Dependence of the element composition of K ... - Wiley Online Library

COMMUNICATIONS TO THE EDITOR Dependence of the Elemental Composition of K. pneumoniae on the Steady-State Specific Growth Rate INTRODUCTION Recently. elemental balances and the application of other macroscopic methods have received much attention in the Macroscopic methods proved to be invaluable tools in all aspects of fermentation and nastewater fields. e.g.. in modeling. process design and development. control. data reduction and correlation, and the like. A prerequisite for the application of these powerful tools is the availability of data on the elemental composition of microorganisms as well as those of limiting substrate and products formed. Of these. usually the most tedious and expensive analysis is the elemental analysis of the dried biomass. Carbon. hydrogen, nitrogen. and oxygen atoms are the major constituents of microorganisms. These four elements constitute about 95% or more of the ash-free bacterial dry weight. Thus consideration of only these four elements is usually sufficient for practical applications. In this article, we present elemental composition data for the bacterium Klebsiella pneumoniue (aerogenes) collected at different steady-state growth rates in a chemostat. Since this organism is a well-known and frequently used bacterium. it is believed that these data might also be useful for other researchers who do not have means for accurate elemental measurements. Moreover, when treated by regression analysis. the data allow the derivation of approximate formulae, relating the elemental composition. and thus the degree of reduction and the molecular ueight. to the steady-state specific growth rate.

MATERIALS AND METHODS Organism Klebsiella pneumoniae (aerogenes) NCTC 418 was used throughout this work. Cultivation Methods Growth medium was prepared according to the formulation given by Evans, Herbert. and Tempest.6 Glycerol was used as the limiting substrate and assayed enzymatically. Feed substrate concentration was 10 kg/m3. The medium was sterilized by membrane filtration through a 0 . 2 - ~ mpore diameter membrane filter under pressure (Sartorious 11307). Equipment The fermentor was a standard 3-L working volume glass vessel (New Brunswick). The culture was maintained at a temperature of 3S°C and pH of 6.8. Airflow rate to the fermentor was controlled at about 60 m3/m3 h. Steady states were verified by simultaneous analysis of oxygen uptake rate, carbon dioxide production rate, dry weight concentration, and acidlalkali addition rate data. Other details of the experimental system were as reported earlier.’.8 Elemental Analvsis While being taken, all samples were cooled down to ca. 5-8°C by an on-line heat exchanger designed and manufactured in the workshop of this department. Typical residence time in the

Biotechnology and Bioengineering. Vol. XXIV. Pp. 1445-1449 (1982) @ 1982John Wiley & Sons. Inc. CCC 0006-3592/82/061445-05~01.SO

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIV (1982)

heat exchanger was ca. 5-10 s. Fifty-milliliter samples were taken and centrifuged immediately at 11,OOO rpm and at 4°C. Samples were then twice washed with ice-cold distilled water and centrifuged. The cell pellet was then transferred to a special marble dish and dried at 105'C for 18-24 h. Then marble grinding balls were put into the dish and the dish was placed in a pulverizing machine (Fritsch, Pulvarisette). In this manner the biomass was pulverized until a very fine homogeneous, cream-colored powder was obtained. The powder was then placed in a desiccator and dried further, for at least one week. Three elements (C, H, N) were determined in a computer-coupled element analyzer (Perkin Elmer). Ash content was determined separately. The element analyzer only needed a few milligrams of the dried biomass; therefore thorough homogenization of biomass was very important. Ash content in every case was found to be ca. 8% of the dry weight. Oxygen was found from the difference. All figures reported in this work are on ash-free basis. The consistency of our elemental analyses was checked and found to be good by comparing the results obtained for the same samples by an independent commercial laboratory.

RESULTS In Table I the ash-free composition of the biomaterial is presented as a function of the specific growth rate in the range of p = 0.035-0.71 hK'. Statistical analysis of data for each element versus the specific growth rate indicates that with the exception of 0%(oxygen) data, all elemental fractions show significant changes in this growth rate range, These data are plotted in Figure 1. As can be seen, data for C. H , and 0 can be represented fairly well by straight lines. N data appear to be best represented by an S-shaped curve but for simplicity they are also fitted by a straight line. The maximum error brought about by fitting a straight line instead of a curve for the N% versus specific growth rate data is calculated to be 7% in N%. Based on these regression approximations, the cell formola can now be evaluated as a function of the steady-state specific growth rate. That is, the elemental formula can be given as I HbNcod

where:

b = (7.282

c = (12.237

-

0.551fi)/z

(2)

+ 3.718p)/(1&)

(3)

d = 26.812/(16z) z

(4)

= (53.304 - 4.100fi)/12

(5)

TABLE I Elemental Composition at Various Specific Growth Rates (wt. 70 Ash-Free Basis)

0.035 0.067 0.16 0.32 0.41 0.54 0.63 0.71

53.22 52.91 52.81 52.01 51.65 50.65 50.84 50.57

7.28 7.23 7.23 7.03 7.10 6.97 6.94 6.91

12.85 12.54 12.46 12.66 13.72 14.94 15.02 14.38

25.51 26.68 26.86 27.65 29.90 26.80 26.56 27.54

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COMMUNICATIONS TO T H E EDITOR

24 molecular weight

23

23

-

2

p

Fig. 1 .

I

I

t

f

I

01

02

03

04

05

(IT-?) I

06

07

01

Elemental composition as a function of the steady-state growth rate.

Since the molecular weight is defined as mol. wt. = l(12)

+ b(1) + ~ ( 1 4 +) d(16)

(6)

it can now be evaluated in term of p by substituting eqs. (2)-(5) in eq. (6). Thus the molecular weight can be shown to be given as a function of p :

mol. wt. =

22.452 1

-

-

0.209~

0.077~

(7)

Another important parameter in bioenergetic calculations. the degree of reduction of the biomass, can also be evaluated as a function of the specific growth rate in a similar manner. Note. however, that here the degree of reduction, y,. is defined for growth with ammonia as the N source, which was the case in our experiments. For N sources other than ammonia the generalized degree of reduction concept, developed by Roels.' has to be used. The degree of reduction for growth with ammonia is given by yx = 4

+b

3c - 2d

(8)

4.290 - 0 . 6 1 1 ~ 1 - 0.077~

(9)

-

yx can now be approximated by

yx =

Equations ( 7 ) and (8) are shown graphically in Figures 2 and 3 . As can be seen. within the experimental range the changes in the molecular weight and the degree of reduction are ca. 5 and 6%. respectively. Therefore, these parameters can be assumed as constants and average values may be used in practical applications when extreme accuracy is not crucial.

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIV (1982) '10 O x y g e n 0

0

=---

.

$ 14 5

1 42

-72

-71

-70

-

p

t

0

I

1

01

02

03

- 69 (hr-ll) I

04

05

0.6

07

08

J

Fig. 2. Molecular weight as a function of the steady-state specific growth rate, drawn according to eq. (7).

Fig. 3. Degree of reduction as a function of the steady-state specific growth rate, drawn according to eq. (8).

COMMUNICATIONS TO THE EDITOR

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References 1. J. A. Roels, Biotechnof. Bioeng., 22, 2457 (1980). 2. J. J. Heijnen and J. A. Roels, Biotechnol. Bioeng.. 23, 239 (1981) 3. J. A. Roels, and N. W. F. Kossen, Progress in Industrial Microbiology. M. J. Bull. Ed.. (Elsevier, Amsterdam, 1978), Vol. 14, p. 95. 4. L. E. Erickson, 1. G. Minkevich, and V. K. Eroshin, Biotechnof. Bioeng.. 20, 1595 (1978). 5. L. E. Erickson, S. E. Selga, and U. E. Viesturs, Biotechnof. Bioeng.. 20, 1623 (1978). 6. C. G. T. Evans, D. Herbert, and D. W. Tempest. in Methods in Microbiology. 1. R. Noms and W. W. Ribbons, Eds. (Academic, London. 1970). Vol. 2. p. 313. 7. A. A. Esener, J. A. Roels, and N. W. F. Kossen, Biotechnof. Bioeng.. 23, 1851 (1981). 8. A. A. Esener, Ph.D. thesis, Delft University of Technology, 1981. A. A. ESENER J. A. ROELS N. W. F. KOSSEN Biotechnology Group Department of Chemical Engineering Delft University of Technology Jaffalaan 9, TH. Delft, The Netherlands Accepted for Publication October 6, 1981