June 1988 B DLOGICAL DEGRADATION CYANIDE BY FIXING … · 2018-06-13 · Based on the total cyanide biodegradation rate constants (Kb) obtained from batch experiments, use of nitrogen-fixing

B

June 1988

DLOGICAL DEGRADATION OF CYANIDE BY NITROGEN- FIXING CYANOBACTERJ A

by

Dr. C.J. Cantzer Dr. W.J. Maier

University of Minnesota Department of Civil and Mineral Engineering

Minneapolis, MN 55455

Project Officer

James S. Bridges Office o f Environmental Engineering and Technology Demonstration

Hazardous Waste Engineering Research Laboratory Cincinnati, OH 4 5 2 6 8

This study was conducted through

Minnesota Waste Management Board St. Paul, MN 55108

and the

Minnesota Technical Assistance Program University of Minnesota Minneapolis, MN 55455

HAZARDOUS WASTE ENGINEERING RESEARCH LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT

U.S. ENVIRONMENTAL PROTECTION AGENCY CINCINNATI, OH 4 5 2 6 8

This project was partially supported with a United States Environmental Protection Agency cooperative agreement through the Minnesota Waste Management Board and the Minnesota Technical Assistance Program.

Although the research described in this report has been funded in part by the United States Environmental Protection Agency through a cooperative agreement, it has not been subjected to Agency review, and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred.

ABSTRACT

This study examined the ability of nitrogen-fixing Anabaena to biodegrade cyanide in batch reactors. Mixed second-order rate constants were obtained that described the biologically-mediated decrease in cyanide for reactors containing initial cyanide concentrations of 3 ppm. For Anabaena cultures not previously exposed to cyanide, the rate constants were a function of pH. Faster rates of cyanide biodegradation were observed at higher pH values. Anabaena cultures acclimated to the presence of cyanide had rate constants that were at least 10 times faster than rate constants for unacclimated cultures.

Mixed second-order rate constants were also obtained for the ability of nitrogenase, the enzyme normally responsible for nitrogen-fixation, to reduce hydrogen cyanide to methane and ammonia. In batch reactors with initial cyanide concentrations of 30 ppb, the rate constants for methane production were at least 10 times faster than expected based on literature values for nitrogen fixation, suggesting that nitrogenase will preferentially use hydrogen cyanide as a substrate as compared to molecular nitrogen. Also, the rate constants for methane production were of the same order of magnitude as the rate constants for total cyanide removal, indicating nitrogenase as an important mechanism for the biodegradation of trace concentrations of cyanide.

The magnitude of the cyanide biodegradation rate constants suggests that the utilization of nitrogen-fixing cyanobacteria in the treatment of cyanide wastes can be a feasible process in some applications, i.e., secondary or tertiary treatment at larger treatment facilities.

iv

i

CONTENTS

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iV Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

1 . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3 . Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4 . Materials and Methods

Experimental Overview . . . . . . . . . . . . . . . . . . . . . . 8

Culture Media . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Experimental Procedures

Growth Rate Experiments . . . . . . . . . . . . . . . . . . 10 Objective and Apparatus . . . . . . . . . . . . . . . 12 Cyanide Mass Balances . . . . . . . . . . . . . . . . 12 Kinetic Parameter Determination . . . . . . . . . . . 13 Objective and Apparatus . . . . . . . . . . . . . . . 14 Kinetic Parameter Determination . . . . . . . . . . . 15

Analytical Procedures . . . . . . . . . . . . . . . . . . . . . 17

Total Cyanide Removal Experiments

Methane Formation Experiments

5 . Results and Discussion

Growth Rate Determination . . . . . . . . . . . . . . . . . . . 19

Limitations of Batch Experiments . . . . . . . . . . . . . 23 Rate Constants for Total Cyanide Removal . . . . . . . . . 24 Effect of Acclimation on Cyanide Removal Rates . . . . . . 32

Total Cyanide Removal Rates

Methane Production Rates Rate Constants for Methane Production . . . . . . . . . . . 33 Comparison of Methane Production with Nitrogen Fixation . . 39

41 Application of Kinetic Data to Cyanide Treatment . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

V

TABLES

Number a 1. Cyanobacteria culture media . . . . . . . . . . . . . . . . . . . . 9

2. Extent of biodegradation and volatilization in selected batch experiments . . . . . . . . . . . . . . . . . . . 25

3. Overall volatilization rate constants (KLa) and mixed second-order rate constants (K ) for total cyanide removal by unacclimate 9 Anabaena cultures in selected batch experiments . . . . . . . . . . . . . 26

4 . Comparison of mixed second-order rate constants (Kb) for total cyanide removal by unacclimated (batch number 4 ) and acclimated (batch number 5) Anabaena cultures in presence of thiosulfate . . . . . . . . . . 33

vi

FIGURES

Number

1. Logarithmic increase in Anabaena biomass (X) as function of time, during a batch experiment . . . . . . . . . . 20

2. Changes in Anabaena biomass (X) versus time after the start of feed flow through the reactor. dotted line represents the expected decrease in X

The

if dilution was the only removal mechanism. . . . . . . . . . . 2 1

3. Decrease in total cyanide concentration (S) during batch test number 3. numerical solution of equation (3) using the

The line represents the

Kb and KLa values on Table 3. . . . . . . . . . . . . . . . . . 27

4. Relationship between log Kb [L/(ug chl hr)] versus

Relationship between log Kb [L/(ug chl hr)] versus

Increase in headspace methane concentration with time.

time-averaged pH . . . . . . . . . . . . . . . . . . . . . . . . 29

5. time-averaged ALPHA . . . . . . . . . . . . . . . . . . . . . . 30

6. For the illustrated batch test, the initial cyanide concentration was 31.2 ug CN/L. . . . . . . . . . . . . . . . . 34

7 . Sensitivity of K obtained from equations (13) and (14) to inputted f values. Both parameters have units of L/(ug chl R r).

Sensitivity of the ratio between Kn and Kb to inputted

. . . . . . . . . . . . . . . . . . . . . . . 37

8. Both parameters have units of

~?($~~h'hr). . . . . . . . . . . . . . . . . . . . . . . . . 38

vii

SECTION 1

INTRODUCTION

The basic p :mi 3 of his study was that the use of nitrogen-fixing

cyanobacteria (blue-green algae) in the biological treatment of small

concentrations of free cyanides (HCN and CN-) can be a cost-effective

alternative to existing treatment processes.

cyanobacteria-based process would be in the secondary treatment of the free

cyanides that escape alkaline-chlorination. Because the extent of cyanide

oxidation in alkaline-chlorination is an equilibrium-driven phenomena, use of

a microbial process to detoxify the last fraction of cyanide should result in

lower alkaline-chlorination operating costs.

cyanobacteria-based process could be in the treatment of the cyanide

associated with metal-cyanide complexes via a two-step process. The first-

step would release cyanide from the metal-cyanide complexes by exposing the

complexes to ultraviolet irradiation. In the second-step, cyanobacteria would

detoxify the released cyanide.

A potential application of a

Another application of a

The use of nitrogen-fixing cyanobacteria in the treatment of cyanide

wastes is a new concept.

with the use of nitrogen-fixing cyanobacteria in the treatment of small

concentrations of cyanide. First, the biological treatment of cyanide with

cyanobacteria should have much lower operating costs than alkaline-

chlorination.

wastes with aerobic heterotrophs can be less than 10% the costs for alkaline-

There are several potential advantages associated

The operating costs for the biological treatment of cyanide

1

c h l o r i n a t i o n (Green and Smith, 1972). The c o s t s a s s o c i a t e d w i t h providing

a e r a t i o n and w i t h provid ing a supplementa l energy source (organic s u b s t r a t e )

f o r t h e maintenance of l a r g e amounts of ae rob ic h e t e r o t r o p h biomass make up a

cons ide rab le p o r t i o n of t h e t o t a l ope ra t ing c o s t s f o r t h e t r a d i t i o n a l

b i o l o g i c a l t r e a t m e n t of hazardous wastes.

pho tosyn the t i c , t hey do no t r e q u i r e a e r a t i o n f o r oxygen and do not r e q u i r e t h e

presence of o rgan ic s u b s t r a t e s t o ma in ta in biomass (Kobayashi and Rittmann,

1982). Thus, in terms of o p e r a t i n g c o s t s , t h e use of n i t rogen-f ix ing

cyanobac te r i a i n t h e t r e a t m e n t of small amounts of cyanide should have an

economic advantage ove r t h e use of h e t e r o t r o p h i c b a c t e r i a , and, consequent ly ,

a s i g n i f i c a n t economic advantage over a lka l ine -ch lo r ina t ion .

Because cyanobacter ia are

Second, n i t rogen- f ix ing cyanobac te r i a have t h e a b i l i t y t o s u r v i v e i n l o w

t o moderate c o n c e n t r a t i o n s of hydrogen cyanide.

because i t i n h i b i t s t h e t e r m i n a l cytochrome ox idase i n r e s p i r a t i o n , which

normally reduces oxygen t o water.

oxidases--some of which are r e s i s t a n t t o cyanide i n h i b i t i o n (Fogg, et al.,

1973; Degn, e t al., 1978; Peschek, 1980; Henry, 1981). Cyanobacter ia a l s o

have s e v e r a l cyanide d e t o x i f i c a t i o n pathways, i.e., enzymat ic pathways t h a t

t r ans fo rm free cyan ides i n t o a less t o x i c form (Castric, 1981; Higgins, et

al., 1984).

pathway i s mediated by t h e enzyme rhodanese, which t r a n s f e r s a s u l f u r from a

donat ing compound (e.&, t h i o s u l f a t e ) t o cyanide t o form th iocyana te (Westley,

1981). Other cyanide d e t o x i f i c a t i o n pathways r e s u l t i n t h e fo rma t ion of amino

a c i d s (Solomonson, 1981). Thus, due t o t h e presence of cyanide d e t o x i f i c a t i o n

Hydrogen cyanide is t o x i c

Cyanobacter ia have s e v e r a l t e r m i n a l

The most s t u d i e d and perhaps t h e most i m p o r t a n t d e t o x i f i c a t i o n

pathways and cyan ide - re s i s t an t r e s p i r a t i o n , cyanobac te r i a can

s o l u t i o n s con ta in ing f r e e cyanides . For example, Howe (1963,

s u r v i v e i n

1965) observed a

2

"luxuriant" growth of cyanobac te r i a on t h e f i l t e r s t o n e s of a b i o l o g i c a l

r e a c t o r t h a t was t r e a t i n g wastes con ta in ing 300 ppm cyanide.

Third, i n a d d i t i o n t o t h e above pathways, t h e n i t rogen- f ix ing

cyanobacter ia can d e s t r o y hydrogen cyanide w i t h t h e enzyme ni t rogenase.

normally r e s p o n s i b l e f o r t h e r educ t ion o f molecular n i t r o g e n (d in i t rogen) t o

ammonia, n i t r o g e n a s e can a l so reduce hydrogen cyanide t o methane and ammonia

(Hardy and Knight, 1967; Hardy and Burns, 1968; Biggins and Kelley, 1970;

Haystead, e t al., 1970; Hwang and B u r r i s , 1972; Hwang, e t al., 1973; Zumft and

Mortenson, 1975; S t e w a r t , 1980; L i , e t al., 1982). In fact, n i t r o g e n a s e w i l l

p r e f e r e n t i a l l y reduce hydrogen cyanide i n s t e a d of i t s normal s u b s t r a t e ,

d i n i t r o g e n ( L i , e t al., 1982). Some r e s e a r c h e r s have proposed t h a t t h e

o r i g i n a l r o l e o f t h e n i t r o g e n a s e system was t o d e t o x i f y t h e cyanide and

cyanogen found i n t h e p r i m i t i v e biosphere when t h e earth had a reducing

atmosphere ( S i l v e r and Pos tga te , 1973; Pos tga te , 1982).

While

Desp i t e t h e c o n s i d e r a b l e amount o f i n f o r m a t i o n i n d i c a t i n g t h a t

cyanobacter ia are a b l e t o s u r v i v e i n the presence o f cyanide and are a b l e t o

d e t o x i f y cyanide, t h e k i n e t i c d a t a r equ i r ed t o assess the f e a s i b i l i t y o f

u t i l i z i n g cyanobacter ia i n t h e t r e a t m e n t o f cyanide wastes does n o t e x i s t .

This s t u d y provides an i n i t i a l assessment of the rate a t which n i t rogen-f ix ing

cyanobacter ia are a b l e t o degrade free cyanide.

second-order rate c o n s t a n t s f o r t h e b io logica l ly-media ted removal of cyanide

by unacc l imated c u l t u r e s of Anabaena were determined i n ba t ch r e a c t o r s .

second set o f ba t ch exper iments determined t h e mixed second-order rate

c o n s t a n t s f o r the r e d u c t i o n o f hydrogen cyanide by ni t rogenase.

I n p a r t i c u l a r , t h e mixed

A

3

SECTION 2

CONCLUSIONS

I n ba t ch reactors w i t h i n i t i a l cyanide c o n c e n t r a t i o n s o f 3 mg/L, t h e

mixed second-order rate c o n s t a n t s (Kb) f o r the removal of cyanide by ni t rogen-

f i x i n g Anabaena cu l tures was a f u n c t i o n of pH.

c o n s t a n t s i n c r e a s e d w i t h i n c r e a s i n g pH f o r unaccl imated Anabaena c u l t u r e s ,

i.e., c u l t u r e s n o t p r e v i o u s l y exposed t o c y a n i d e .

t empera ture of 2SoC, the observed ra te c o n s t a n t i n terms of c h l o r o p h y l l (ch l )

c o n c e n t r a t i o n was 5.0.10-6 L/(ug c h l hr).

Kb i n c r e a s e d t o 2.2*lC1-~ L/(ug c h l hr).

and pH sugges ted t h a t t h e r a t i o o f hydrogen cyanide t o t o t a l free cyanide

c o n c e n t r a t i o n s , [HCN]/([HCN]+[CN-I), i n f luenced b iodegrada t ion rates. Because

HCN i s much more t o x i c and i n h i b i t o r y than CN-, and because a n increase i n pH

would reduce HCN c o n c e n t r a t i o n s , t h e faster b iodegrada t ion rates observed a t

h ighe r pH v a l u e s was probably due t o a r educ t ion i n i n h i b i t i o n .

The b iodegrada t ion rate

A t a pH o f 8.4 a n d a

When the pH was i n c r e a s e d t o 9.5,

The observed r e l a t i o n s h i p between Kb

When previous ly exposed t o cyanide, Anabaena biodegraded cyanide a t a

The Kb va lue f o r an a c c l i m a t e d Anabaena c u l t u r e was 10 times faster rate.

g r e a t e r than tha t for an unaccl imated Anabaena c u l t u r e . Thus, r e a c t o r s

o p e r a t i n g under s t e a d y - s t a t e c o n d i t i o n s should have faster cyanide removal

rates than t h o s e observed i n t h e batch experiments.

The mixed second-order rate c o n s t a n t f o r t h e r e d u c t i o n o f cyanide t o

methane by n i t r o g e n a s e (K,) was determined i n small gas - t igh t ba t ch r e a c t o r s

with i n i t i a l cyanide c o n c e n t r a t i o n s of 30 ug/L. A t y p i c a l va lue o f Kn was

4

2.6*10-4 L/(ug ch l h r ) , which was a t least a n o r d e r o f magnitude g r e a t e r t han

expected based on e x i s t i n g n i t r o g e n - f i x a t i o n k i n e t i c data.

expected Kn suppor ted t h e i n v i t r o observa t ion t h a t n i t r o g e n a s e w i l l

p r e f e r e n t i a l l y reduce hydrogen cyanide r a t h e r t han i t s normal s u b s t r a t e of

molecular n i t rogen .

tests, n i t r o g e n a s e a c t i v i t y reduced cyanide c o n c e n t r a t i o n s from 30 ug/L down

t o 20 ug/L.

cyanide a t such low c o n c e n t r a t i o n s , t h e u t i l i z a t i o n o f n i t rogen-f ix ing

cyanobacter ia i n the t r e a t m e n t of t r a c e - l e v e l s of cyanide is worth f u r t h e r

examination.

The l a r g e r than

Based on t h e amount of methane produced du r ing t h e ba t ch

Because few cyanide d e s t r u c t i o n p rocesses are a b l e t o a t t a c k

Based on t h e t o t a l cyanide b iodegrada t ion ra te c o n s t a n t s (Kb) obta ined

from ba tch exper iments , use of n i t rogen-f ix ing cyanobacter ia i n t h e secondary

o r t e r t i a r y t r e a t m e n t of cyanide wastes could be a f e a s i b l e p rocess , provided

t h a t t h e treatment p rocess h a s adequate mean cell r e s i d e n c e time. With a ne t

s p e c i f i c growth rate of 0.8 d-l, t h e unaccl imated Anabaena c u l t u r e can have

faster growth k i n e t i c s and t race-contaminant removal rates than e x i s t i n g

b i o l o g i c a l treatment processes . Because cyanobacter ia are pho tosyn the t i c , the

amount o f biomass i n a r e a c t o r is n o t a f u n c t i o n of o r g a n i c s u b s t r a t e

concen t r a t ion .

low c o n c e n t r a t i o n s o f cyanide would be small compared t o t r a d i t i o n a l a e r o b i c

p rocesses for t r e a t i n g d i l u t e wastes.

c o n s e r v a t i v e r a t e and biomass parameters p r e d i c t e d t h a t a h y d r a u l i c r e t e n t i o n

time of o n l y 5 days is r e q u i r e d t o reduce an i n f l u e n t cyanide c o n c e n t r a t i o n of

4 mg/L by 93 percent .

i n f l u e n t BOD c o n c e n t r a t i o n i n a n a e r o b i c chemostat would r e q u i r e a h y d r a u l i c

r e t e n t i o n time of 21 days, based on t y p i c a l BOD k i n e t i c parameters (Metcalf

Thus, t h e v o l u m e t r i c s i z e of t h e r e a c t o r s r e q u i r e d t o treat

For example, a chemostat model u s ing

In comparison, t h e 90 pe rcen t r e d u c t i o n of a 10 mg/L

5

and Eddy, 1979). However, t h e r e q u i r e d size of t h e BOD chemostat would be

reduced i f supplementa l s u b s t r a t e s were added t o the r e a c t o r t o i n c r e a s e

biomass concen t r a t ions .

Because of t h e p o t e n t i a l t o a t tack t race-concent ra t ions of cyanide and of

t h e p o t e n t i a l f o r low o p e r a t i n g c o s t s , t h e use of n i t rogen-f ix ing

cyanobacter ia i n the b i o l o g i c a l t r e a t m e n t o f cyanide wastes may be an

a t t r a c t i v e a l t e r n a t i v e t o e x i t i n g cyanide t r e a t m e n t technologies . However,

t h e c a p i t a l c o s t s and t h e requi rement f o r t r a i n e d pe r sonne l a s s o c i a t e d w i t h

b i o l o g i c a l t r e a t m e n t processes , may make t h e cyanobacter ia p rocess u n s u i t a b l e

f o r small-volume cyanide-waste gene ra to r s .

probably b e t t e r s u i t e d f o r l a r g e r cyanide treatment facilities.

The cyanobacter ia p rocess is

6

SECTION 3

RECOMMENDATIONS

This s tudy demonstrated t h e a b i l i t y of n i t rogen-f ix ing cyanobacter ia t o

biodegrade low concen t r a t ions of f r e e cyanides i n ba tch r eac to r s .

s t u d i e s need t o examine t h e a b i l i t y of t h e cyanobacter ia t o degrade cyanide

under s t e a d y - s t a t e c o n d i t i o n s and t o de termine t h e s t a b i l i t y of t h e s teady-

state reactors t o s l i g h t p e r t u r b a t i o n s i n cyanide concent ra t ions . If such

l abora to ry - sca l e expe r imen t s con t inue t o demons t r a t e t h e a t t r a c t i v e n e s s of

u t i l i z i n g n i t rogen- f ix ing cyanobac te r i a i n t h e t r e a t m e n t of low cyanide

concen t r a t ions , then a p i l o t - s c a l e s tudy should be performed t o de te rmine t h e

economic and t e c h n i c a l f e a s i b i l i t y of t h e process .

Fu tu re

7

SECTION 4

MATERIALS AND METHODS

EXPERIMENTP OVERVIF!

The o b j e c t i v e s of t h e s tudy were 1) t o de t e rmine t h e rates a t which a

cyanobac te r i a c u l t u r e removed f r e e cyanides (HCN + CN-), and 2) t o de te rmine

t h e rate a t which HCN was reduced t o methane by t h e a c t i v i t y of t h e enzyme

n i t rogenase .

r e a c t o r under ba t ch condi t ions .

cyanide removal rates under cont inuous-feed cond i t ions , i.e., i n a chemostat .

The methane product ion expe r imen t s were conducted i n small 0.037-l i ter , gas-

t i g h t v i a l s under ba tch condi t ions .

Rates of t o t a l cyanide removal were de termined i n a 1.3-liter

At tempts were a l s o made t o de t e rmine t o t a l

CULTURE MEDIA

The cyanobac te r i a used i n t h e cyanide deg rada t ion expe r imen t s were

Anabaena sp. ob ta ined from Caro l ina B i o l o g i c a l Supply Co. (Burl ington, NC,

c a t a l o g number 151710).

i.e., none were f r e e o f b a c t e r i a .

None of t h e c u l t u r e s used i n t h e s tudy were axenic ,

The media used t o ma in ta in Anabaena c u l t u r e s and used i n t h e cyanide

deg rada t ion exper iments was t h e Hughes-Gorham-Zehnder media desc r ibed i n Al len

(1973). The chemica l composi t ion of t h e media is shown on Table 1.

i n c r e a s e t h e b u f f e r i n g c a p a c i t y of t h e media a t pH va lues s l i g h t l y g r e a t e r

than t h e pKa f o r hydrogen cyanide f o r media used i n t h e ba tch expe r imen t s

0.186 grams of HBO3 was added t o each l i t e r of media (3 mM) . pH were performed by t h e a d d i t i o n of e i t h e r 1 M NaOH or HC1.

To

Adjustments i n

P r i o r t o its use,

8

TABLE 1. CYANOBACTERIA CULTURE MEDIA

Macronutrients gramdliter ~~ ~~

K2HP04

M ~ S O ~ - 7n20 CaC12 2H20

Na~C03

NaSi03*H20

Citric Acid

EDTA

Ferric Citrate

Micronutrient Solution

Micronutrient Solution

0.369

0.075

0.036

0.020

0.058

0.006

0.001

0.006

0.08 mL

grandliter

3.10

1.69

0.287

0.088

0.0167

0.33

0.119

0.083

0.154

0.138

0.018

0.474

9

t h e media was autoclaved.

rest of t h e media t o prevent t h e p r e c i p i t a t i o n of ferric phosphates.

no f ixed-n i t rogen was added t o t h e media, t h e Anabaena had h e t e r o c y s t s and

relied on t h e f i x a t i o n of d i n i t r o g e n f o r t h e i r n i t r o g e n needs.

F e r r i c c i t r a t e was autoc laved s e p a r a t e l y from t h e

Because

Maintenance c u l t u r e s were grown i n au toc laved 100-mL Erlenmeyer f l a s k s

t h a t were s toppered w i t h cheese-cloth-wrapped c o t t o n plugs.

c u l t u r e s were i l l u m i n a t e d by cool-white f l u o r e s c e n t l i g h t , and were no t

cont inuous ly a g i t a t e d nor was a i r bubbled through t h e media.

co2 l imi ta t ions , t h e Anabaena i n t h e maintenance c u l t u r e s had slow growth

rates.

The maintenance

Probably due t o

The Anabaena used i n t h e t o t a l cyanide removal and t h e methane product ion

ba tch tests were not d i r e c t l y t r a n s f e r r e d from maintenance culture t o

r e s p e c t i v e ba t ch r e a c t o r s . I n s t e a d , an inoculum from a maintenance cu l ture

f lask was placed i n t o a 500-mL gas wash b o t t l e t h a t conta ined f r e s h media.

The media was a g i t a t e d by t h e i n t r o d u c t i o n of f i l t e r e d a i r pass ing through a

porous g l a s s d i f f u s e r . I n a d d i t i o n t o provid ing a g i t a t i o n , t h e a i r bubbles

r e l e a s e d by t h e d i f f u s e r provided a cont inuous supply of C02 t o t h e system,

Anabaena i n t h e g a s wash b o t t l e had r a p i d growth rates (doubling times on t h e

o r d e r of 1 day).

p o r t i o n s of t h e gas-wash-bottle c u l t u r e s were t r a n s f e r r e d to t h e ba tch

exper iment reactors.

Upon r each ing a d e s i r e d biomass ( ch lo rophy l l ) concen t r a t ion ,

EXPERIMENTAL PROCEDURES

Growth Rate Experiments

Anabaena growth ra tes were de termined i n a New Brunswick S c i e n t i f i c

(Edison, NJ) B i o f l o Chemostat Model C32 wi th a Pyrex r e a c t i o n v e s s e l t h a t held

1.3 l i t e r s of media. During t h e i n i t i a l growth rate exper iments , t h e r e a c t o r

10

was opera ted i n ba t ch mode (no cont inuous feed) and no cyanide was present .

Temperatures were maintained a t 25OC.

I n c r e a s e s i n biomass were determined by monitor ing t h e i n c r e a s e i n

chlorophyl l -a (ch l ) c o n c e n t r a t i o n s w i t h r e s p e c t t o time. The rate of i n c r e a s e

i n biomass was assumed t o be f i r s t - o r d e r w i th r e s p e c t t o biomass, i.e,

Air was supp l i ed a t a rate of 15 L/hr.

dX - = u x d t

i n which X is the chlorophyl l -a concen t r a t ion (up chl/L), t is time (hr), and

u is t h e ne t s p e c i f i c growth rate (hr-l).

de te rmining t h e s l o p e ( l ea s t - squa res f i t ) of the l i n e produced when the

n a t u r a l logar i thm of X ( In X) was p l o t t e d v e r s u s time.

r e q u i r e d f o r the va lue o f X t o double was determined from

The va lue of u was obtained by

The amount of time

0.693 t d = -

U

i n which t d is t h e doubling time ( h r ) .

Subsequent growth rate exper iments were a t t e m p t e d w h i l e o p e r a t i n g the

reactor as a chemostat (once-through r e a c t o r wi th cont inuous feed).

Regard less of t h e f e e d flow rate, a g i t a t i o n speed, a i r f l o w rate, l i g h t

i n t e n s i t y , and method of r e a c t o r s t a r t -up , s t e a d y - s t a t e c h l o r o p h y l l

c o n c e n t r a t i o n s were never obtained. As d i s c u s s e d i n t h e f o l l o w i n g s e c t i o n , X

v a l u e s a l w a y s decreased at a rate faster than would be p r e d i c t e d based on t h e

d i l u t i o n rate ( f eed flow rate d iv ided by reactor volume).

S ince the a v a i l a b l e flow-through r e a c t o r was unable t o suppor t steady-

s ta te p o p u l a t i o n s of Anabaena i n e i t h e r t h e absence and presence of cyanide,

t h e k i n e t i c c o e f f i c i e n t s f o r t h e degrada t ion of cyanide r epor t ed i n t h i s s t u d y

were de termined from short- term ba tch experiments.

11

T o t a l Cyanide Removal Experiments

Objec t ive and Apparatus--

The o b j e c t i v e of t h e t o t a l cyanide removal experiments was t o determine

t h e mixed-second order rate cons t an t s f o r t h e b i o l o g i c a l l y mediated removal of

free cyanides from t h e 1.3-liter r e a c t o r opera ted i n a ba tch mode.

batch exper iments were of s h o r t du ra t ion ( 4 t o 5 hours) , SO t h a t during t h e

course of t h e experiment any i n c r e a s e s i n Anabaena biomass due t o growth would

be neg l ig ib l e .

ug CN/L. Water tempera tures were maintained a t 25OC.

t o t h e r e a c t o r at a rate of 15 L/hr , and a i r e x i t i n g t h e r e a c t o r was drawn

through a gas-wash b o t t l e con ta in ing a 1 N s o l u t i o n of NaOH t o cap tu re any HCN

t h a t was v o l a t i l i z e d from t h e r eac to r .

Cyanide Mass Balances--

These

I n i t i a l t o t a l cyanide concen t r a t ions were approximately 3000

F i l t e r e d air was added

The two mechanisms r e spons ib l e f o r t h e decrease i n t o t a l f r e e cyanide

concen t r a t ions i n t h e 1.3-L r e a c t o r were biodegradat ion of HCN and CN- and

v o l a t i l i z a t i o n of HCN.

equa t ion f o r t h e batch r e a c t o r i s

Based on t h e s e two mechanisms, t h e mass ba lance

dS - = - Kb Xo S - KLa ALPHA S d t

(3)

i n which S i s t h e concen t r a t ion of f r e e cyanide (HCN + CN-) i n t h e r e a c t o r

(up CN/L), Kb is t h e mixed second-order rate cons t an t f o r t h e b iodegrada t ion

of free cyanides by t h e microorganisms (L/(ug c h l hr)) , Xo i s t h e

concen t r a t ion of Anabaena i n t h e r e a c t o r a t t h e start of t h e batch experiment

(up chl/L), KLa i s t h e o v e r a l l mass transfer c o e f f i c i e n t f o r t h e movement of

HCN from t h e l i q u i d phase t o t h e gas phase ( l / h r ) , and ALPHA is the r a t i o of

HCN t o t h e t o t a l amount of f r e e cyanide i n s o l u t i o n ,

12

[ HCN I [HCN] t [CN-]

ALPHA = ( 4 )

The ALPHA parameter must be included i n t h e v o l a t i l i z a t i o n term, because HCN

i s t h e only form of free cyanide t h a t i s s u b j e c t t o v o l a t i l i z a t i o n .

a func t ion of t h e pKa of HCN and of t h e pH of t h e so lu t ion .

of HCN a t 250C i s 9.3, va lues of ALPHA can be obtained from (Snoeyink and

Jenkins , 1980)

ALPHA is

Because t h e pKa

10-PH ALPHA =

10-pH + 10-9.3

i n which pH is -log[@] and log is t h e base 10 logari thm.

The rate of i n c r e a s e i n t h e mass of HCN c o l l e c t e d i n t h e gas-wash b o t t l e

a s CN- can be descr ibed by

dM - = v KLa ALPHA S d t

( 6 )

i n which M is t h e t o t a l mass of f r e e cyanide i n t h e gas-wash b o t t l e (up CN)

and V is t h e volume of t h e b i o l o g i c a l batch r e a c t o r (1.3 L).

K ine t i c Parameter Determination--

The data obta ined from each batch experiment included 1) t h e i n i t i a l free

cyanide concen t r a t ion (So), 2) t h e i n i t i a l Anabaena biomass i n terms of

chlorophyl l -a concen t r a t ion (Xo), 3) t h e t o t a l cyanide concen t r a t ion (S) and

pH of each s a m p l e c o l l e c t e d from t h e r e a c t o r , and 4) t h e mass of cyanide found

i n t h e gas-wash b o t t l e a t t h e end of t h e batch experiment (Mf).

t h e o v e r a l l mass transfer c o e f f i c i e n t (KLa) f o r s t r i p p i n g HCN out of t h e batch

r e a c t o r was not known. Batch v o l a t i l i z a t i o n experiments conducted w i t h

s ter i le media i n d i c a t e d t h a t KLa va lues changed s i g n i f i c a n t l y between

replicates, d e s p i t e t h e c o n t r o l s f o r t h e a i r f low rate and t h e a g i t a t i o n rate

The v a l u e of

13

being set a t t h e same values .

t h e expe r imen ta l d a t a f o r each ba tch test--So, Xo, S( t ) , pH(t), and M f .

Thus, both Kb and KLa had t o be de te rmined from

The va lues of Kb and KLa f o r each batch test were ob ta ined v i a a t r i a l

and e r r o r a l g o r i t h m u t i l i z i n g t h e Runge-Kutta method t o so lve equa t ion (3).

F i r s t , a n approximate va lue of KLa was determined by s e t t i n g Kb t o z e r o and

f i n d i n g t h e KLa va lue t h a t p red ic t ed t h e amount of cyanide collected i n t h e

gas-wash bot t le a t t h e end of t h e ba t ch experiment (Mf).

v a l u e of KLa accounted for changes i n pH.

KLa was i n c r e a s e d and Kb va lues were inc reased u n t i l t h e Runge-Kutta model

approximated t h e observed decrease i n t o t a l cyanide concen t r a t ion (S) w i t h

time.

d e s c r i p t i o n of observed blf and S ( t ) values .

Methane Formation Experiments

Ob jec t ive and Apparatus--

Th i s approximate

Second, t h i s approximate v a l u e of

Thi rd , v a l u e s of KLa and Kb were then a d j u s t e d t o y i e l d t h e b e s t

The o b j e c t i v e of t h e methane exper iments was t o de te rmine t h e mixed-

second o r d e r rate c o n s t a n t f o r t h e convers ion of HCN by n i t rogenase t o methane

and ammonia by moni tor ing t h e i n c r e a s e i n headspace methane c o n c e n t r a t i o n s i n

gas - t igh t r e a c t o r s t h a t were operated i n a ba tch mode.

of s h o r t d u r a t i o n (1 t o 2 hours), so t h a t i n c r e a s e s i n Anabaena biomass o r

changes i n envi ronmenta l c o n d i t i o n s du r ing t h e course of t h e experiment would

be i n s i g n i f i c a n t .

not p rev ious ly been exposed t o cyanide.

were approx ima te ly 30 ug CN/L.

pH v a l u e s were a d j u s t e d t o 10.

25OC.

headspace g a s i n t o a Hewlett-Packard 5840A g a s chromatograph w i t h a f lame-

The ba tch tests were

The Anabaena used i n t h e methane product ion expe r imen t s had

I n i t i a l t o t a l cyanide c o n c e n t r a t i o n s

To minimize t h e v o l a t i l i z a t i o n of HCN, i n i t i a l

:dater t empera tu res dur ing t h e expe r imen t s were

Methane c o n c e n t r a t i o n s were determined by i n j e c t i n g 100 uL samples of

14

i o n i z a t i o n d e t e c t o r (FID).

The reactors used i n methane expe r imen t s c o n s i s t e d of a 40-mL Wheaton

The s t o p p e r was a serum b o t t l e s f i t t e d w i t h a Supelco Mini-Valve s topper .

combinat ion va lue and septum t h a t provided a means of sampling t h e headspace

of the b o t t l e , w h i l e being gas - t igh t when t h e headspace was n o t being sampled.

To ma in ta in a uniform d i s t r i b u t i o n of Anabaena i n t h e media, a small Teflon-

coa ted magnet ic s t i r - b a r was p laced i n s i d e t h e b o t t l e and t h e b o t t l e was

placed on t o p of a s t i r r i n g p l a t e .

conta ined 25 mL of media and had a headspace volume of 12 mL.

s t o p p e r occupied t h e remaining 3 mL.

K i n e t i c Parameter Determination--

During t h e ba t ch exper iments , t h e b o t t l e

The stem of t h e

Each mole of methane appear ing i n t h e headspace of t h e serum bo t t l e was

assumed t o be produced from t h e r educ t ion of one mole of hydrogen cyanide t o

methane and ammonia by n i t rogenase .

i n t h e headspace cor responds t o a mass c o n c e n t r a t i o n of cyanide t h a t was

reduced by n i t rogenase ,

Thus, t h e molar c o n c e n t r a t i o n of methane

M w ~ ~ , Vgas

~ C N "water P [CHq] - (7 )

i n which P is the cyanide c o n c e n t r a t i o n i n the water phase cor responding t o

t h e mass of methane produced (up CN/L), [CHq] i s t h e molar c o n c e n t r a t i o n of

methane i n t h e headspace g a s (umoles CH4/L of gas), MWCH4 is t h e molecular

weight of methane, MWCN i s t h e molecular weight of t h e cyanide f u n c t i o n group,

Vgas is t h e volume of t h e headspace (0.012 L), and Vwater i s t h e volume of t h e

water phase i n t h e serum b o t t l e (0.025 L).

of d e s c r i b i n g methane c o n c e n t r a t i o n (the product of n i t r o g e n a s e a c t i v i t y ) , t h e

v a l u e of P i n c r e a s e d w i t h time dur ing t h e methane ba tch tests.

Because P r ep resen ted ano the r way

15

The rate of methane product ion by n i t rogenase was assumed t o f o l l o w mixed

second-order k i n e t i c s ,

dP xo s - = Kn

d t

i n which P is t h e water-phase concen t r a t ion of cyanide corresponding t o t h e

mass of methane produced (ug CN/L), t is time (hr) , Kn is t h e mixed second-

order r a t e cons t an t f o r t h e product ion of methane by n i t rogenase expressed i n

terms of chlorophyl l -a concen t r a t ion (L/(ug c h l hr)), Xo is t h e concen t r a t ion

of Anabaena a t t h e s tar t of t h e ba tch tes t (up chl/L), and S is t h e t o t a l

cyanide concen t r a t ion a t time t (up CN/L).

Because n i t rogenase was not t h e only b i o l o g i c a l mechanism f o r reducing

cyanide concen t r a t ions (S) i n t h e v i a l s , t h e d e t e r m i n a t i o n of Kn r equ i r ed t h a t

t h e decrease i n S i n equat ion (8) reflect t h e s e a d d i t i o n a l mechanisms. The

t o t a l r a t e a t which S dec reases w i t h time due t o b i o l o g i c a l a c t i v i t y can be

descr ibed by

dS Kb xo - = -

d t ( 9 )

i n which Kb is t h e mixed second-order rate c o n s t a n t f o r t h e biodegradat ion of

free cyanides by Anabaena cultures (L/(ug c h l hr)).

(9) y i e l d s

I n t e g r a t i o n of equat ion

i n which So is t h e i n i t i a l cyanide concen t r a t ion (up CN/L).

equat ion (IO) f o r S i n equat ion (8) y i e l d s t h e fo l lowing equation:

S u b s t i t u t i o n of

dP - = Kn Xo So exp[-Kb Xo t] . d t

I n t e g r a t i o n of equat ion (11) from t = O and P=Po=O t o t=t and P=P produces

16

which desc r ibes methane concen t r a t ion as a func t ion of time.

The goa l of t h e methane batch experiments was t o de te rmine t h e va lue of

Kn f o r t h e n i t rogen-f ix ing Anabaena c u l t u r e .

parameters i n equat ion (12) were known, except f o r Kn.

o v e r a l l biodegradat ion r a t e cons t an t (Kb) was determined from t h e batch

exper iments examining t o t a l cyanide removal.

(RHS) is def ined f o r each sampling time by

For each batch test, a l l of t h e

The v a l u e of t h e

When an independent v a r i a b l e

SO RHS = - (1 - eXp[-Kb Xo t ]) ,

‘b

equat ion (12) i s reduced t o a linear r e l a t i o n s h i p ,

P = Kn RHS . (14)

Thus, t h e va lue of Kn f o r each batch test was c a l c u l a t e d from t h e s lope of a

l i n e a r l ea s t - squa res f i t of t h e P( t ) and RHS(t) d a t a p o i n t s c o l l e c t e d dur ing

t h e batch test. The y- in te rcept f o r equat ion (14) should equal zero , because

P0=O,

ANALYTICAL PROCEDURES

Cyanide and ch lo rophy l l measurements fo l lowed t h e procedures presented i n

Standard Methods (APHS, 1980).

Prior t o a n a l y s i s , cyanide samples were d i s t i l l e d i n a commercially-

a v a i l a b l e cyanide d i s t i l l a t i o n apparatus . I n t h e b o i l i n g f l a s k , t h e cyanide

sample was sub jec t ed t o a c i d i c c o n d i t i o n s and t o a magnesium c h l o r i d e reagent.

Th i s s o l u t i o n was re f luxed f o r 1 hour.

c o l l e c t e d i n a gas-wash b o t t l e con ta in ing 1.25 N NaOH so lu t ion .

The v o l a t i l i z e d hydrogen cyanide was

For samples w i t h cyanide concen t r a t ions g r e a t e r than 1 mg CN/L, t h e

17

cyanide concen t r a t ions i n t h e a l k a l i n e d i s t i l l a t e were determined by t h e

t i tr imetric method, The d i s t i l l a t e was t i t r a t e d w i t h a s tandard si lver

n i t r a t e (AgN03) s o l u t i o n t o form a s o l u b l e cyanide complex, Ag(CN-)Z.

as a l l t h e CN had been complexed and a small excess concen t r a t ion of AgC had

been added, t h e excess Agf was de tec t ed by t h e s i l v e r - s e n s i t i v e i n d i c a t o r ,

paradimethylaminobenzalrhodanine, which turned from a yel low t o salmon c o l o r

(APHS, 1980).

A s soon

For samples w i t h concen t r a t ions less than 1 mg CN/L, t h e c y a n i d e con ten t

F i r s t , of t h e a l k a l i n e d i s t i l l a t e was determined by t h e c o l o r i m e t r i c method.

t h e pH of a d i s t i l l a t e sample was ad jus t ed t o approximately 8 w i t h t h e

a d d i t i o n of a phosphate buf fer . Second, chloramine-T was added, which

converted t h e CN- in the d i s t i l l a t e t o CNC1. Third, w i t h t h e a d d i t i o n of a

py r id ine -ba rb i tu r i c r eagen t , t h e CNCl formed a red-blue dye. The absorbance

of t h e aqueous dye a t 578 nm was l i n e a r l y p ropor t iona l t o CN concen t r a t ion i n

t h e d i s t i l l a t e (APHS, 1980).

The concen t r a t ion of chlorophyl l -a i n a sample was used as an i n d i c a t o r

of Anabaena biomass concent ra t ion .

determined by t h e co ld ace tone e x t r a c t i o n method.

cyanobacter ia suspension w i t h a small amount of MgC03 was drawn through a

0.45-um membrane filter. The f i l ter was placed was placed i n t o a c e n t r i f u g e

tube , and a small known volume of a 90% ace tone (v/vwater ) s o l u t i o n was added

t o t h e c e n t r i f u g e tube.

ch lo rophy l l from t h e Anabaena f o r 24 hr a t a tempera ture of 4OC.

c e n t r i f u g a t i o n , t h e chlorophyl l -a concen t r a t ion i n t h e supe rna tan t was

c a l c u l a t e d from t h e supe rna tan t ' s absorbances a t 663, 645, and 630 nm (APHS,

1980).

Chlorophyll-a concen t r a t ions were

A known volume of t h e

The ace tone s o l u t i o n was al lowed t o extract t h e

Af te r

Absorbances were measured wi th a Beckmann Spectrophotometer.

18

SECTION 5

RESULTS AND DISCUSSION

GROWTH RATE DETERMINATION

For ba tch growth exper iments conducted a t pH va lues ranging from 8.0 t o

9.5 i n t h e absence of cyanide, t h e average n e t specific growth rate (u) of t h e

Anabaena was 0.832 d-l , which corresponded t o a doubl ing time ( td ) of 20 hr.

The fastest observed n e t growth r a t e was 1.07 d-l ( ~ ' 1 5 . 6 hr) ; t h e growth

curve f o r t h i s ba tch experiment i s shown on Figure 1.

va lues were comparable t o growth rates descr ibed by o t h e r s (S tewar t , 1977;

Fay, 1983; Bo the , e t . a l . , 1984) .

These growth rate

The Anabaena d id no t s u r v i v e i n maintenance c u l t u r e s buf fered a t pH

va lues of 10 o r greater.

While t h e ba t ch growth exper iments y ie lded a c t i v e l y growing Anabaena

popu la t ions , a t t e m p t s t o develop s t eady- s t a t e popu la t ions of Anabaena i n a

chemostat f a i l e d .

and under t h e same envi ronmenta l c o n d i t i o n s as t h e ba tch growth experiments,

except t h a t a cont inuous f eed was added t o t h e r eac to r .

sub jec t ed t o a cont inuous f e e d , t h e concen t r a t ion of ch lo rophy l l i n t h e

r e a c t o r decreased a t a rate f a s t e r than would be p red ic t ed based on t h e

hydrau l i c d i l u t i o n rate (D) f o r t h e r eac to r .

The chemostat s t u d i e s were performed i n t h e same r e a c t o r

When t h e r e a c t o r was

A t y p i c a l response between c h l o r o p h y l l concen t r a t ion and time af ter t h e

s t a r t of feed f low i s shown on Figure 2.

concen t r a t ion i n t h e once-through r e a c t o r i nc reased from 220 t o 300 ug chl/L.

Af t e r 22 h r of f low, t h e ch lo rophy l l

19

1000,

n -I \ z " 100- 0 s U

x

IO -

In X = 1.07 t + 2.12 r2= 0.981

0 2 4 6 t (day)

Figure 1. Logarithmic increase in Anabaena biomass (X) as function of time, during a batch experiment.

20

0 Time (hr)

Figure 2. Changes i n Anabaena biomass (X) v e r s u s time after t h e s t a r t o f f eed flow through t h e r eac to r . dec rease i n X i f d i l u t i o n was t h e only removal mechanism.

The d o t t e d l i n e r e p r e s e n t s t h e expected

21

This biomass increase was equa l t o t h e i n c r e a s e p r e d i c t e d by t h e average n e t

s p e c i f i c growth rate (u) minus t h e d i l u t i o n rate (D), i.e.,

Xt = Xo exp[(u-D) t ] (15)

i n which Xt is t h e c h l o r o p h y l l concen t r a t ion a t time t (ug/L), X, is t h e

ch lo rophy l l concen t r a t ion when f low was s t a r t e d (ug/L), u i s t h e average n e t

s p e c i f i c growth rate f o r Anabaena obta ined from t h e ba tch growth exper iments

(0.0347 hr- l ) , D is t h e d i l u t i o n rate f o r t h e r e a c t o r (0.0211 hr- l ) , and t is

t h e time s i n c e start of f low through t h e reactor (hr).

22 h r of f eed a d d i t i o n , t h e t r a n s i e n t ch lo rophy l l c o n c e n t r a t i o n s fo l lowed t h e

p r e d i c t e d growth cu rve f o r a once-through r eac to r .

Thus, f o r t h e f i r s t

After 22 h r , t h e t r a n s i e n t ch lo rophy l l c o n c e n t r a t i o n s no l onge r followed

I n s t e a d of con t inu ing t o i n c r e a s e o r of p l a t e a u i n g a t a t h e p r e d i c t e d t rend .

s t e a d y - s t a t e va lue , t h e c o n c e n t r a t i o n of c h l o r o p h y l l i n t h e once-through

reactor decreased w i t h time (Figure 2).

1.5 times g r e a t e r t han t h e d i l u t i o n rate (D).

The rate of c h l o r o p h y l l d e c r e a s e was

P o s s i b l e e x p l a n a t i o n s f o r t h e observed d e c r e a s e i n c h l o r o p h y l l

c o n c e n t r a t i o n s inc luded an Anabaena die-off o r t h e presence of a s e l e c t i v e

mechanism f o r removing t h e Anabaena t r ichomes from t h e reactor.

die-off could have occurred , due e i t h e r t o cel l l y s i s caused by t h e lack of

n u t r i e n t s o r t o a r a p i d i n c r e a s e i n t h e concen t r a t ion of grazers . Because

Anabaena d i e - o f f s d i d n o t occur du r ing t h e ba t ch growth exper iments , and

because t h e ba t ch growth exper iments were of longer d u r a t i o n and obta ined

h ighe r c h l o r o p h y l l c o n c e n t r a t i o n s than t h e s i t u a t i o n d i sp layed in Figure 2,

t h e die-off hypo thes i s was n o t cons idered a s t r o n g p o s s i b i l i t y .

An Anabaena

The s e l e c t i v e removal mechanism exp lana t ion was based on t h e fact t h a t

t h e f low e x i t e d from t h e r e a c t o r through an over f low weir. Any mechanism t h a t

LL

accumulated Anabaena i n t h e neus t ron o r i n t h e upper r e g i o n s of t h e r e a c t o r

would have a l lowed t h e c h l o r o p h y l l c o n c e n t r a t i o n s t o dec rease a t a ra te faster

than p r e d i c t e d by t h e d i l u t i o n rate.

e n t r a i n m e n t of t h e f i l a m e n t o u s Anabaena on r i s i n g a i r bubbles and t h e buoyancy

confe r r ed on Anabaena due t o t h e presence of g a s v e s i c l e s . An i n t e r e s t i n g

o b s e r v a t i o n was t h a t s i g n i f i c a n t p o r t i o n s of t h e Anabaena c o l l e c t e d i n t h e

over f low-col lec t ion v e s s e l were buoyant. However, the observed buoyancy could

have a r i s e n from t h e d i f f e r e n t environmental c o n d i t i o n s provided i n t h e

over f low-col lec t ion vesse l .

P o s s i b l e mechanisms included t h e

Regardless of t h e mechanism, it was n o t p o s s i b l e t o e s t a b l i s h steady-

state c o n d i t i o n s i n t h e once-through r e a c t o r e i t h e r i n t h e presence o f o r i n

t h e absence of cyanide.

faster than p r e d i c t e d by t h e d i l u t i o n rate, d e s p i t e v a r i a t i o n s i n f e e d f l o w

rate, a g i t a t i o n speed, a i r f low rate, l i g h t i n t e n s i t y , and method o f r e a c t o r

s tar t -up. Because t h e a v a i l a b l e r e a c t o r could n o t f u n c t i o n as a chemostat f o r

t h e Anabaena c u l t u r e s , t h e k i n e t i c c o e f f i c i e n t s f o r the degrada t ion of cyanide

were determined from short- term ba tch experiments.

Chlorophyl l c o n c e n t r a t i o n s always decreased a t a rate

TOTAL CYANIDE REMOVAL RATES

L i m i t a t i o n s o f Batch Experiments

The mixed second-order rate c o n s t a n t s (Kb) d e s c r i b i n g t h e b i o l o g i c a l l y -

mediated d e c r e a s e i n t o t a l cyanide c o n c e n t r a t i o n s by t h e Anabaena c u l t u r e s

were de termined v i a short- term ba tch experiments.

the b a t c h exper iments was t o provide k i n e t i c i n f o r m a t i o n f o r t h e s e t u p o f the

chemostat experiments.

t o e s t a b l i s h s t e a d y - s t a t e c o n d i t i o n s , t h e results of t h e ba t ch experiments , as

t h e on ly s o u r c e of k i n e t i c i n f o r m a t i o n , became more impor tan t t han o r i g i n a l l y

The i n i t i a l o b j e c t i v e of

With i n a b i l i t y of t h e a v a i l a b l e once-through r e a c t o r

23

intended.

The short- term ba tch exper iments were performed w i t h Anabaena c u l t u r e s

Because the c u l t u r e s were t h a t had n o t prev ious ly been exposed t o cyanide.

n o t a c c l i m a t e d t o t h e presence of cyanide (i.e., t h e o p t i m a l c o n c e n t r a t i o n s of

cyanide degrading enzymes were not developed), t h e r e p o r t e d rate c o n s t a n t s f o r

t o t a l cyanide removal are probably s lower than would be observed f o r

a c c l i m a t e d Anabaena c u l t u r e s .

underes t imate cyanide removal p o t e n t i a l i s tha t the maximum i n h i b i t o r y effects

of cyanide on r e s p i r a t i o n and enzyme a c t i v i t y were expressed du r ing a ba t ch

test (i.e., p r o t e c t i o n mechanisms and pathways were n o t a l lowed t o become

f u l l y ope ra t iona l ) . Thus, a s t e a d y - s t a t e t e r t i a r y cyanide t r e a t m e n t p rocess

u t i l i z i n g Anabaena should have faster rate c o n s t a n t s f o r the degrada t ion of

cyanide than the r e p o r t e d values.

Rate Cons tan ts for T o t a l Cyanide Removal

Another r eason why t h e r e p o r t e d rate c o n s t a n t s

The two mechanisms r e s p o n s i b l e f o r reducing t o t a l cyanide c o n c e n t r a t i o n s

du r ing a ba tch test were b iodegrada t ion and v o l a t i l i z a t i o n .

ba t ch tests l i s t e d on Table 2 was 4 hours , w i t h t h e except ion o f ba t ch tes t 1

which was 5 hours long.

v a r i e d c o n s i d e r a b l y between t h e batch tests.

1, the e x t e n t o f cyanide l o s s due t o v o l a t i l i z a t i o n i n c r e a s e d as the time

averaged pH v a l u e decreased.

averaged pH v a l u e of 8.44, 1112 ug L" was v o l a t i l i z e d , w h i l e t h e amount of

cyanide v o l a t i l i z e d du r ing ba tch test 3 w i t h a time-averaged pH of 9.5 was 138

ug CN. This was the expected resul t , because as pH dropped t h e f r a c t i o n of

cyanide e x i s t i n g as v o l a t i l e hydrogen cyanide increased. In c o n t r a s t , the

e x t e n t o f b iodegrada t ion i n c r e a s e d a s t h e time averaged pH va lue i n c r e a s e d

The length of the

The e x t e n t o f b iodegrada t ion and v o l a t i l i z a t i o n

With t h e except ion of ba t ch test

For example, f o r ba t ch tes t 2 with a time-

24

TABLE 2. EXTENT OF BIODEGRADATION AND VOLATILIZATION I N SELECTED BATCH EXPERIMENTS

I n i t i a l To ta l V o l a t i l i z e d Biodegraded Biomass Mass CN Mass of Mass of

Time XO Removed Cyanide Cyanide Batch Ave . Number PH (ug chl/L) (ug CN) (up CN) (ue CN)

1 9.59 412 752 534 218

2 8.44 4926 1188 1112 76

3 9.50 203 443 138 305

4* 9.00 560 360 324 36

5** 9.00 367 583 410 173

* **

media f o r t h i s ba tch tes t included 0.5 ppm t h i o s u l f a t e

re-exposure of biomass i n batch test #4 t o cyanide and t h i o s u l f a t e

( f o r t h i s observa t ion ignore batch test 5, which is a batch test examining t h e

p o t e n t i a l f o r acc l imat ion) . For example, a t a time-averaged pH of 8.44 (batch

test 2), biodegrada t ion accounted f o r t h e removal of 76 ug CN.

averaged pH of 9.5 (batch test 3), biodegradat ion accounted f o r t h e removal of

305 ug CN.

test 2, d e s p i t e it having only 4Z of t h e biomass.

t h a t pH played an impor t an t r o l e i n de te rmining rate of t o t a l cyanide removal,

whether t h e removal mechanisms was predominantly by v o l a t i l i z a t i o n or by

biodegradat ion.

A t a time-

Batch test 3 had a greater e x t e n t of biodegradat ion than batch

These obse rva t ions sugges t

The biodegrada t ion and v o l a t i l i z a t i o n r a t e c o n s t a n t s i n equat ion (3 ) were

determined from a computer a lgori thm. The r e s u l t i n g r a t e cons t an t s provided

25

t h e s imul taneous b e s t fit of two sets of data:

concen t r a t ion w i t h time i n t h e r e a c t o r and t h e mass of cyanide c o l l e c t e d i n

t h e gas-wash b o t t l e . The c a l c u l a t e d Kb and KLa va lues f o r batch tests 1

through 4 a r e l i s t e d on Table 3. As shown on Figure 3 f o r batch test 3, t h e

c a l c u l a t e d va lues of Kb and KLa descr ibed t h e decrease i n t o t a l cyanide

concent ra t ion . S i m i l a r f i t s of t h e d a t a were obtained f o r t h e o the r batch

tests.

t h e dec rease i n cyanide

TABLE 3. OVERALL VOLATILIZATION RATE CONSTAWS (KT a) AND MIXED SECOND-ORDER RATE CONSTANTS (K ) FOR TOTAL CYANIDE REMOVAL BY +ACCLIMATED ANABAENA

&TURES IN SELECTED BATCH EXPERIMENTS

Time Time KLa Kb Batch Averaged Averaged Number PA ALPHA (h r - l ) [L/(ug c h l h r ) ]

1 9.59 0.339 0.17 8.5

2 8.44 0.879 0.22 5.0.

3 9.50 0.387 0.052 2.2.10-4

4* 9.00 0.666 0.075 8.0.

* media f o r t h i s ba tch test included 0.5 ppm t h i o s u l f a t e

Examination of Table 3 i n d i c a t e s t h a t t h e f i t t e d va lues of Kb and KLa

va r i ed cons ide rab ly between t h e 4 ba tch tests.

changes i n pH on v o l a t i l i z a t i o n r a t e s were accounted f o r by t h e ALPHA term in

equa t ion (3), t h e &fo ld v a r i a t i o n i n t h e c a l c u l a t e d KLa va lues was due t o

d i f f e r e n c e s i n a e r a t i o n rates and a g i t a t i o n i n t e n s i t i e s . These d i f f e r e n c e s

e x i s t e d , d e s p i t e t h e mechanical s e t t i n g s on t h e exper imenta l r e a c t o r being t h e

same f o r each ba tch test.

Because t h e e f f e c t of temporal

26

3300'

-- n

Figure 3. Decrease in total cyanide concentration (S) during batch test number 3. (3) using the Kb and KLa values on Table 3.

The line represents the numerical solution of equation

I I I I

0 aample - equation (3) -

27

- v)

-- -

2500 I I I I

I I

I I

The mixed second-order b iodegrada t ion rate c o n s t a n t s (Kb) f o r t h e

unaccl imated Anabaena c u l t u r e s ranged from 5.0*10-6 t o Z.Z.10-4 L/(ug c h l hr).

This observed 44-fold v a r i a t i o n i n t h e Kb va lues appeared t o b e a func t ion of

pH.

time-averaged pH f o r each ba tch test increased .

between Kb and t h e time-averaged pH va lue was approximated by t h e fo l lowing

linear r eg res s ion :

A s shown on F igure 4 , t h e b iodegrada t ion rate c o n s t a n t i nc reased as t h e

The observed r e l a t i o n s h i p

log Kb - 1.44 - 17.7 (16)

r2 = 0.836

i n which Kb has t h e u n i t s L/(ug c h l h r ) , s i s t h e time-averaged pH va lue f o r

t h e ba tch test , l o g is t h e base 10 logar i thm, and r2 i s t h e squa re of t h e

linear r e g r e s s i o n c o e f f i c i e n t .

Because Kb va lues inc reased as t h e time-averaged pH inc reased , t h e

observed Kb v a l u e s were probably responding t o ALPHA,

Since t h e i n i t i a l t o t a l cyanide concen t r a t ion f o r t h e ba tch tests were similar

(aprox. 3000 ug CN/L), comparing Kb t o t h e time-averaged ALPHA value would

i n d i c a t e t h e effect of HCN on b iodegrada t ion r a t e s .

between Kb and t h e time-averaged ALPHA value suggested t h a t f o r s m a l l e r

c o n c e n t r a t i o n s of HCN t h e rate of t o t a l cyanide b iodegrada t ion by t h e Anabaena

c u l t u r e s inc reased (F igure 5 ) .

approximated by t h e fo l lowing l i n e a r regress ion:

The observed r e l a t i o n s h i p

The r e l a t i o n s h i p between Kb and ALPHA was

l o g Kb = -2.91 ALPHA - 2.88 (17)

r2 = 0.862

i n which ALPHA was c a l c u l a t e d from t h e time-averaged pH f o r each ba tch test.

28

-3

-4

-6- I 9 I PH

Figure 4 . Relationship between log K b [L/(ug c h l h r ) ] versus time-averaged pH.

29

-3

-4

n X 0 0 -

-5.

-6-

IOg Kbx-2.9IALPHA -2.88

r 2 = 0.862

0 0.4 0.8 ALPHA

Figure 5. Relationship between log K b [L/(ug chl hr)] versus time-averaged ALPHA.

30

The rate a t which unacclimated Anabaena c u l t u r e s reduced t o t a l c y a n i d e

concen t r a t ions was opt imized by reducing t h e f r a c t i o n of t h e cyanide t h a t

e x i s t e d as HCN (reduced ALPHA value).

concept t h a t HCN is more t o x i c and i n h i b i t o r y than CN- (Doudoroff, e t a l . ,

1966; Doudoroff, 1976). The ALPHA value is smallest a t pH va lues greater t h a n

t h e pKa f o r HCN.

reduces t h e r a t e of v o l a t i l i z a t i o n . These f a c t o r s sugges t t h a t t h e

b iodegrada t ion e f f i c i e n c y of a cyanide- t reatment process based on ni t rogen-

f i x i n g cyanobacter ia would i n c r e a s e a s t h e pH of t h e s y s t e m is increased.

This improvement i n r e a c t o r efficiency w i t h pH would occur only up t o pH

v a l u e s of 10, because Anabaena c u l t u r e d i d no t su rv ive a t pH va lues g r e a t e r

t h a n 10.

This observa t ion i s c o n s i s t e n t wi th t h e

Operat ing a r e a c t o r a t pH va lues above t h e pKa f o r HCN a l s o

The maximum observed Kb f o r t h e degrada t ion of c y a n i d e by unaccl imated

Anabaena c u l t u r e s is l a r g e r than t h e mixed second-order rate c o n s t a n t s f o r

e x i s t i n g b i o l o g i c a l treatment processes .

test 3 i n t o u n i t s of L/(mg VSS d) where VSS i s v o l a t i l e suspended s o l i d s , t h e

maximum observed rate cons tan t was 0.0195 L/(mg VSS d). In comparison, t h e

b iodegrada t ion of acetate by methanogens (i.e., anaerobic he t e ro t rophs t h a t

produce methane) can have a mixed second-order r a t e c o n s t a n t of 0.0054

L/(mg VSS d) (Rittmann and McCarty, 1980). Thus, t h e b iodegrada t ion of

cyanide by unaccl imated Anabaena c u l t u r e s can have faster rate cons t an t s than

t h e b iodegrada t ion of a c e t a t e by methanogens.

growth rate f o r Anabaena under optimum cond i t ions was 0.832 d-l , whi le t h e

maximum s p e c i f i c growth rate f o r t h e a c e t a t e - u t i l i z i n g methanogens was

r epor t ed as 0.25 d-l (Rittmann and McCarty, 1980).

faster growth rates and contaminant removal rates than methanogens, and

Converting t h e observed K b f o r ba tch

The observed n e t specific

Because Anabaena can have

31

because s u c c e s s f u l wastewater t r ea tmen t f a c i l i t i e s have been developed us ing

methanogens, t h e r e appears t o be no k i n e t i c l i m i t a t i o n f o r t h e development of

b i o l o g i c a l treatment processes u t i l i z i n g Anabaena.

l i m i t p rocess development, such as cons t ruc t ion cos ts .

t r ea tmen t of wastes w i t h suspended microorganisms, large r e a c t o r s may be

requi red t o provide adequate cyanide removal and t o ensure process s t a b i l i t y .

E f f e c t of Acclimation on Cyanide Removal Rates

However, o t h e r f a c t o r s may

A s wi th t h e anaerobic

By re-exposing an Anabaena cu l ture t o cyanide, t h e impact of acc l ima t ion

on biodegrada t ion rates was assessed.

4 (Table 2). T h i o s u l f a t e (0.5 ppm) was added t o t h e cu l ture media used i n

ba tch t es t 4 t o induce t h e product ion of rhodanese, t h e enzyme t h a t catalyzes

t h e format ion of t h iocyana te from f r e e cyanide and t h i o s u l f a t e . The a d d i t i o n

of t h i o s u l f a t e was assumed t o have no e f f e c t on t h e Kb va lue f o r ba tch t es t 4.

However, t h e induc t ion of rhodanese and o the r d e t o x i f i c a t i o n pathways dur ing

ba tch tes t 4, were expected t o i n c r e a s e t h e r a t e of t o t a l cyanide removal

dur ing ba tch test 5. Comparison of t h e Kb va lues f o r ba tch tests 4 and 5

would i n d i c a t e t h e d i f f e r e n c e i n cyanide biodegradat ion k i n e t i c s between

unaccl imated and acc l ima ted Anabaena cu l tu re s .

Batch test 5 was a r epea t of ba tch test

Seve ra l hours after complet ion of batch test 4 , a e r a t i o n and a g i t a t i o n

were s h u t o f f i n t h e exper imenta l r eac to r .

overnight .

w i t h new c u l t u r e media con ta in ing t h i o s u l f a t e .

r e s t a r t e d .

a second cyanide ba tch experiment.

The Anabaena was al lowed t o se t t le

The next morning t h e supe rna tan t was siphoned o f f and rep laced

Aerat ion and a g i t a t i o n were

Two hours later, t h e a c c l i m a t e d Anabaena c u l t u r e was sub jec t ed t o

The a c c l i m a t e d Anabaena c u l t u r e reduced t o t a l cyanide concen t r a t ions 12

times faster than t h e unacclimated Anabaena cu l ture (Table 4). The acc l ima ted

32

TABLE 4. COMPARISON OF MIXED SECOND-ORDER RATE CONSTANTS fKL) mi? TnTbi ~ -. . - -. .-

CYANIDE REMOVAL BY ~ACCLIMATED (BATCH NUMBER 4) AND A C C L X + E D (BATCH M E R 5) ANABAENA CULTURES I N PRESENCE OF THIOSULFATE

Time KLa Kb Time Batch Averaged Averaged Number PH ALPHA (hr - l ) [L/(ug c h l h r ) ]

4 9.00 0.666 0.075 8.0 1 0-6

5 9.00 0.666 0.085 1.0.10-4

Anabaena c u l t u r e (ba tch test 5) had a mixed second-order rate c o n s t a n t (Kb) of

l.0.10-4 L/(ug c h l hr ) , wh i l e Kb f o r t h e unacc l imated Anabaena c u l t u r e (ba tch

t e s t 4) was 8.0.10-6 L/(ug c h l h r ) .

Thus, when an Anabaena c u l t u r e h a s time f o r the induc t ion of rhodanese

and o t h e r enzymat ic pathways, t h e ra te c o n s t a n t s for cyanide b iodegrada t ion

increase .

would be acc l ima ted t o a s t eady- s t a t e cyanide concen t r a t ion , t h e rate

c o n s t a n t s f o r cyanide b iodegrada t ion would l i k e l y be f a s t e r t h a n t h o s e l i s t e d

on T a b l e 3.

Because t h e Anabaena i n a n ope ra t ing cyanide- t reatment p rocess

MTHANE PRODUCTION RATES

Rate Cons tan ts for Methane Product ion

For t h e methane product ion d a t a shown on F igu re 6, a n unaccl imated

Anabaena c u l t u r e (X0=1146 ug chl/L) produced 14 ug C H ~ / L (gas phase) i n 1.75

hours when t h e i n i t i a l t o t a l cyanide concen t r a t ion i n t h e v i a l was 31.2

ug CN/L (water phase).

r e d u c t i o n of 11.0 ug CN/L (water phase) by n i t rogenase i n 1.75 hours.

a b i l i t y of n i t rogen- f ix ing cyanobac te r i a t o reduce cyanide c o n c e n t r a t i o n s

T h i s amount of methane product ion corresponded t o t h e

The

33

Time (hr)

Figure 6. Increase in headspace methane concentration with time. illustrated batch test, the initial cyanide concentration was 31.2 ug CN/L.

For the

34

below 20 ug/L may be s i g n i f i c a n t , because no cyanide d e s t r u c t i o n p rocess

h a s demonstrated t h e a b i l i t y t o reduce t o t a l cyanide c o n c e n t r a t i o n s t o

l e v e l s less than 25 ug/L (Brunker, 1980). Thus, n i t rogen-f ix ing

cyanobac te r i a may be well s u i t e d f o r use i n t h e secondary o r t e r t i a r y

t r e a t m e n t of cyanide wastes.

The s u i t a b i l i t y of u t i l i z i n g t h e n i t rogen-f ix ing c a p a c i t y of

cyanobac te r i a t o treat small concen t r a t ions of cyanide can be r e l a t e d to t h e

mechanism by which n i t rogenase s y n t h e s i s i s regula ted .

is induced by t h e lack of ammonia (S tewar t , 1977; 1980). Thus, t h e

i n t r a c e l l u l a r concen t r a t ion of n i t rogenase is not a f u n c t i o n of s u b s t r a t e

c o n c e n t r a t i o n (N2 or HCN), but i s a f u n c t i o n of product c o n c e n t r a t i o n

(ammonia). By promoting t h e a c t i v e growth of cyanobacter ia , ammonia i s k e p t

i n s h o r t supply, which r e s u l t s i n t h e maintenance of maximal l e v e l s of

n i t rogenase (S tewar t , 1977), r e g a r d l e s s of cyanide concent ra t ion . In

c o n s t r a s t t o n i t rogenase , t h e s y n t h e s i s and maintenane of h igh i n t r a c e l l u l a r

c o n c e n t r a t i o n s of rhodanese is a func t ion of cyanide c o n c e n t r a t i o n (Atkinson,

1975; Atkinson, e t al . , 1975; Brunker, 1980). The a b i l i t y t o ma in ta in maximal

a c t i v i t y l e v e l s of n i t rogenase a t low cyanide c o n c e n t r a t i o n s i m p l i e s t h a t

n i t rogenase may be more e f f e c t i v e i n t h e removal of low l e v e l s of cyanide than

cyanide-induced enzymat ic pathways.

Nitrogenase s y n t h e s i s

The d e t e r m i n a t i o n of t h e rate c o n s t a n t f o r t h e r educ t ion of cyanide by

n i t r o g e n a s e r equ i r ed t h e c a l c u l a t i o n of t h e mixed second-order rate c o n s t a n t

f o r t h e o v e r a l l removal of cyanide from t h e v i a l . A s t h e pH i n t h e v ia l was

10, t h e va lue of mixed second-order rate cons t an t f o r t h e t o t a l cyanide

removal rate (Kb) c a l c u l a t e d from equa t ion (17) was 4 . 3 3 ~ 1 0 - ~ L/(ug c h l hr).

T h i s Kb v a l u e was used i n equa t ions (13) and (14) t o c a l c u l a t e t h e mixed

35

second-order rate c o n s t a n t (K,) f o r t h e reduct ion of cyanide by n i t rogenase .

For t h e methane product ion shown i n F igure 6, t h e c a l c u l a t e d Kn va lue was

2.58*10d4 L/(ug c h l hr).

A quick comparison of t h e c a l c u l a t e d Kn and Kb va lues would s u g g e s t t h a t

f o r an i n i t i a l cyanide concen t r a t ion (So) Of 31.2 ug CN/L, t h e rate of HCN

r educ t ion by n i t rogenase accounted f o r 60% o f t h e t o t a l cyanide removal rate.

One problem w i t h t h i s comparison is t h a t t h e c a l c u l a t i o n of Kb from equat ion

(17) assumes t h a t so is approximately 3000 ug CN/L. Because t h e v a l u e of Kb

was found t o increase w i t h decreas ing HCN concent ra t ion , t h e use of equat ion

(17) i n t h e a n a l y s i s of above methane product ion experiment may g r e a t l y

underes t imate Kb. For tuna te ly , t h e Kn v a l u e obta ined from equa t ions (13) and

(14) was r e l a t i v e l y i n s e n s i t i v e t o changes i n Kb. For i n p u t t e d t o t a l cyanide

b iodegrada t ion rate c o n s t a n t s (Kb) ranging from 2.58.10- 4 ( a l l cyanide removal

due t o n i t rogenase) t o l.0*10-3 L/(ug c h l hr ) (only 5% of o r i g i n a l cyanide

remained i n r e a c t o r after 1.75 hr), t h e Kn va lues obta ined from equa t ions (13)

and (14) on ly va r i ed by a f a c t o r of 2 (Figure 7). For t h e i n p u t t e d range of

Kb va lues , t h e ratio of n i t rogenase a c t i v i t y t o t o t a l cyanide b iodegrada t ion

rates (Kn/Kb) ranged from 0.39 t o 1.00 (Figure 8). These c a l c u l a t i o n s sugges t

t h a t t h e r educ t ion of HCN t o methane by n i t rogenase is a s i g n i f i c a n t mechanism

of cyanide b iodegrada t ion f o r unaccl imated Anabaena c u l t u r e s exposed t o low

cyanide c o n c e n t r a t i o n s (S0=31.2 ug CN/L).

A s t h e i n i t i a l concen t r a t ion of cyanide increased , t h e mixed second-order

rate cons tan t f o r n i t rogenase a c t i v i t y (K,) appeared t o decrease.

methane product ion experiment w i t h an i n i t i a l cyanide concen t r a t ion (So) of

400 ug CN/L and a pH of 9.9, t h e c a l c u l a t e d Kn va lue was 2.62*10-6

L/(ug c h l hr).

For a

Th i s Kn va lue was 2 o r d e r s of magnitude s lower than t h e Kn

36

r Y

.0004 --

I 1 I

.0002

--

0 -3.75

--

--

I I I I I I

-3.35 -2.95

Figure 7. S e n s i t i v i t y of Kn ob ta ined from e q u a t i o n s (13) and (14) t o i n p u t t e d K b values. Both parameters have u n i t s of L/(ug c h l hr).

37

n Y \

Y c

-3.75 -2.95

Figure 8. S e n s i t i v i t y of t h e r a t i o between Kn and Kb t o i n p u t t e d K b va lues . Both parameters have u n i t s of L/(ug c h l hr).

38

va lue c a l c u l a t e d when So was 31.2 ug CN/L.

So was probably due t o HCN i n h i b i t i o n of t h e ATP gene ra t ing pathways i n t h e

he t e rocys t s , because cyanide a t t h e exper imenta l concen t r a t ions has no e f f e c t

on t h e rate of e l e c t r o n f low through n i t rogenase ( L i , e t al., 1982). Thus,

t h e e f f e c t of cyanide concen t r a t ions on Kn may d iminish wi th acc l imat ion .

Comparison of Methane Product ion wi th Nitrogen F ixa t ion

The decrease i n Kn wi th i n c r e a s i n g

The observed rate of HCN reduct ion (methane product ion) by n i t rogenase

was compared t o l i t e r a t u r e v a l u e s of d in i t rogen (N2) r educ t ion rates.

d i n i t r o g e n reduct ion rates were converted t o HCN r educ t ion r a t e s based on

t h r e e assumptions.

independent of HCN concen t r a t ion , so t h a t t h e rate of e l e c t r o n f low f o r

n i t rogen f i x a t i o n is assumed equal t o e l e c t r o n f low f o r HCN reduct ion (Li , et

al., 1982).

descr ibed above.

form of HCN.

he t e rocys t i s 7.3 (Stewart , 1977), two pH u n i t s below t h e pKa f o r HCN. Thi rd ,

f o r each mole d i n i t r o g e n reduced there is t h e s imul taneous reduct ion of 2

pro tons t o make 1 mole of hydrogen gas , i.e., 8 e l e c t r o n s must f low through

n i t rogenase f o r each mole of N2 fixed.

moles of H2 produced per mole of N2 reduced by n i t rogenase (Bur r i s and

P e t e r s o n , 1978; Lean, e t a l . , 1975; P o s t g a t e , 1982; Fay, 1983; B o t h e , e t a l . ,

1984).

The

F i r s t , t h e rate of e l e c t r o n f low through n i t rogenase is

This is a reasonable assumption f o r t h e exper imenta l c o n d i t i o n s

Second, a l l of t h e cyanide i n s i d e t h e h e t e r o c y s t is i n t h e

This is a reasonable assumption, because t h e pH i n s i d e of a

This is a conse rva t ive estimate of t h e

Ramos, e t al., (1987) r epor t ed t h a t an Anabaena c u l t u r e had a maximum

s p e c i f i c d i n i t r o g e n r educ t ion r a t e of 30 umole N2/(mg c h l hr).

above assumptions, t h i s corresponded t o a c a l c u l a t e d maximum s p e c i f i c HCN

r educ t ion rate (k) of 1.08 ug HCX/(ug c h l hr).

Based on t h e

This maximum r a t e was ad jus t ed

39

t o account f o r t h e e f f e c t of ambient HCN concen t r a t ions by t h e fo l lowing

modified Monod equat ion:

k S r~~~ =

K, + S

i n which rHCq is t h e s p e c i f i c HCN reduct ion rate rug HCN/(ug c h l h r ) ] , k is

t h e maximum s p e c i f i c HCN reduct ion rate [LO8 ug HCN/(ug c h l h r ) ] , S is t h e

ambient HCN concen t r a t ion (up HCN/L), and K, i s t h e Michaelis-Menten cons t an t

f o r t h e product ion of methane from HCN by n i t rogenase (121,500 ug HCN/L) a s

r epor t ed by L i , et al., (1982). Th i s K, va lue included both t h e effect of

enzyme a f f i n i t y f o r HCN and of H2 product ion on rHCw

larger than t h e ambient cyanide concen t r a t ions used i n t h e methane product ion

experiments , equa t ion (18) can be reduced t o

Because K, is much

k r~~~ = - S

Km

i n which k/Km has t h e va lue 8.9.10-6 L/(ug c h l hr).

equ iva len t t o t h e fo l lowing rearrangement of equat ion (8):

Equation (19) i s

1 dP Kn S - _ =

Xo d t

i n which Kn is t h e experimental ly-der ived mixed second-order rate cons t an t f o r

t h e product ion of methane by n i t rogenase [L/(ug c h l hr)] . The c a l c u l a t e d k/K,

va lue was compared t o t h e experimental ly-der ived Kn values .

The observed va lues of K, ranged from 2.62.10-6 L/(ug c h l hr ) when So was

400 ug CN/L t o 2.58*10-4 L/(ug c h l h r ) when S o was 31.2 ug CN/L. The

c a l c u l a t e d k/Km value of 8.9*10-6 L/(ug c h l h r ) l i e s between t h e two observed

Kn values .

compared t o known i n v ivo n i t rogen- f ixa t ion rates and in v i t r o HCN r educ t ion

Thus, t h e observed rates of methane product ion were reasonable

40

rates.

The observed Kn va lue f o r S0=31.2 ug CN/L was a l m o s t 30 times l a r g e r t h a n

t h e c a l c u l a t e d k/Km value.

c o n c e n t r a t i o n s of HCN a t a more r a p i d ra te than d in i t rogen .

a g r e e s w i t h t h e obse rva t ion by L i , e t al., (1982) t h a t t h e a c t i v a t i o n

r equ i r emen t s f o r n i t rogenase t o reduce HCN are lower t h a n t h e a c t i v a t i o n

r equ i r emen t s t o reduce d in i t rogen .

t r a n s l a t e i n t o faster k i n e t i c s , n i t rogenase w i l l p r e f e r e n t i a l l y reduce HCN.

Thus, i f n i t rogenase ' s r equ i r emen t s f o r ATP and e l e c t r o n s can be cont inuous ly

s a t i s f i e d , t h e n t h e enzymatic a p p a r a t u s i n Anabaena normal ly r e s p o n s i b l e f o r

n i t r o g e n - f i x a t i o n may p lay an impor t an t r o l e i n de t e rmin ing t h e rate a t which

low c o n c e n t r a t i o n s of cyanide are biodegraded i n a t r e a t m e n t process . I n t h e

methane product ion exper iments w i t h S0=31.2 ug CN/L, t h e rate of n i t r o g e n a s e

a c t i v i t y was r e s p o n s i b l e f o r a t least 39% of t h e t o t a l cyanide b iodegrada t ion

ra te ( F i g u r e 8).

mis s u g g e s t s t h a t Anabaena may reduce low

Such a conclus ion

Because lower a c t i v a t i o n e n e r g i e s u s u a l l y

APPLICATION OF KINETIC DATA TO CYANIDE TREATMENT

The purpose of t h i s s e c t i o n is t o p r e d i c t t h e s t r e n g t h of a cyanide waste

t h a t can t r e a t e d by a p rocess u t i l i z i n g suspended c u l t u r e s of n i t rogen- f ix ing

Anabaena.

through competely-mixed r e a c t o r .

s tudy were n o t ob ta ined under s t e a d y - s t a t e c o n d i t i o n s , a p p l i c a t i o n of t h e

f o l l o w i n g d i s c u s s i o n t o t h e des ign of a n o p e r a t i n g cyanide t r e a t m e n t p rocess

should be done w i t h g r e a t cau t ion .

T h i s p r e d i c t i o n w i l l assume s t eady- s t a t e c o n d i t i o n s i n a once-

Because t h e k i n e t i c d a t a c o l l e c t e d i n t h i s

Assuming mixed second-order b iodegrada t ion k i n e t i c s and ignor ing

v o l a t i l i z a t i o n , t h e mass ba lance e q u a t i o n f o r cyanide i n a completely-mixed

once-through r e a c t o r is as fo l lows :

41

dS v - = Q S i - Q S - Kb X S

d t

i n which V is t h e volume of t h e r e a c t o r (L), S i s t h e concen t r a t ion of cyanide

i n t h e e f f l u e n t and i n t h e r e a c t o r (up CN/L), Si is t h e cyanide concen t r a t ion

i n t h e i n f l u e n t (ug CN/L), t is time (hr) , Q is t h e volumetr ic f l o w rate

through t h e r e a c t o r (Llhr) , X i s t h e concen t r a t ion of Anabaena i n t h e r e a c t o r

(up chl /L) , and Kb is t h e mixed second-order rate c o n s t a n t f o r t h e

b iodegrada t ion of cyanide by Anabaena [L/(ug c h l hr)].

cond i t ions (Le., dS/dt=O and dX/dt=O), t h e i n f l u e n t concen t r a t ion can be

desc r ibed by

Under s t eady- s t a t e

si = s [1 + Kb x 01 (22)

i n which 0 i s t h e hydrau l i c r e t e n t i o n time (hr ) and is equal t o V/Q.

a s s ign ing va lues of S, Kb, X and 0, t h e s t r e n g t h of t h e cyanide waste can be

e s t i m a t e d from equat ion (22).

Thus, by

I n p r e d i c t i n g t h e i n f l u e n t cyanide concen t r a t ions t h a t can be t r e a t e d by

an Anabaena process , t h e fo l lowing parameters were assumed:

pH = 9.5

X = 500 ug chl /L

0 = 120 h r

temperature = 25OC

S = 300 ug CN/L

Kb = 0.00022 L/(ug c h l h r )

The va lues of t h e above parameters are considered t o be conserva t ive .

va lue of S is an o rde r of magnitude smaller than t h e cyanide concen t r a t ion a t

which Kb was determined. The above Kb va lue assumes an unaccl imated Anabaena

c u l t u r e , under s t e a d y - s t a t e c o n d i t i o n s Kb should be l a r g e r . The biomass

concen ta t ion (X) i s an o rde r of magnitude smaller than t h e h ighes t

concen t r a t ion observed i n t h e experimental r eac to r .

p rovides a safety f a c t o r of 10 above t h e average net s p e c i f i c growth rate f o r

The

A 0 of 120 h r ( 5 days)

42

Anabaena.

of 4260 ug CN/L (4.26 mg CN/L).

concen t r a t ion of 500 ug chl /L can be maintained i n a chemostat wi th a

hydrau l i c r e t e n t i o n time of 5 days, then t h e r e a c t o r should be able t o reduce

an i n f l u e n t cyanide concen t r a t ion of 4.26 mg/L t o an e f f l u e n t concen t r a t ion of

0.3 mg CN/L, a 93 percent reduct ion. As t h e rate of cyanide b iodegrada t ion is

f i r s t - o r d e r wi th r e s p e c t t o cyanide concen t r a t ion (equat ion 22), similar

removal e f f i c i e n c i e s should be observed f o r lower i n f l u e n t cyanide

concen t r a t ions .

S u b s t i t u t i o n of t h e above parameters i n t o equat ion (22) y i e l d s a Si

Thus, i f a s t eady- s t a t e Anabaena

The above c a l c u l a t i o n s p r e d i c t t h a t a chemostat w i t h a hydrau l i c

r e t e n t i o n time (e) of 5 days is requ i r ed t o achieve a 93Z reduc t ion i n

cyanide.

c o n s t r u c t i o n of a r e a c t o r l a r g e enough t o hold t h e volume of wastes genera ted

i n 5 days may be p roh ib i t i ve . However, t h e vo lumet r i c s i z e of a cyanobacter ia

r e a c t o r could be reduced by using methods t o make t h e mean cell r e s idence time

(ec) l a r g e r than t h e hydrau l i c r e t e n t i o n time (e).

For some cyanide-waste gene ra to r s , t h e c o s t s a s s o c i a t e d w i t h t h e

One p o s s i b l e method f o r i n c r e a s i n g 0, invo lves t h e r ecyc l ing of

cyanobacter ia biomass i n t h e e f f l u e n t l i n e t o t h e reac tor .

algae r e a c t o r w a s o r i g i n a l l y proposed by McGriff and McKinney (1971).

success of t h e ac t iva t ed -a lgae r e a c t o r is dependent on t h e a b i l i t y t o s e p a r a t e

a l g a l cells from t h e r e a c t o r e f f l u e n t by s e t t l i n g . Despi te t h e presence of

gas v e s i c l e s , cyanobacter ia w i l l s i n k when t h e i r growth is l i m i t e d by f i x e d

n i t rogen a v a i l a b i l i t y (Klemer, et al., 1982). Because n i t rogen-f ix ing

cyanobacter ia on ly produce one mole of ammonia per cyanide reduced i n s t e a d of

t h e two ob ta ined from d i n i t r o g e n , t h e a v a i l a b i l i t y of n i t rogen may l i m i t

cyanobacter ia growth rates i n r e a c t o r s used t o treat cyanide wastes.

The ac t iva t ed -

The

While

43

cyanobacter ia may s i n k , t h e poten t ia l ly-s low s e t t l i n g v e l o c i t i e s of t h e

f i l amen tous n i t rogen- f ixe r s might r e q u i r e c l a r i f i e r s w i t h l a r g e surface areas .

Thus, t h e r educ t ion i n cons t ruc t ion c o s t s f o r t h e ac t iva ted-a lgae r e a c t o r

compared t o a chemostat could be par t ia l ly negated by t h e cons t ruc t ion c o s t s

f o r a clarifier.

Another method of i nc reas ing QC would be t h e use of attached-growth

r e a c t o r s i n s t e a d of suspended-growth chemostats.

a t t ached media s t eady- s t a t e concen t r a t ion of biomass becomes less dependent on

hydrau l i c f low rates.

a s s u r i n g t h a t s u f f i c i e n t l i g h t i n t e n s i t y reaches a l l of t h e cyanobacter ia

b io f i lms .

combination of t h e l a r g e Qc a s s o c i a t e d w i t h b io f i lm r e a c t o r s and t h e a b i l i t y

of n i t rogenase t o de tox i fy t race-concent ra t ions of cyanide sugges t s t h a t a

n i t rogen-f ix ing cyanobacter ia b io f i lm r e a c t o r would be well s u i t e d f o r t h e

secondary o r tert iary t r ea tmen t of cyanide wastes.

By growing cyanobacter ia on

One problem w i t h a cyanobacter ia b i o f i l m r e a c t o r is

If t h e l i g h t a v a i l a b i l i t y problem can be solved, then t h e

In a d d i t i o n t o providing adequate mean ce l l r e t e n t i o n times, ano the r

concern is p r o t e c t i n g t h e cyanobacter ia from f l u c t u a t i o n s i n cyanide

concent ra t ion .

cyanide concen t r a t ion , small short- term i n c r e a s e s i n cyanide concen t r a t ions

can be i n h i b i t o r y t o t h e microorganisms and, t hus , d i s r u p t t h e mic rob ia l

p rocess (Gaudy, e t al., 1982). One means of p r o t e c t i o n i s t o dampen t h e

magnitude of t h e i n f l u e n t cyanide concen t r a t ion f l u c t u a t i o n s by p r i m a r y

t r e a t m e n t of t h e cyanide was tes before b i o l o g i c a l treatment.

t r ea tmen t of t h e cyanide wastes would a l s o reduce t h e cyanide load t o t h e

cyanobacter ia process. Thus, t h e s teady and small concen t r a t ions of cyanide

i n t h e e f f l u e n t of an a l k a l i n e - c h l o r i n a t i o n o r o the r p r i m a r y t r e a t m e n t process

Despi te t h e acc l ima t ion of a mic rob ia l p rocess t o a given

Primary

44

would be conducive t o t h e maintenance of cyanobacter ia i n a secondary

treatment process.

t h e last f r a c t i o n o f cyanide, i n s t e a d of a t t e m p t i n g t o treat a l l of t h e

cyanide by the pr imary process , should resul t i n lower o p e r a t i n g c o s t s f o r

larger cyanide t r e a t m e n t facil i t ies.

U t i l i z a t i o n of n i t rogen- f ix ing cyanobac te r i a t o d e t o x i f y

45

REFERENCES

Allen, M.M. 1973. Methods f o r Cyanophyceae. In: J.R. S t e i n (ed.), Handbook of Phycologica l Methods - Cul tu re Methods and Growth Measurements, Cambridge Un ive r s i ty P res s , Cambridge, pp. 127-138.

APHS. 1980. Standard methods f o r t h e examination of water and wastewater , 16 th ed i t i on . American Pub l i c Heal th Assoc ia t ion , Washington, D.C.

Atkinson, A. 1975. Bacterial cyanide d e t o x i f i c a t i o n . Biotech. Bioeng. 17(3): 457-460.

A t k i n s o n , A., C.G.T. Evans, and R.G. Yeo. 1975. B e h a v i o r of B a c i l l u s s t e a r o t h e r m o u h i l i s grown i n d i f f e r e n t media. J. Appl. Bact. 38: 301- 304.

Biggins , D.R., and M. Kelley. 1970. I n t e r a c t i o n of n i t rogenase form K l e b s i e l l a uneumoniae wi th A T P o r cyanide. Biochimica et Biophysica Acta 205: 288-299.

Bothe , H., H. Nelles, K.P. Hager , H. Papen, and G. Neuer. 1984. P h y s i o l o g y and b iochemis t ry of n i t rogen- f ixa t ion by cyanobacter ia . In: C. Veeger and W.E. Newton (eds.), Advances i n Nitrogen F i x a t i o n Research, Dr. W. Junk Pub l i she r s , Boston, pp. 199-210.

Brunker, R.L. 1980. The b i o l o g i c a l degrada t ion of cyanides by a u t o t r o p h i c organisms. Conference, pp. 146-151.

Proceedings of t h e 12th Mid-Atlantic I n d u s t r i a l Waste

Bur r i s , R.H., and R.B. Peterson. 1978. Nitrogen-f ixing blue-green algae: t h e i r H metabolism and t h e i r a c t i v i t y i n f r e shwa te r lakes . In: U.

and Asymbiotic Bacteria. Ecol. Bull. (Stockholm) 26: 28-40. Granhal 1 (ed.), Environmental Role of Nitrogen-Fixing Blue-Green Algae

Castric, P.A. 1981. The metabolism of hydrogen cyanide by bacteria. In: B. V e n n e s l a n d , E.E. Conn, C.J. Knowles , J. Westley, and F. W i s s i n g (eds . ) , Cyanide i n Biology, Academic Press , London, pp. 233-261.

Degn, H., D. L l o y d , and G.C. H i l l (eds.) . 1978. F u n c t i o n s o f a l t e r n a t i v e t e r m i n a l oxidases. Pergamon Pres s , Oxford, 196 pp.

D o u d o r o f f , P., G. Leduc, and C.R. S c h n e i d e r . 1966. Acute t o x i c i t y t o f i s h of s o l u t i o n s con ta in ing complex metal cyanides , i n r e l a t i o n t o concen t r a t ions of molecular hydrocyanic ac id . Trans. Am. Fish. SOC. 95: 6-22.

46

Doudoroff, P. 1976. Toxicity of fish to cyanide and related compounds: a review. Research Series EPA-600/3-76-038, 154 pp.

USEPA Office of .Research and Development, Duluth, MN, Ecological

Fay, P. 1983. The blue-greens. The Institute of Biology's Studies in Biology No. 160, Edward Arnold, Baltimore, Maryland, 88 pp.

Gaudy, A.F., E.T. Gaudy, Y.J. Fenn, and G. Bruennemann. 1982. Treatment of ~. - . -- cyanide waste by the extended aeration process. Fed. 54: 153-164.

J. Wat. Pollut. Contr.

Fogg, G.E., W.D.P. Stewart, P. Fay, and A.E. Walsby. 1973. The blue algae. Academic Press, London, 459 pp.

Green, J., and D.H. Smith. 1972. Processes for the detoxification of waste cyanides. Metal Finish. Journal, August issue, pp. 229-232.

Hardy, R.W.F., and R.C. Burns. 1968. Biological nitrogen fixation. Ann. Rev. Biochem. 37: 331-358.

Hardy, R.W.F., and E. Knight, Jr. 1967. ATP-dependent reduction of azide and HCN by nitrogen-fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum. Biochimica et Biophysica Acta 139: 69-90.

Haystead, H., R. Robinson, and W.D.P. Stewart. 1970. Nitrogenase activity in extracts of heterocystous and non-heterocystous blue-green algae. Mikrobiol. 74: 235-243.

Arch.

Henry. M.F. 1981. Bacterial cyanide-resistant respiration: a review. In: B. Vennesland, E.E. Conn, C.J. Knowles, J. Westley, and F. Wissing (eds.), Cyanide in Biology, Academic Press, London, pp. 415-436.

Higgins, I.J., D. Scott, and R.C. Hammond. 1984. Transformation of C1 compounds by microorganisms. In: D.T. Gibson (ed.), Microbial Degradation of Organic Compounds, Marcel Dekker, Inc., New York, pp. 43- 87.

Howe. R.H.L. 1963. Proceedings of pp. 690-705.

Howe, R.H.L. 1965. disadvantages.

Recent advance in cyanide waste reduction practice. the 18th Industrial Waste Conference at Purdue University,

Bio-destruction of cyanide wastes--advantages and Int. J. Air Water Pollut. 9: 463-478.

Hwang, J.C., and R.H. Burris. 1972. Nitrogenase-catalyzed reactions. Biochimica et Biophysica Acta 283: 339-350.

Hwang,'J.C., C.H. Chen, and R.H. Burris. 1973. Inhibition of nitrogenase- catalyzed reductions. Biochimica et Biophysica Acta 292: 256-270.

47

Klemer, A.R., J. Feui l lade , and M. Feui l lade. 1982. Cyanobacteria blooms: carbon and n i t rogen l i m i t a t i o n have oppos i t e e f f e c t s on t h e buoyance of O s c i l l a t o r i . Science 215(4540): 1629-1631.

Kobayashi, H., and B.E. Rittmann. 1982. Microbia l removal of hazardous o rgan ic compounds. Environ. Sci . Tech. 16(3): 170a-183a.

Lean, D.R.S., C.F.H. L i a o , T.P. Murphy, and D.S. P a i n t e r . 1978. The importance of n i t rogen f i x a t i o n i n lakes . In: U. Granhal l (ed.), Environmental Role of Nitrogen-Fixing Blue-Green Algae and Asymbiotic Bacteria. Ecol. Bull . (Stockholm) 26: 41-51.

L i , J.G., B.K. B u r g e s s , and J.L. Corb in . 1982. N i t r o g e n a s e r e a c t i v i t y : cyanide as s u b s t r a t e and i n h i b i t o r . Biochem. 21: 4393-4402.

McGriff, E.C., and R.E. McKinney. 1971. Act ivated algae: a n u t r i e n t removal process. Water and Sewage Works 118(11): 377-379.

Metcalf and Eddy, Inc. 1979. Wastewater engineering: t r ea tmen t , d i s p o s l , reuse. McGraw-Hill, New York, 920 pp.

Peschek, G.A. 1980. E lec t ron t r a n s p o r t r e a c t i o n s i n r e s p i r a t o r y p a r t i c l e s of hydrogenase-induced Anacvst is nidulans. Arch. Microbiol. 125: 123-131.

Pos tga te , J.R. 1982. Fundamentals of n i t rogen f i x a t i o n . Cambridge Un ive r s i ty P res s , Cambridge, 252 pp.

Ramos, J.L., M.G. Guerrero, and M. Losada. 1987. F a c t o r s a f f e c t i n g t h e photoproduction of ammonia from d i n i t r o g e n and water by t h e cynobacterium Anabaena sp. s t r a i n ATCC 33047. Biotech. Bioeng. 29: 566-571.

Rittmann, B.E., and P.L. McCarty. 1980. Design of f ixed- f i lm processes w i t h s t e a d y - s t a t e b i o f i l m model. Prog. Wat. Tech. 12: 271-281.

S i l v e r , W.S., and J.R. Postgate . 1973. Evolut ion of asymbiot ic n i t rogen f i x a t i o n . J. Theor . B i o l . 40: 1-10.

Snoeyink, V.L., and D. Jenkins . 1980. Water Chemistry. John Wi ley & Sons, N e w York, 463 pp.

V e n n e s l a n d , E.E. Conn, C.J. Knowles , J. Westley, and F. W i s s i n g (eds . ) , Cyanide i n Biology, Academic Press, London, pp. 11-28.

Solomonson, L.P. 1981. Cyanide a s a metabol ic i n h i b i t o r . In: B.

S t e w a r t , W.D.P. 1977. B l u e - g r e e n a l g a e . I n : R.W.F. H a r d y and W.S. S i l v e r (eds.), A T r e a t i s e on Din i t rogen Fixation--Section 111. Biology, Wiley- I n t e r s c i e n c e , New York, pp. 63-123.

S tewar t , W.D.P. 1980. Some a s p e c t s of s t r u c t u r e and Function i n n i t rogen- f i x i n g cyanobacter ia . Ann. Rev. Microbiol. 34: 497-536.

48

\

Westley, J. 1981. Cyanide and s u l f a n e su l fu r . In: B. Vennesland, E.E. Conn, C.J. Knowles, J. Westley, and F. W i s s i n g (eds . ) , Cyan ide i n Biology, Academic P res s , London, PP. 29-49.

Z u m f t , W.G., and L.E. Mortenson. 1975. The n i t rogen-f ix ing complex of bac te r i a . Biochimica e t Biophysica Acta 416: 1-52.

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June 1988 B DLOGICAL DEGRADATION CYANIDE BY FIXING … · 2018-06-13 · Based on the total cyanide biodegradation rate constants (Kb) obtained from batch experiments, use of nitrogen-fixing