a Biotechnology Letters Yol.3, No.l 15-20 (1981) Dear Colleaque, The Biotechnology Letters doesnot provide re-prints. We therefore enclose a photocopy of the article you have requested. Thank you for your interest in our work A. A. Esener , April 1981 COMMENTS ON THE DESCRIPTION OF THE MAINTENANCE METABOLISH DURING ANAEROBIC GROWTH WITH PRODUCT FORMATION AL AL Bi eners J. A. Roels and N. W. F. Kossen Biotechnology Group, Department of Chemical Engineering, Delft University of Technology, Jaffalaen 9, P.0.Box 5029 2600 GA Delft , The Netherlands SUMMARY Product formation during anaerobic growth in the absence of any other electron acceptor has been shown to be linked to the energy production processes. It has been demonstrated that when this fact is not taken into account, experimental data results in an incorrect description of the maintenance metabolism. RESULTS AND DISCUSSION Maintenance metabolism during anaerobic qrowth has recently received much attention in the literature. Some of these articles, in our opinion have drawn a very misleading picture of the energetic status of anaerebic systems (Gromie and Doelle,1980; Gona et. al-,1979). Based on incorrect theoretical treatments and biased estimates, it has been claimed that the maintenance requirements could be reduced effectively to zero by merely varying the chemical composition of the cultivation medium (Cromie and Doelle,1980). We therefore feel obliged to express our opinion about this apparently ill-treated system in order to promote a constructive discussion which may be useful for further research and a better understanding of the maintenance metabolism. In this work, first, it will be shown that in anaerobic systems with product formation in the absence of external electron acceptors, energy generation is coupled to the product formation process and secondly exanples of misinterpretation of data from such systems will be pointed out. ‘The mathematical treatment to be used follows from the work of Roels(1980) and Roels and Kossen (1978). The reader is referred to the original articles for a detailed analysi Starting from macro-balancing principles , a general stoichiometric equation can be written for microbial growth on a single carbon and energy source with ammonia as the nitrogen source. For simplicity only one product will be considered. 2 CB Oe Ma, + $20, tf Ny oe OCH ON, 4456, HON, +%6C0,+6,H,0 (1) Pea ay! °csNa, 2 Ls 16For a system at steady state, if the above description holds, flows of all components can be expressed in terms of any three known flows. The Linear law of substrate consumption, introduced after the postulation of maintenance mechanism (Herbert, 1958), can be generalized to account for the product formation during growth (Huuphrey and Jefferis,1973; Roels,1980; Roels and Kossen, 1978). Fyn! Vogt tpl Yep ty Sy @ A similar expression can now be shown to hold for the oxygen consumption rate: a a 3) 707 Fal Yo * Tel Yop * orSx ) Equations (1)-(3) describe the general case. The anaerobic case, which is a special case, can also be described by this set of equations when the oxygen uptake rate is put equal to zero. i.e. when r, = 0, from equation (3) one obtains 7 Yop Te! Yor ~ Po"% ? @ Substituting this result in equation (2) and rearranging, the following expression is obtained for the substrate consumption rate. Fy TO Tog ~ Yop! lon hep))* Het CBE Bo Lopl FopeOe 3D The terms given in parenthesis in the above equation are all constants and hence it becomes evident that there renains no sepatate contribution for Tein the substrate consumption equation. For the case of anaerobic sBorh with product formation, 1, can hence be expressed by rH YL, + ml. G 6) Combining this resule wich s degree of reduction balance ( Erickson et-al., 1978; Roels,1980),of the form : Yer Fs Yr Fx * pr Tp © an expression for the rate of product formation can be formulated : BOOT Wyeth, MO I Rt Os YMG @ Equation (6) shows that, for this special case (anaerobic growth with the excretion of one product or a mixture of them in constant ratio) no separate tern for r, appears in the r, expression. This implies that the energy generation i8 directly coupied®to the product formation. Equation (8) shows that the rate of product formation thus contains two contributions ; ‘The Eirse proportional to the bionass production, and the second to the amount of biomass present. This type of expressions have already been used for sone processes (Roels,1980). Therefore, if r, is expressed simply as : Fp ty! Yo tp + Sy @) ‘The following relationships can be obtained by comparing eqs. (8) and (9) + yh Cy, > a wh) ao) 16my = Cg! 1p) 85 ap These relationships are shown as solid lines in figures 1. and 2., assuming average values of y," 4.2 and the molecular weight of the organism as 25.0 , for the “case of ethanol production from glucose anaerobically. It can be noticed that the solid line in fig. 1. is well approximated by the straight line; ¥ = (¥, / Y,)- YS since vy. ¥! ey, (see eq.(10)). a 2 an PaenefSrs Sy! , Y., m! and m, were determined by performing linear regression analy$is &n tie data?reported by Cromie and Doelle (1980) using equations (6) and (9). Although there is considerable scatter in fig. 2., generally the above presented argument seens to hold for this system. i.e. product formation is indeed associated with the energy generation in the system. This type of energy generation was not taken into account by Goma et. al. (1979) who have calculated zero maintenance during ethanol production by Saccharomyces cerevisiae growing on glucose, aneerobically. Using eq. (2) these workers have calculated m_ to be zero towerds the end of the fermentation. However, their andlysis obviously takes no account of the energy produced during product formation. Energy produced in the form of ATP during ethanol production can naturally be channelled to satisfy the maintenance demands. A simple block diagran (Fiq. 3.) illustrates the possible mechanisns for the distribution of the substrate energy. Recently another claim of zero maintenance cane from Cronie and Doelle (1980). These authors clained that they have succeeded in reducing the maintenance requirements effectively to zero by changing the chemical composition of the cultivation medium they have used for ethanol production from glucose by Zymononas nobilis, anaerobically. However, an analysis of their original data by the above presented procedure revealed that their conclusions vere incorrect. When further analysis were car out it becane obvious that their conclusions were entirely based on theie biased parameter estimation technique. When their data were subjected to linear regression analysis , it was found out that all their paraneter estimations with the exception of one ( experiment no: 10, Table I) were in great errors. i.e. see Piqure 4. For the two cases for vhich Cromie and Doelie (1980) claimed zero maintenance, a Linear regression procedure yielded the values of 1.89 and 1.25 g/g/hr for m, (experiments no: 6 and 9 in table I ). A negative value for a, obviogsly does not make sense, however it does not allow the experimefiter to assune and report it zero. Al! chat can be said is that, either the model does not hold for this situation or/and experimental data are not accurate. Since it has been shown that the product formation was directly coupled to energy generation an energy balance for this system can now be given. Considering AIP aa the energy currency, ATP consumption, in terms of the linear law, is given by (Stouthaner,1973) Sere 7 Yarp * ane aay Moreover, as one mole of ATP is produced for every two moles of ethanol produced via the Enthner-Doudorof pathway (for the case of Z. mobilis) Sgehanot 72° CH / Yarp * arp) a3) If this equation i Fiteed to the data of experiment no: 6 (for which a zero maintenance claim has been made) an m,z, value of ” |10 mnoles ATP/g-bionass/hr. can be obtained (Fig. 5.). This value is quite high when compared with sone of the data collected and reported fy Stouthaner (1977) as shown in Table IT. One can therefore conclude that maintenance demands were never reduced to zero as claimed by Gromie and poelle (1980). In fact ic is quite probable thet their organism ‘nobilis, is a good ethanol producer because of its high maintenance energy demands and since it cannot generate sufficient energy via any other metabolic route, to satisfy them. CONCLUSIONS Product formation ( a single product or a constant ratio mixture) during anacrobic growth in the absence of external electron acceptors, is linked to the energy producing processes. This fact must be taken into Account when data obtained from such systems are to be interpreted. The statement of Cromie and Doelle (1980) " In varying not only the dilution rate but also the angonium sulphate (nitrogen source) and magnesium sulphate concentrations, the maintenance coefficient m Was successfully reduced to zero at all dilution rates " is based fon their incorrect conceptual and mathematical treatment. {a fact it can be shown that a high maintenance value for an ethanol producing organism is a desirable property as this will increase the efficiency of the conversion of the substrate to the product. NOMENCLATURE, Ct 0, 8g, bionaas elemental formula c, H0.'N) substrate elemental formla agi bse, dy 678,702 8) product elemental formla yeas ¢, biomass concentration clea. carbon equivalent a; maintenance coefficient on the i'th component ty rate of consunption/production of the i'th component vay maximal yield of j, on the i'th component ep flow of the i'ch component 4 degree of reduction of the the i'th component y, = 4a tb ~ 2c ~34 « sbecifie growth rate subscripts ° oxygen P product 8 substrate x biomass REFERENCES Cromie, S. and Doelle, H.W. (1980) Biotechnol. Lett., 2, (8), 357- Erickson, L-E., Minkevich, T.G. & Eroshkin, V.X. (1978) Biotechnol. Bioeng. 20,1595. Goma, G., Moletta, R., and Novak, M. (1979) Biotechnol. Zett., 1(10), 415 Herbert, D. (1958) in'Recent Progress in Microbiology’ Symp.7th Int. Congr Microbial., Ed. Tunewall, G., p-381, Almqvist, Stockholm. poels, JA. (1990) Tue application of macroscopic principles to microbial hetatolisn., (accepted for publication in Biotechnol. Bioeng.) poels, J-A. and Kossen, N.W.F. (1978) in Progress in Industrial Microbiology, Hd. BULL, M.d., 14, p95, Elsevier, Amsterdam. Stouthaner, AH. (1977) Symp-Soc-Gen.Wicrobiol., 27, 287 Stouthaner, AsH, and Bettenhaussen, C. (1973) Biochim.Biophys.Acta., 301, 53 ®Table I. Reported and calculated maintenance coefficients. Mg, (Ceomie and Doelle , m, (linear regression)" | 1980) g/g/hr a/a/e (this study) 0.15 5.9 1.55, 0.30 2.9 1.05 0.45 3.2 2.35 0.15 3.9 5.80 0.30 5.8 2.95 0.45 0.0 1.85 0.15 18 6.75 0.30 1.0 4205 0.45 0.0 -1.25 0.30 3.0 3.00 0.45 5.4 5.00 E> Ng = Hg50, 1 data reported b; 4 Cromie and Doelle (1980). v Table II. my. values for some organisms grown anaerobically in chemostat~ ‘ATP. ‘7 aa ae : Organism Growth limiting factor tmq% mmole ATP/g/t L, caset glucose 1S E. aerogenes glucose (minimal) 6.8 glucose (complex) 9.9 tryptophan 38.7 B. coli glucose 18.9 S. cerevisiae glucose 0.5 glucose 0.25 S. cerevisiae glucose 0.70 C. panapaitosis glucose 0.21 2, mobilis glucose 10.3 § “weferences as quoted by Stouthaner (1977) §Calculated fron data reported by Cromie and Doelle(1980) for exp. not cee eed ont. fe eons bey po are a5 | Fig. 3. Distribution of the substrate energy in microbial metabolisn. (oels and Kossen, 1978) 19 |oosof. {ea/c-ea) 0.020} mh (Cea /Ceqsnry eal ° BOms S016 Fig. 2. mvs. mz (—) seq. (11) (Ce) 5 data by Cromie and Doel1e(1980) ° ro 075 036 Figst. Yo vs. Yt px (—) 5 eq. (10) (©) sexperimental data (Cromie and Doelle,1980) (mole ATP/g biamascinr) , OS intarcant repories by poo L 1 of L L 1 L ass 3s he ° 305 oe os abs Fig. 4. Regression Line for Fig. 5. m, ion for exp.no th exp: no: 6 by eq. (12) 2