 

 

 

  

 

 

 

 

 

 

 

   
  

 

 

 

 

 

 

  

   

 

 

 

 

 

 

 

.v V
..V. .A er'... A ‘ AI .. ‘l c
A . c . V
_ . L V . u _ V . H V . A
. A . _ .A . . V “A . A. > o. —
o _ L . . . A m _ . V M u .V . .
J . 1| . . A v 1.1% . . 0 0A . A! AA u..~ . .A A H V 9V A V A V a L
. L L . L . . . . . V . “ +V . V _ V
w . A M A A V. . Va. . _ . p . W A . _ . .
. . A A A . V «V A . V . _ H . . A . .V » .
V . V . A V . . . V . o . . .H A . V .. 0 V m A
.-V.-.-V..1.!..I. ..Vx--:li..V...}-i-Vlel1113.3-I! . IL}: V V A V V V LVV L A 4 1 #1-..- 51:13
. . . . . V AI V A . A A A . . . V .
A m . .. Aom . . L. V M . — A V. . 0 a V~ . ~V A
A V . A V A V ~ V A 1 A V .V V A A .. oV . A .
. ... a . A . A. V. V .c A h . V . V ~V A . . . .
. .-. A ‘1‘. A .. A .1. .31. ..AA .A--- ..o... AAA-$A||17..A A) ...; . A. .A. . 7..-». | IA... A- t - -s. AAAA .o- . .a A . V . . . I . y
V. Va. . . V .+. _ V . .V. V A .. V V V V. V V. A. . — M V
V . . . . H A o A A o A . fl V . _ A . .
. r . Vv A . ... V Lfi . V . o . V . . .. p A. . V+ . . w A V
. . n _ a .A . V ..V. V,. _ Va V V . ._ V . p . .
[ I I Q .71 ‘I‘ ..b‘lQ.LfiI'lIC . LV. I‘ll AI”. '0‘ .IQIA‘ A LYfilrl'QI-\.+lvlo"uy"i|j .ll'lv'lurl': tifllc‘V IfntQIIAQr i ‘0‘) 'VI‘AIII Al APO"..- OAT I 0.... ‘ NIIIIIIIQ .AOIAII‘IAVl.“ A AI'L‘I l IIOI ii IAA I A .0. V *AtvuA CIA! A lAlv‘ All ..A V. w- . A_ AI .A A a A
V. .. . .. _ V . . .V V V. . . . V V . A . . . V. V . .
. . V A _ A p .H . V V .V A. w L . _ A Vv . . A V n V A u a
. u a. .u . . V“ — . . . A . a .. vA « . V * .
A V a V. V . . . .— A . . .. VV . V .. . b . A .
. AA A AA... A AALAIA. lvl ..... .?!AA. ..AA. A A. V VvA A IV. .l. v AAA. AAV A595. A Av|.A.QAA ..‘u.& .A. A AAA- V . V A ..v. I vVIr‘. A . ... o
. .+ . u H L . n n . .V . .» AL . A. A . . 4 n
A”. . . I . a .v > . V ~ .. V A. . A A . . .
_ . V. . A ' . Q . . A o r .. Y. . p . o A
V . _ . V . . . VV. _ A V A _
it _ A «V1 r W 4r VI ( W 1‘ AW 1.1 *1 a} 14 r Lr ? 1 IIAIIAVIIUAPIIVAIIA- V- A
V m . V H W 7 M. f r... U. .V .7 V. ...”. VV . . .V V _ V
V . V
\ . A 4 AA 5 v . A V AV — .A . V A A A A. o A o A
. A . V . .V . A . . . . . . V . V V. V . V .. A . V m. .V V. . . . . .
- v 0.. _A A W .VA VA...69A AA‘+’A ..4 A- A 0!. o. A A.” A AIV>1u r. v 9. .4- .DL rVVv V AA ..VI..- IVAr."-A+!LAOAA AQI'A- H“ 0.0- OI‘H '9 V vAA... AIIAA :A*§ . AAA.) .{OA V ... A A r}: A A A .1 A V
. V . . a A A AA A . A V V A V V... V A . . A o.. A A. . . .
. . V .p . VA . A _ . ..H . v .... V~. H V. .. ..V.. + A. V w . V V a
. . . a .A AAA n h A +V v ~ A _ V ¢. A... .I. A. ..V.. V.V A V . . A
. . . .V AAA ... . A A A V . .V A A . V v V . . . A. V. . A A A s .
A 4 ’ QIYIIVO .lfv‘dl’lAr 4 A All IHOI'OAVOIIIIAV it qulg'o‘.’ JV r r A } 5|“ 5|“! IIOAIOI?AIIIA? VIOIAIQIA f-‘ IA. 1.,‘AIJlIxAA'ioAPAAI L A AA A 11A T. A A} Ar
V. . +.. A . V mV .V I V V. ..V. “V V A A A V . AA. fl V 0. V V . A .
. V 7.:-. ..V. A A. . o. . VV. A A. . A VA, .A .. pAA .V 9 . . A o .
V A . .m w .... . AL. n A A... . . * V. ~..~ 4.. ..L . .AOAA.. * A M _ .
V . V . .u. A A . . . .. .A V ... A ...V.. . V.- VV . >A . . v n
V. H A A A V VI. AA..1vo.+u A rAAoIVV . AAA... ...! AVL.I A.v”. A LAIA.‘56.9V . AIAIMIAAIAPQIXA’L. A... AACAMA. 1+..AA9 l A . + .I V. A . A . .. . -
V . .. V . H. .A A. ¢ A. F * . . .. . . * .Vv .. .
a V r A A . L V A A. V V. ... A A . . m .V— . ~ A . . A. .A ... . .
.V A. .. . . .9 . .A V. n A . . .n . V .V A . . . V
V . A A _ . . Va V . . V _ V
I Vii. 71’ij . J A-r fl ‘ W r A1! D .r th r 1+ I !A4lA\III( {I
N V a V .A . . . M d . . A.— o . A V
V . _ V . . ..V. . V V V a . V V V V
V . L n V h V . . . V V ..L . V 0
. A .. V .Avv AA. .AA A A of .AA. VA... Al. LA! ... IA- A1Ib||¢l .. o I! . o .A A
A a 4.V # . . V V_. . 4A . _ m . .. . . . _ V
H m w .. A. . A A u 4 A + L n . m
A .. . V. V ..A a . A A A A . .
A n 0 A A V ”A. . V A .V . V o_ . h c
I r. J o I DA Av‘ IE VIA I I A F A A 0A D.IAIVIVYII‘I PLV All], On! .I"?! loL’ I I A 1.? 14‘ A
. . H A . _ V . L. u A a
. . H V . V . .V A A n V . .
. .L A A V. . A . . ... fl _ m
A “V I . a A v -
V A. V. V, r qAIoAA JIAAA v 1...:on A‘xrAV .. .1‘A&.A,1A AVOV 9.5 t- .H. a . . V L
V. V L. V. V V V V . .V . . L . .
o . _ V. .. A fi.. _ 5 V A A v V. _
A .. uh . n . m . c L- A . . «V
A. in-‘ VAOAI.\‘- V J I» 7 V I!” 10 l 1.!!! 1...!!! -AOIA IIA‘A
V H. 1.1.x 4. .V V V V V V . fl. . V V
V L . V.” V L . . V L V _ . V. n V V .
t. V A. A .. . V . . . . .
A V . A V . o . .V . A P _ .
V... V .. VHV- -.V.. .---..V..&VV . . l1}. + . l. V .V ....... A .. ...V 4.--: . .-u. - -
. . w . . . V. .VV a L . .
I A . A A. . . V A A .A.>A . A . V“ VV 0 A .
5 V . _ . . . V V V V V V I. V .
_ V . . . . . . .. .V . .. . ._
II .p Y: ._A ?' | 0|. AVItAOIQ'AVIOAOIl’IAOIALrPAAIIDAYAII'IAK VIAIIIAL luvllv v,’lV|+I0VAI.' 1'4. 01.0 ACKA +1.1. AAAV I}..- .AAQIIA f LYO‘QOAOIV1. 1AIV
n _ m V . . V L . V n . . V _ V I . _
. . . .V V.
. V _ L
. g .. . . V V v1 V
_ V V V V . . . . V V V _ L V .
. 4 Av ‘0 4A-! _ V AAOQ AVV VulbllVAKAAAOA . ll 5: 4| V A t! V V. .+ . .‘ ... . A
. L n V . L ..V. V .V V . . V V V V H _ H
.0 . . .a . O _ u
. _ V . V . _. V. . V V V . V . H
. . .V V. .H A . h . F U
I‘. .".Y.. J? r tLl» 1 . 'fi 1% . _ L} r!» .AAEI'II I-‘i‘l4l&1¢l-li".i
L _ . V . o . .
L w V V V L . . H V _ . L V V .V V
. V . . . . . A V V . . V . V _ . V
. V . W V V V _ . . V V
y. A V V. . . .1.. A.. AA--.» 11». A. 121* .A ..+V . A w -i. .Tv-t..-oAA~..Vu A .V. -. ... . .rv AA. ..
. . . V V. V .V . V .
. V V A . m . A L V _ L V . A . M V . V“ a
A V V V . . .
. V V . . .V _ V. V n u
A I -fi- Ill-A1“Atl’ AFAII‘OIAAltlJVYAIUIO.II..Il.‘IIA--..AIOI'- Tl‘.’..AFAVA;‘ u ’lllvlvlu'.#lfllll. A0 IAA'I.r11| -1.}; AJIVIfVLorll.A-V At IAVIJAI. .VAV" AI‘A AAAAMAO {All 0 IA? ‘ u
V . A V . Ao . V V . . a .
V . V . w V AV . . A . . A. L . V A . . o .
o .. . m V . V A. . . . ¢ . .. V . A
u .. ......L --. .‘yvV.. -IAA 4.v A..A. ..-! --A V.V .V ...- *..V-.. 15» ..Av IAA+ .0.A.v.uw;. A. -V VI. VA A ... 1V - . V- -13 Ar
V . V A. . . . V A A . . L V .
. . 4 L . AA . . H V V V . . .. _ . _ . V A . A .
L A A A .. . . V. V. A . . A . . . A V >
V . . .V V. P . . V . V . V V
‘1‘ L AW ? < Adr > ’ 4 Fl. AP A‘¥|»Y!.tlla|llv‘" A‘Al AIQAA ‘l‘luw A I.
V L V . . V .. . V . V . .. V m V
A . V V L . A w A V A V. . . A .. n A . A. . .
V . V . . .V _. V . . A * . . o . u . A
V. . o . . . V H. . *. .VA. . V . . . A V a . V
A A vAV .. A in AA. A A Vt! . AVA. +Av AAA A AAAA .+ .A.§A-.AVI In .AAA.VA1 o AAA.-. LA.AI‘»IA '0'4 I «I ¢A 1V AI. v V. VAA AA A AIQCAA.»V .I A A A AA.» . . .A . .
. A . . V . V o A . . . A..AV.AA a .. AV AV A~ V. A A . .A V V . V A .
m . A A . . AA. . n A .V.. V. . .1 AV . V . A V . _
V U a . . . .M . u A ..L.. m . . . M .V A ._ A. L . .
. V _ V A . . V V . V o . A A V A . h . .
1!- V.. AA. v I A ---VVAAAW.O 0".{TAIAV Volt!!! 4 AI} Iofillo.019'ilp {If I'Ao .V-..‘|.HAAIIAI.HIVVAII. VIAIAI AAII‘IQIO'A'A1!.AIVI.IvII1¢L.QAIi 011.13-} A’Alo. YAAAiAA I.‘ .0 IIAVA- .A... o! 0.. ’ A- V4. | . 4
V V V .ufld . V on? V .5 ad 3 V . V
L . A . A A . A _ o . V
u V L V V _ .Hn Vow” . H a V V “a v V V . .r H.
V b L .V V . . A .. L A . . .AA A . . A L . .
v V . A ..-V. .. A V. V. .v.-Avo .91.-.t .1”: 6.LI.AL.1.O. to»- V- AILVAAf -.tvr.-vA .iAAAA-AAV -H. (V.4'AH.A
L w v. A . V V A A "A . L . V A . AVA v o . . V.
w w u b . o ..A o n w. 0.. V .VAV . 4 M A .
V V V .A . .. A .A V .V V r . . V . .
m A V V A 4 A .H a .9 1 . _ .. V P V” A P A A v c H!
A. D r u
A ... . V . . M V L . . .. AV.V. V. A N. A V. . .
u. ”T 5 V A. . N . A“ V L 3 . . .V .
' . A. . A . . V A V . . .
V L .V. a V V I. . a V V .0 V L L . V A V,
A IV 1. .4. IA; A A. A Al. ‘4 A AAIA.‘ AAVAAAAA in. AA . VIA.AA .. AA IA .iA .1.. ..V . V AA-
. _ V. * . . . [4 V. . A H. a V“ A .. V
V V V _ . . _. . . V V w ,
V V V . u n. . H V V . V L V.
.A V?V I. U. IAL \ALVIA ‘Alol‘4 [14(0V0'4 .IITAv-"T... I:1..’A¢-§IO.VIIIC+|'AIOOVO "l A A‘AI [1991’ ’I . 9AA} ..IIIA'V A
. L V . . V L . V ... {1. V ”I" A. iAttrA . I. ....A OVT Qtlvft. A
. .V . . V A. . V .
V m 0 V d k . V, . m u H . ,
A A . 7

 

 

 

........

«‘91..¢ou

.
W
* .

wm-w-w

1..

6
. .
o

o .

I 1'...» 1‘“

o

-l

O.
L . I!» '1

o-—-‘-~¢o

 

VV
I!

‘Cr. ul—

 

 

 

MICHIGAN STATE UNIVER ITY L

S

IBRARIES

3 1293 (LO'IO 8964