Chapter 39 M i c r o b i a l Calorimetric Analysis Lignin-Related Compounds in Micromolar Concentrations Rex E . Lovrien , Mark L. Ferry , Timothy S. Magnuson , 1
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and Robert A. Blanchette
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Biochemistry Department, Gortner Laboratory, University of Minnesota,
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St. Paul, M N 55108 2
Plant Pathology and Forestry Department, University of Minnesota, St. Paul, M N 55108
Microbial combustion of lignin fragments and lignin model compounds produces heat which is measurable via heat conduction calorimetry, even with samples ranging in size from 5-200 nanomoles. The general method is called microbial calorimetric analysis (MCA). First, microbes (Pseudomonas or soil bacteria) are grown on lignin fragments or model compounds as a carbon source. The adapted bacteria, so obtained, then function as rapid-acting, specific reagents for metabolizing such compounds. The technique has the following advantages: (i) cells (1-2 mg) are capable of aerobically combusting many kinds of C -C compounds in 5-10 minutes; (ii) interfering compounds can be removed by stripping; (iii) detection limits are comparable to spectrophotometric methods (e.g., to micromolar levels for sugars and phenols); and (iv) chromogenic groups are not required for detection. The sensitivity of the technique is based on the large aerobic heats of such compounds, and the velocities with which adapted cells can drive metabolism. 1
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common b a c t e r i a have been adapted t o g r o w i n g o n l i g n i n fragments, these c o m p o u n d s essentially undergo c o m b u s t i o n p r o d u c i n g heat. C u r rent heat c o n d u c t i o n calorimeters c a n easily measure s u c h heats o f aerobic m e t a b o l i s m , even i n q u a n t i t i e s r a n g i n g f r o m 5-100 n a n o m o l e s (1,2). B o m b c o m b u s t i o n c a l o r i m e t r y converts samples c o m p l e t e l y t o c a r b o n d i o x i d e a n d w a t e r . A e r o b i c b a c t e r i a l m e t a b o l i s m o n l y produces h a l f as m u c h heat, e.g., sugars, phenols, alcohols a n d a l i p h a t i c acids l i b e r a t e 100-600 K c a l / m o l , a n d l i g n i n a n d C s - 1 2 p e t r o l e u m s generate ~ 1000 K c a l / m o l " . T h e lower c a l o r i m e t r i c measurement ( ± 3 % precision) is ~ 2-30 m i l l i c a l o r i e s . D i v i d i n g s u c h ranges b y c a . 100-500 K c a l h e a t / m o l e gives ~ 5-200 nanomoles o f - 1
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c o m p o u n d needed for m i c r o b i a l c a l o r i m e t r i c a n a l y s i s ( M C A ) . S i n c e s a m ple volumes are 1-2 m l , m i c r o m o l a r c o n c e n t r a t i o n s can be used for t h i s m e t h o d . Besides the c o n c e n t r a t i o n ranges w h i c h a n y a n a l y t i c a l m e t h o d m a y be e x p e c t e d to cover, four f u r t h e r aspects are also i m p o r t a n t : n a m e l y , d e t e c t a b i l i t y of the a n a l y t e ; whether a s a m p l e c a n be used " r a w " or has to be i s o l a t e d ; i n t e r f e r i n g c o m p o u n d s a n d s p e c i f i c i t y ; a n d cost. C o n c e r n i n g d e t e c t a b i l i t y of l i g n i n a n d cellulose d e r i v e d fragments, M C A has considerable advantages. S u c h c o m p o u n d s are often p o o r l y c h r o m o g e n i c ; some have no chromogens at a l l , or are p o o r l y p r o c h r o m o g e n i c , i.e., are difficult t o a t t a c h a c h r o m o p h o r e . B u t these sorts o f c o m p o u n d s " b u r n " w i t h large heats i n M C A , so t h a t a l a c k of c h r o m o p h o r e s for o p t i c a l d e t e c t i o n is not at a l l a h i n d r a n c e . M C A c a n also u t i l i z e t u r b i d , r a w or p i g m e n t e d s a m p l e s , w h i c h are i m p o s s i b l e for s p e c t r o p h o t o m e t r y . M i c r o b i a l c a l o r i m e t r y was recently reviewed b y B a t t l e y (3). W h e r e a s earlier c a l o r i m e t e r s r e q u i r e d c a . 100 m l of m i c r o b i a l s u s p e n s i o n , heat c o n d u c t i o n c a l o r i m e t e r s u s i n g the Seebeck effect a n d P e l t i e r p u m p c o n t r o l require o n l y 0.2 t o 2 m l of s u b s t r a t e or a n a l y s i s s o l u t i o n a n d of cell s u s p e n s i o n . A 100200 m l overnight c u l t u r e p r o v i d e s enough cells for 10-20 measurements for MCA. M i c r o b i a l s t r i p p i n g adds m u c h to M C A ' s scope, a n d s i m p l i f i e s i t . S t r i p p i n g uses i n d u c e d b a c t e r i a for g e t t i n g r i d of i n t e r f e r i n g c o m p o u n d s . F i g u r e 1 o u t l i n e s direct c a l o r i m e t r y , a n d s t r i p p i n g , together w i t h average p a r a m eters for l i g n i n m o d e l c o m p o u n d s a n a l y s i s . S t r i p p i n g b y E. coli was first used i n c e l l u l o l y s i s , q u a n t i t a t i n g cellobiose vs. glucose (4). It was d e v e l o p e d f u r t h e r for p h e n o l i c m a t e r i a l s , u s i n g Pseudomonas and bacteria from s o i l isolates. M C A takes advantage of b a c t e r i a l a b i l i t y t o synthesize large a m o u n t s of enzymes necessary for c a r r y i n g c a r b o n sources t h r o u g h o x i d a t i v e m e t a b o l i s m , thereby i n d u c i n g the enzymes (5). T h e y are analogous to the classic e x a m p l e , the lac o p e r o n of E. coli. ( G r o w n o n glucose, E. coli have 0.5 t o 5 /?-galactosidase e n z y m e molecules per cell. B u t g r o w n o n lactose, E. coli c a n synthesize 1000 to 1,500 /?-galactosidase m o l e c u l e s / c e l l (6).) T h u s , M. irichosporium produces a l m o s t 1 7 % of i t s soluble p r o t e i n as a m e t h a n e m o n o h y d r o x y l a s e (7). A Pse udom on as synthesizes a b o u t 3 % of i t s p r o t e i n as p r o t o c a t e c h u a t e m o n o h y d r o x y l a s e , w h e n g r o w n o n p - h y d r o x y b e n z o a t e (8). M o s t of the p r o - o x i d a t i v e enzymes of b a c t e r i a are s t a b i l i z e d i n s i d e the c e l l , b u t are v e r y fragile outside the cell. T h e r e f o r e , the v i e w t h a t a n a l y s i s m a y be c a r r i e d out v i a i s o l a t e d enzymes for a r o m a t i c processing, p e r h a p s c o u p l e d t o a n electrode o f some k i n d , appears q u i t e i m p r a c t i c a l . M C A takes advantage of w h a t b a c t e r i a l cells can a c t u a l l y do, n a m e l y to s t a b i l i z e a n d p r o t e c t e n z y m e s , besides the i n i t i a l synthesis. Hence, M C A is l i k e l y to be far m o r e p r a c t i c a l t h a n any " b i o e l e c t r o d e " m e t h o d for a n a l y s i s . Bacterial Growth and Adaptation Pseudomonas putida A T C C 11172 was used for b o t h m i c r o b i a l c a l o r i m e t r y a n d for s t r i p p i n g p h e n o l a n d lower cresols. S e v e r a l isolates f r o m l o c a l soils, w h i c h were able to c o m b u s t larger a r o m a t i c s s u c h as c i n n a m i c a n d s y r i n g i c
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acids, were o b t a i n e d by a c i r c u l a t i n g e n r i c h m e n t a p p a r a t u s s i m i l a r t o t h a t described b y A u d u s (9). L i g n i n m o d e l c o m p o u n d s , 0 . 0 3 % , p l u s 0 . 0 1 % g l u cose were i n c u b a t e d w i t h ~ 100 g o f soil for 2 d a y s , w i t h a i r c i r c u l a t i o n at r o o m t e m p e r a t u r e . A f t e r 2 days, enricher i n o c u l a were transferred to plates w i t h 0 . 5 % yeast e x t r a c t a n d the c o m p o u n d . ( F o r c i n n a m i c a n d h y d r o x y c i n n a m i c a c i d c a r b o n sources, the yeast e x t r a c t was left o u t . ) P l a t e isolates were g r o w n at r o o m t e m p e r a t u r e for 12-48 h . C o l o n i e s f r o m plates were used t o i n o c u l a t e shake-flask l i q u i d cultures w i t h A s h w o r t h - R o m b e r g salts (10) c o n t a i n i n g 0 . 0 5 % b y weight o f a c a r b o n source a n d 0 . 0 3 % yeast e x t r a c t . L i q u i d c u l t u r e g r o w t h u s u a l l y o c c u r r e d below 0 . 0 5 % c o m p o u n d , b u t some w h a t larger concentrations tended t o k i l l even w e l l a d a p t e d Pseudomonas. A f t e r g r o w t h (at 30°) the o r g a n i s m s were d i l u t e d w i t h either m i n i m a l salts or i s o t o n i c saline a n d c e n t r i f u g a l l y washed t w i c e . C e l l c o n c e n t r a t i o n s were a d j u s t e d u s i n g the s p e c t r o p h o t o m e t r y t u r b i d i t y factor 2.0 x 1 0 x Α β β ο * c m " " p r e v i o u s l y described (1) to measure n u m b e r s of c e l l s / m l , ± 1 0 % for Pseudomonas. 9
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Stripping A d a p t e d cells able to b i n d u n w a n t e d , interfering c o m p o u n d s were washed t w i c e a n d adjusted i n c o n c e n t r a t i o n . A p p r o x i m a t e l y 1 m l of suspension c o n t a i n i n g 5 χ 1 0 cells was u s u a l l y sufficient to s t r i p 1 m l samples c o n t a i n i n g 50-100 nanomoles of i n t e r f e r i n g c o m p o u n d w i t h over 9 0 % r e m o v a l effectiveness. A f t e r m i x i n g the cells a n d samples b y v o r t e x i n g a n d i n c u b a t i n g for a m i n u t e , the s t r i p p e r cells were s p u n d o w n i n a m i c r o c e n t r i f u g e . A n a l i q u o t o f the s u p e r n a t a n t was t a k e n for c a l o r i m e t r y or other means o f a n a l y s i s , e.g., F o l i n a n a l y s i s . 1 0
Spectrophotometric Analysis; Folin Phenol P h e n o l a n d p h e n o l i c derivatives, cresol a n d related c o m p o u n d s were a n a l y z e d by F o l i n - C i o c a l t e a u reagent at 700 m / i , s t a n d a r d i z i n g each c o m p o u n d separately b y i t s F o l i n response. T h e i r slopes ( m o l a r a b s o r p t i o n coefficients) were 8000-10,000 M cm . Interestingly, m e t h o x y l a r o m a t i c s w i t h no free h y d r o x y l groups were not reactive. _
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Heat Conduction Calorimetry, Batch M i x i n g F i g u r e 2 i l l u s t r a t e s the arrangement o f a t h r e e - c h a n n e l b a t c h m i x i n g c a l o r i m e t e r u s i n g L i p s e t t - J o h n s o n - M a a s vessels (11) for a 1 m l a n a l y s i s s a m p l e , 2 m l m i c r o b i a l suspension, a n d 3 m l headspace. T h e f o u r t h vessel i n F i g u r e 2, together w i t h i t s Seebeck thermosensors, is a reference m o d u l e . U s u a l l y i t is loaded w i t h 2 m l m i c r o b i a l suspension a n d 1 m l of solvent ( m i n i m a l s a l t s ) . T h e reference m o d u l e voltage opposes a l l three s a m p l e m o d u l e s so t h a t each measurement is a difference or net power measurement. I n effect, the heat of m i x i n g the s y s t e m , or the heat of r e s i d u a l m i c r o b i a l ac t i v i t y w i t h o u t c a r b o n , is s u b t r a c t e d f r o m the samples. T h e voltage signals o f heat c o n d u c t i o n calorimeters a c t u a l l y measure power. P o w e r integrated over t i m e equals heat, w h i c h is d i r e c t l y p r o p o r t i o n a l to the area m e a s u r e d
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Direct Calorimetry
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Adapted cells 1-2 mg. able to metabolize the carbon source or analvte Adapted cells, 2-4 mg., able to ^ bind interfering compound.
Mix with 1-2 ml. of ~" sample, aerobic
Measure heat proportional to the amount carbon, 2-100 nanomoles, 1-100 meal, 5-10 min.
Mix with 1-3 ml sample, vortex, centrifuge, retain supernate
Stripped supernate, 1-2 ml. for calorimetry
F i g u r e 1. M e t h o d s o f M C A : D i r e c t c a l o r i m e t r y b y m i c r o o r g a n i s m m e t a b o l i s m o f samples f o r a n a l y s i s ; s t r i p p i n g b y m i c r o o r g a n i s m b i n d i n g o f i n t e r fering compounds.
F i g u r e 2. H e a t c o n d u c t i o n (Seebeck effect) b a t c h m i x i n g c a l o r i m e t e r for three samples a n d one reference c h a n n e l . A f t e r l o a d i n g a n d e s t a b l i s h i n g baselines, t h e assembly is i n v e r t e d t o m i x react ants a n d s t a r t heat p r o d u c t i o n . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 2. © 1983, A l a n R . L i s s , Inc.)
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u n d e r each t h e r m o g r a m as s h o w n i n F i g u r e 3. Headspaces i n each vessel c o n t a i n 3-6 m l o f a i r , t h a t i s , w i t h a p p r o x i m a t e l y 50-100 f o l d excess o x y g e n for aerobic m e t a b o l i s m o f 5-100 nanomoles of most substrates u p t o C12 sizes. M o r e d e t a i l e d p a r a m e t e r s , response t i m e s , noise g e n e r a t i o n , figure o f m e r i t , etc., have been p u b l i s h e d (2) a n d i n c o r p o r a t e d i n B R I C m a n u f a c t u r e d i n s t r u m e n t s . T h e u n i t s h o w n i n F i g u r e 2 is inside a m e t a l s h e l l , s u r r o u n d e d b y three m e t a l boxes. T h e outer two boxes are P e l t i e r p u m p e d , for p r o t e c t i n g against t h e r m a l fluctuations f r o m outside. T h e s y s t e m c a n be o p e r a t e d at a n y chosen t e m p e r a t u r e between 10-50°. T h e m i c r o b i a l c a l o r i m e t r i c analyses were c a r r i e d out at 25°. H e a t c o n d u c t i o n c a l o r i m e ters derive t h e i r signals f r o m heat flow, generated by the samples after m i x i n g . T h e y do not rely o n measurement of s m a l l t e m p e r a t u r e changes. T h e heat c o n d u c t i o n p r i n c i p l e (12) is far easier a n d more sensitive, also less expensive, t h a n n e a r l y a n y means i n v o l v i n g t h e r m i s t o r s or t h e r m o m e t r y . C a l i b r a t i o n is c a r r i e d out i n two ways: electric resistance h e a t i n g w i t h a s m a l l probe inserted i n each vessel; a n d " w e t " c a l i b r a t i o n u s i n g T r i s - H C l n e u t r a l i z a t i o n (2). Microbial Calorimetric Analysis Pseudomonas putida A T C C 11172
(MCA)
of C o m p o u n d s
Using
Pseudomonas g r o w n o n p h e n o l a n d various cresols c o m b u s t s t h e m at 25° i n 300-600 sec i f there are excess cells, ~ 1-2 m g d r y weight, a n d l i m i t e d carb o n , ~ 5-30 nanomoles. F i g u r e 3 shows t h a t w h e n Pseudomonas is a d a p t e d t o o-cresol, i t combusts o-cresol a n d p h e n o l t o c o m p l e t i o n i n a b o u t 6 m i n utes. G r o w n o n v a n i l l i c a c i d , Pseudomonas metabolizes l i m i t e d a m o u n t s of v a n i l l i c a c i d , albeit s o m e w h a t more s l o w l y t h a n i n the o-cresol or p h e n o l u t i l i z i n g s y s t e m . Nevertheless, t h i s occurs i n far shorter times t h a n c a n p o s s i b l y be observed b y a g r o w t h response. T e n to twenty n a n o m o l e s of v a n i l l i c a c i d m e t a b o l i z e c a . 3 0 - 5 0 % as r a p i d l y as o-cresol or p h e n o l . G r o w t h o n glucose allows the b a c t e r i a to combust glucose. B u t g r o w t h o n glucose does not equip t h e m to h a n d l e a n y of the l i g n i n m o d e l c o m p o u n d s as i n d i c a t e d b y the glucose g r o w n cells m i x e d w i t h o-cresol (lower trace o f F i g u r e 3). S u c h t h e r m o g r a m s , besides m e a s u r i n g the n a t u r e of v a r ious o r g a n i s m s ' responses to c a r b o n sources, also measure m e t a b o l i c rates. I n the case of glucose a n d glucose-grown cells, when c o m p a r e d w i t h d a t a f r o m B e r k a et al. w h o used P. cepacia a n d a s p e c t r o p h o t o m e t r i c m e t h o d to follow the disappearance o f sugar (13), i t was f o u n d t h a t our rates of u t i l i z a t i o n agreed w i t h i n a factor o f less t h a n two. S u c h rates f r o m c a l o r i m e t r y are s i m p l y a d i v i s i o n of the m o l a r heat o f aerobic glucose m e t a b o l i s m , 300 K c a l / m o l e glucose (1), by the power averaged over the t i m e i n t e r v a l for c o m b u s t i o n a n d the n u m b e r of moles of glucose c o n s u m e d . T h e quotient is a c o m b i n e d u p t a k e a n d m e t a b o l i c rate. S t a n d a r d i z a t i o n plots for M C A have the same n a t u r e as such p l o t s for other a n a l y t i c m e t h o d s . T h e y are plots of " r e a d o u t " (in t h i s case, heat) vs. c o n c e n t r a t i o n of s t a n d a r d . F i g u r e 4 shows a s t a n d a r d i z a t i o n p l o t for M C A response to o-cresol, c o m p a r e d w i t h a p l o t for F o l i n spect r o p h o t o m e t r y w h i c h is also for o-cresol. T h e question t h a t then arises is
F i g u r e 3. M i c r o b i a l c a l o r i m e t r y of 10-40 nanomoles of c o m p o u n d s u s i n g P. puiida a d a p t e d (upper four traces), a n d not a d a p t e d (lower trace) to the c o m p o u n d s , 2 5 ° C , m i n i m a l salts solvent. R M = r e m i x i n g i n m i d - r u n to ensure aerobicity. O r d i n a t e is p r o p o r t i o n a l to power. I n t e g r a t e d areas are p r o p o r t i o n a l to o v e r a l l heat of c o n s u m i n g a l l m e t a b o l i z a b l e c a r b o n .
0.22
50|
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OS
F i g u r e 4. C o m p a r i s o n o f M C A a n d F o l i n s p e c t r o p h o t o m e t r y for a n a l y s i s o f o-cresol s p a n n i n g t h e lower p r a c t i c a l ranges o f i n s t r u m e n t a l r e a d o u t .
Ε υ 0.18 h-
ο ο
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the s e n s i t i v i t y o f M C A c o m p a r e d w i t h other m e t h o d s i n a n a l y s i s o f one l i g n i n - r e l a t e d c o m p o u n d . S u c h a c o m p a r i s o n depends l a r g e l y o n the lower h e a t s s u i t a b l e for Seebeck c a l o r i m e t r y , a n d the lower absorbancies s u i t a b l e for s p e c t r o p h o t o m e t r y at o p t i m u m w a v e l e n g t h n o r m a l l y u s i n g a 1 c m c u v e t t e . A b s o r b a n c e s of c a . 0.10-0.30 i n s p e c t r o p h o t o m e t r y a n d heats o f 3-40 m i l l i c a l i n c a l o r i m e t r y were t a k e n as reasonable c r i t e r i a for F i g u r e 4. I m p o r t a n t p a r a m e t e r s w h i c h c o n t r o l s u c h a c o m p a r i s o n are the m o l a r a b s o r p t i o n coefficient for o - c r e s o l - F o l i n color, £700 = 8 1 4 0 M ~ c m ~ , a n d the m o l a r heat o f aerobic m e t a b o l i s m for o-cresol, 520 K c a l / m o l e o-cresol u s i n g aerobic Pseudomonas. T h e p a r a m e t e r s are the slopes o f the p l o t s o f F i g u r e 4. C l e a r l y the c r i t e r i a for two c o m p l e t e l y different m e t h o d s c a n be d r a w n closer together or f u r t h e r a p a r t , d e p e n d i n g o n w h a t i n s t r u m e n t a l " r e a d o u t s " are t a k e n as reasonable lower ranges. However, even w h e n a r a t h e r intensely colored s a m p l e for a n a l y s i s is c o m p a r e d , as F o l i n - c r e s o l conjugates are, M C A is at least as sensitive as F o l i n s p e c t r o p h o t o m e t r y i n the present case. T h e c o m p a r i s o n accrues because o f the large heat o f aerobic m e t a b o l i s m , a n d use o f excess cells, w e l l a d a p t e d , a n d l i m i t e d for c a r b o n ( l i m i t e d a n a l y t e ) . I f real samples are v e r y t u r b i d , or l a d e n w i t h F o l i n - r e a c t i v e p i g m e n t s as m a n y l i g n i n c o m p o u n d s are, m i c r o b i a l c a l o r i m e t r y has a f u r t h e r a d v a n t a g e . It is far less sensitive t o s u c h interference t h a n spectrophotometry. 1
1
S t r i p p i n g Efficiency a n d S t r i p p i n g Specificity A s w i t h a n y other a n a l y t i c a l m e t h o d , M C A ' s c a p a c i t y is enhanced i f i t is easy t o remove i n t e r f e r i n g c o m p o u n d s . M i c r o b i a l a d a p t a t i o n confers s p e c i f i c i t y i n b i n d i n g u n w a n t e d c o m p o u n d s so they can be swept o u t w i t h o u t l o s i n g the s a m p l e for a n a l y s i s . F i g u r e s 5 a a n d 5b show the d a t a for p h e n o l a n d v a n i l l i c a c i d s t r i p p i n g u n d e r convenient c o n d i t i o n s , n a m e l y , w i t h easily a c q u i r e d a m o u n t s o f s t r i p p e r cells (5 x 1 0 cells), a n d c o n d i t i o n s where i n terfering c o m p o u n d c o n c e n t r a t i o n s are r e l a t i v e l y large, i.e., u p to 10 t i m e s the a m o u n t o f s a m p l e for a n a l y s i s . 1 0
P h e n o l - g r o w n cells n e a r l y q u a n t i t a t i v e l y b i n d a n d c a r r y d o w n p h e n o l a n d some of the cresols i n c o n c e n t r a t i o n s ~ 8 x 1 0 ~ m g / m l (ca. 6 x 10"" M). P h e n o l a n d ο-, ρ - , a n d m-cresol are s t r i p p e d as s h o w n i n F i g u r e 5 a . I n c o n t r a s t , l i t t l e v a n i l l i c a c i d is removed b y p h e n o l - g r o w n P. putida, i.e., v a n i l l a t e r e m a i n s i n the s u p e r n a t a n t , whereas p h e n o l a n d the cresols are b o u n d a n d s p u n d o w n w i t h the s t r i p p e r cells. 3
5
W h e n the same P. putidaare grown on vanillic acid, Figure 5b, vanil late efficiently b i n d s a n d is s t r i p p e d , whereas v e r y l i t t l e p h e n o l is removed (see F i g u r e 5a). I n d e e d , F i g u r e s 5 a a n d 5b i l l u s t r a t e the efficiency a n d s p e c i f i c i t y w i t h w h i c h P. putida A T C C 11172 b i n d s phenols, a n d as c a n be seen is s h a r p l y dependent o n the c a r b o n source used for g r o w t h . W i t h i n cogeners, p h e n o l i t s e l f a n d o-, m - , a n d p-cresol, there is n o t m u c h d i s c r i m i n a t i o n i n the a b i l i t y o f p h e n o l - g r o w n cells t o s t r i p t h e m b e l o w levels o f c a . 8 x 1 0 ~ m g / m l . A b o v e t h i s c o n c e n t r a t i o n , differences between the i n d i v i d u a l c o m p o u n d s are seen b u t even these d i s a p p e a r w h e n cell c o n c e n t r a t i o n s are m a d e even larger. F o r 50 x 1 0 p h e n o l - g r o w n P. putida/ml, up to 3
1 0
3
VANILLIC ACID (mg/ml χ 10 )
1 0
F i g u r e 5. S t r i p p i n g p h e n o l , cresols a n d v a n i l l i c a c i d , dependent o n Pseu domonas a d a p t a t i o n t o c o m p o u n d s t o be s t r i p p e d ; 5 x 1 0 c e l l s / m l d u r i n g s t r i p p i n g . A r r o w i n F i g u r e 5 a : C o n c e n t r a t i o n o f c o m p o u n d s below w h i c h s t r i p p i n g is q u a n t i t a t i v e (see t e x t ) .
3
PHENOL (mg/ml χ 10 )
39.
553
Microbial Calorimetric Analysis
LOVRIEN ET A L
30 χ 1 0 ~ m g / m l of these c o m p o u n d s a l l b i n d a n d therefore get s t r i p p e d . H o w e v e r , for p r a c t i c a l purposes, one prefers not to have to use s u c h a large n u m b e r of cells a n d o r d i n a r i l y there is no need to. A t least for t h i s o r g a n ism, usually 5 χ 1 0 c e l l s / m l are enough to s t r i p c o m p o u n d s o f t h i s k i n d i n the 1 0 ~ t o 1 0 " M range. 3
1 0
6
5
G l u c o s e - g r o w n P . putida s t r i p glucose, b u t g r o w n o n the cresols they do not b i n d glucose efficiently i n m i c r o m o l a r sugar c o n c e n t r a t i o n s n o r m e t a b o l i z e i t r a p i d l y . F r o m results o f diverse e x p e r i m e n t s , we favor E. coli as s t r i p p e r s a n d m e t a b o l i z e r s o f c a r b o h y d r a t e s i n M C A (4). H o w e v e r , as m i g h t have been expected f r o m S t a n i e r , P a l l e r o n i a n d D o u d o r o f f ' s research (14), Pseudomonas provides the best prospects for l i g n i n m o d e l c o m p o u n d a n d p e t r o l e u m c o m p o u n d b i n d i n g a n d m e t a b o l i s m . A n arrow i n F i g u r e 5 a i n d i c a t e s a n abscissal v a l u e , below w h i c h essentially 1 0 0 % b i n d i n g a n d s t r i p p i n g occurs. C a l c u l a t i o n s analogous t o those c a r r i e d o u t earlier (15) leads to the e s t i m a t i o n t h a t , given such a n a m o u n t of p h e n o l a n d s u c h a n u m b e r o f cells (8 x 1 0 ~ m g / m l , 5 χ 1 0 c e l l s / m l ) , o n l y between 0.5 a n d 1.0% of m e m b r a n e l i p i d s are r e q u i r e d to a c c o m m o d a t e s u c h a n a m o u n t of p h e n o l . T h a t e s t i m a t i o n does not i n d i c a t e the m e c h a n i s m o f b i n d i n g . B u t i t does i n d i c a t e t h a t once the cells are a d a p t e d , t h e y are a d e q u a t e l y large "sponges" for such a n a m o u n t of c o m p o u n d . 3
1 0
S t r i p p i n g is s i m p l e , r a p i d , a n d g r e a t l y e x p a n d s M C A for these k i n d s o f c o m p o u n d s . M i c r o b i a l s t r i p p i n g of t h i s k i n d l i k e l y w o u l d be h e l p f u l i n a n a l y t i c a l m e t h o d s other t h a n c a l o r i m e t r y b u t o d d l y t h i s has not been developed. T a b l e I s u m m a r i z e s the m a i n p o i n t s f r o m n u m b e r s of p l o t s s i m i l a r to F i g u r e s 5 a a n d 5 b . T h e c o n d i t i o n s were: 5 x 1 0 c e l l s / m l , 6 to 8 x 1 0 ~ m g c o m p o u n d to be s t r i p p e d / m l , 30 second m i x i n g . G l u c o s e was s t r i p p e d f r o m 15 x 1 0 ~ m g g l u c o s e / m l . 1 0
3
3
T a b l e I. Efficiency of S t r i p p i n g (Percent R e m o v e d ) C o m p o u n d s D e p e n d e n t o n the C a r b o n Source for G r o w t h , P. putida A T C C 11172 C a r b o n Source o n w h i c h G r o w n ( a d a p t e d ) C o m p o u n d to be S t r i p p e d
o-Cresol
m-Cresol
p-Cresol
Vanillic Acid
Phenol
Glc*
97 100 100 0 100 0
100 100 100 1 100
93 100 100 4 99
73 87 92 84 5
100 100 100 0 100
6
o-Cresol m-Cresol p-Cresol Vanillic Acid Phenol Glucose *Glc =
Glucose.
7 56
554
PLANT C E L L W A L L P O L Y M E R S
Calorimetric Combustion of o-Cresol and Vanillic A c i d Mixtures i n 5-100 N a n o m o l e s R a n g e P. putida A T C C 11172 g r o w n o n o-cresol efficiently s t r i p s a n d also c o m b u s t s o-cresol. F i g u r e 6 shows M C A a n d s t r i p p i n g performance for o-cresol (lower abscissa) w h e n considerable v a n i l l i c a c i d is present ( u p p e r abscissa). T h e d a t a i l l u s t r a t e s h o w a d a p t a t i o n confers specificity. F i r s t , u s i n g o-cresol g r o w n cells for c o m b u s t i n g a n e q u i m o l a r m i x t u r e o f o-cresol a n d v a n i l l i c a c i d y i e l d s v i r t u a l l y the same p l o t as o-cresol alone, d o w n to 8 nanomoles o-cresol. Secondly, after o-cresol s t r i p p i n g , n e a r l y a l l the o-cresol is removed a n d v a n i l l i c a c i d interferes m i n i m a l l y (lower p l o t o f F i g u r e 6). T h i r d l y , a d a p t a t i o n to o-cresol enables the cells to combust a n d t o s t r i p o-cresol. B u t i t disables t h e m f r o m u s i n g v a n i l l i c a c i d at a n y finite rate o n M C A ' s t i m e scale. However, v a n i l l i c a c i d - g r o w n P. putida is f u l l y capable of s t r i p p i n g a n d c o m b u s t i n g v a n i l l a t e i n a few m i n u t e s . I n s h o r t , a d a p t a t i o n confers reasonable specificity d o w n to m i c r o m o l a r concentrations o f o-cresol p l u s the a b i l i t y to s t r i p o-cresol. Lignin Model
Compounds
I n order t o o b t a i n r a p i d m e t a b o l i s m a n d r a p i d heat generation f r o m a r o m a t i c c o m p o u n d s related to l i g n i n , several s o i l isolates were used. P. putida A T C C 11172 d i d not grow easily o n c o m p o u n d s such as s y r i n g i c a c i d a n d c e r t a i n other c o m p o u n d s m o r e l i g n i n - l i k e . However, soils collected l o c a l l y y i e l d e d b a c t e r i a , first f r o m e n r i c h m e n t cultures a n d t h e n g r o w n o n plates or defined l i q u i d c u l t u r e , were able t o c o m b u s t the c o m p o u n d s l i s t e d i n T a b l e II. T h e T a b l e r e p o r t s concentrations o f the c a r b o n source used i n M C A , the t i m e intervals needed for c o m p l e t e l y m e t a b o l i z i n g 10-50 nanomoles of c o m p o u n d , a n d the a p p a r e n t m o l a r heats o f m e t a b o l i s m i n low c a r b o n c o n c e n t r a t i o n s ( Δ ° Η ) . C i n n a m i c a c i d a n d 3 , 4 - d i m e t h o x y c i n n a m a t e evolved r a t h e r e x t r a o r d i n a r y heats, over 800 K c a l / m o l e . S u c h heats were not ex pected i n t h a t 3 , 4 - d i m e t h o x y c i n n a m a t e is a l r e a d y p a r t l y o x i d i z e d . M i c r o b i a l c o m b u s t i o n o f n a p h t h a l e n e gave 810 K c a l / m o l e , 2 - m e t h y l n a p h t h a l e n e gave 652 K c a l / m o l e , u s i n g P. putida i n earlier w o r k (1). P l a i n l y the r e l ative state o f o x i d a t i o n or r e d u c t i o n o f c a r b o n sources does not p r e d i c t h o w m u c h heat c o m p o u n d s generate i n m i c r o b i a l c o m b u s t i o n , i n c o n t r a s t t o c o n v e n t i o n a l oxygen b o m b c a l o r i m e t r i c c o m b u s t i o n . R a t h e r , there is a dependence o n h o w w e l l , or h o w p o o r l y , catabolites fit e x i s t i n g m e t a b o l i c p a t h w a y s of o r g a n i s m s (16). If there is p o o r fitting, m o r e c a r b o n has to be used i n e x o t h e r m i c p a t h w a y s e m i t t i n g C O 2 to generate needed i n t e r mediates such as N A D H r e q u i r e d t o process w h a t e v e r fragments t h a t c a n be r e t a i n e d . O n c e cells (isolates) are a d a p t e d to these c o m p o u n d s they c a n d r i v e m e t a b o l i s m t h r o u g h i n 10-20 m i n u t e s , i f c a r b o n is l i m i t e d a n d there are excess cells a n d oxygen. T h u s , M C A does not need to complete a g r o w t h cycle as i n g r o w t h assays, b u t s i m p l y takes u p c a r b o n a n d gets t h r o u g h the l i n e a r , early c a t a b o l i c stages.
40
50
60
1
NANOMOLES O-CRESOL/ml
30
1
70
1
80
1
72
F i g u r e 6. C a l i b r a t i o n o f heat generation f r o m o-cresol b y Pseudomonas m e t a b o l i s m as a s t a n d a r d p l o t for a n a l y s i s o f o-cresol alone a n d i n ocresol, v a n i l l i c a c i d m i x t u r e s , dependent o n Pseudomonas adaptation i n both calorimetry and stripping.
20
1
10
36 1
1
Ί
NANOMOLES VANILLIC ACID/ml 18
9
90
1
81
556
PLANT C E L L W A L L P O L Y M E R S
Table II. L i g n i n M o d e l C o m p o u n d Calorimetry w i t h Soil Cultures
Compound p-Hydroxyphenyl acetate Syringic acid C i n n a m i c acid Hydrocinnamic acid 3,4-Dimethoxycinnamic acid
Sample Concentration (micromolar)
T i m e for C o m p l e t e C o m b u s t i o n of 5-50 Nanomoles (min)
M o l a r H e a t of Aerobic Metabolism, K c a l
6-40 21-42 2-30
16 18 10
805 470 830
12-30
15
710
9-36
20
850
Conclusions M C A of o x i d i z e d l i g n i n fragments (such as the h y d r o x y c i n n a m i c acids i n T a b l e II) appears to be as sensitive as s p e c t r o p h o t o m e t r i c analyses f r o m results seen so far, even c o m p a r i n g c o m p o u n d s t h a t are b r i g h t l y colored or w h i c h c a n be m a d e so (chromogenic, prochromogenic c o m p o u n d s ) . M C A ' s specificities are d e t e r m i n e d b y those c o m p o u n d s w h i c h b a c t e r i a can u p t a k e a n d m e t a b o l i z e . A d a p t e d b a c t e r i a are quite selective i n t h i s r e g a r d . A l t h o u g h M C A requires a n excess of cells to drive a n a l y t i c a l reactions r a p i d l y , the r e q u i r e d a m o u n t s u s u a l l y are easily o b t a i n e d once the i n o c u l a are a v a i l able. A b o u t 1-5 m g of cells are needed for each c o m b u s t i o n , a n d 2-10 m g for m o s t s t r i p p i n g o p e r a t i o n s . Therefore, 50-300 m l of overnight c u l t u r e is sufficient to p r o d u c e cells for 10-30 combustions or s t r i p p i n g s . C u r r e n t l y we are d e v e l o p i n g means for freezing cells so they m a y be stored. Because M C A ranges d o w n to 5-50 μΜ analytes a n d a i r s o l u b i l i t i e s are ~ 1 . 3 m M (dissolved O 2 , 0.3 m M (1)), there is plenty of oxygen a v a i l a b l e at 25° t o ensure aerobicity. Nevertheless, we use one or two r e m i x i n g s i n m i d - r u n ( F i g . 3) t o ensure a e r o b i c i t y i f s a m p l e concentrations are above c a . 50 μ Μ . O x y g e n r e s p i r o m e t r y has sometimes been used as a basis for a n a l y s i s . I n i t i a l l y one m i g h t t h i n k the two m e t h o d s , o x y g e n r e s p i r o m e t r y a n d m i c r o b i a l c a l o r i m e t r y , are equivalent. However, there are very large differences between t h e m i n practice w h e n samples are m i c r o m o l a r i n c o n c e n t r a t i o n . T o a n a l y z e a few m i c r o m o l a r concentrations v i a disappearance of o x y g e n , the oxygen baseline concentrations have to be held i n very n a r r o w toler ances, i.e., i n 0.1 t o 5 or 10 μ Μ d u r i n g a n a l y s i s . H o l d i n g t h a t against a b a c k g r o u n d w h i c h (i) is sufficient to m a i n t a i n f u l l aerobicity, a n d (ii) l i k e l y to fluctuate for various reasons, is not easy i n p r a c t i c e . M C A operates o n very different grounds. A n y r o u g h l y fluctuating oxygen c o n c e n t r a t i o n w i l l do, as l o n g as there is a n oxygen s u r p l u s . M C A is c a r b o n - l i m i t e d , not oxygen difference-limited. T h e s t r e n g t h of M C A is t h a t i t uses a r o u g h excess of cells, a r o u g h excess of oxygen, a n d produces a s i g n a l d i r e c t l y
39.
LOVRIEN ET AL.
Microbial Calorimetric Analysis
557
proportional to "carbon" (the sample). It is not dependent on detection of small changes in oxygen concentration against fluctuating, large, oxygen concentrations. Lignin itself usually requires days, months, or years to biodegrade. However, oxidized lignin fragments can biodegrade rapidly if adapted bac teria are available. Over the years, dozens of microbial calorimetric studies of carbon utilization as a correlate of microbial growth have been carried out by Dermoun et al. (17). Many such papers leave the impression that heat generation commonly requires 8-24 hours to peak. In fact, however, any organism having a doubling time from 20-60 minutes must transport and metabolize carbon in 2-20 minutes. Therefore, oxidized fragments of lignin, and perhaps other lignin degradation products, whatever their source, can mostly be expected to "burn" rapidly, if cells are adapted and there are enough of them to bind all available carbon. A disadvantage of M C A is that it is new and not familiar. However, M C A takes advantage of several ancient processes and is congruent with them, namely bacterial processing in soil, silage, sewage, and digestion. M C A simply measures their heats using organisms that propel these pro cesses on a large scale. A cknowledgment This work was supported by the University of Minnesota Agricultural Ex periment Station and by the University Bioprocess Technology Institute. Literature Cited 1. Lovrien, R.; Jorgenson, G.; Ma, M.; Sund, W. Biotech. Bioeng. 1980, 22, 1249-69. 2. Hammerstedt, R. H.; Lovrien, R. E . J. Exp. Zool. 1983, 228, 459-69. 3. Battley, Ε. H. Energetics of Microbial Growth; Wiley-Interscience: New York, 1987; 322-51. 4. Lovrien, R.; Williams, Κ. K.; Ferry, M . L.; Ammend, D. A. Appl. En viron. Microbiol. 1987, 53, 2935-41. 5. Parke, D. V. Enzyme Induction; Plenum Press: New York, 1975; Ch. 1-3. 6. Zubay, G . Biochemistry; Addison-Wesley Publ: Reading, M A , 1983; Ch. 26. 7. Fox, B. G.; Lipscomb, J. D. Biochem. Biophys. Res. Comm. 1988, 154, 165-70. 8. Fujisawa, H.; Hayaishi, O. J. Biol. Chem. 1968, 243, 2673-81. 9. Audus, L. J . Nature 1946, 158, 149-50. 10. Ashworth, J . M.; Kornberg, H. L. Proc. Roy. Soc. London Sec. Β 1966, 165, 179-88. 11. Lipsett, S. G.; Johnson, F. M . G.; Maas, O. J. Am. Chem. Soc. 1927, 49, 925-43. 12. Barisas, B. G.; Gill, S. J . Ann. Rev. Phys. Chem. 1978, 29, 141-46. 13. Berka. T . R.; Allenza, P.; Lessie, T. G . Curr. Microbiol. 1984, 11, 143-48.
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14. Stanier, R. Y.; Palleroni, N. J.; Doudoroff, M. J. Gen. Microbiol. 1966, 43, 159-79. 15. Lovrien, R.; Hart, G.; Anderson, K. J. Microbios 1977, 20, 153-72. 16. Anderson, J. J.; Dagley, S. J. Bacteriol. 1980, 143, 525-28. 17. Dermoun, Z.; Boussand, R.; Cotten, D.; Belaich, J. P. Biotech. Bioeng. 1985, 996-1004. RECEIVED April 28, 1989
