Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

Journal of General Microbiology (1986), 132, 779-788. Printed in Great' Britain 779

The Energetics and Kinetics of Extracellular Polysaccharide Production From Methanol by Micro-organisms Possessing Different Pathways of

C, Assimilation

By J . D. L I N T O N , ' * P . D. WATTS, ' R . M . A U S T I N , ] D. E . H A U G H '

Shell Research Limited, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, UK

Gist-Brocades N V, Delft, The Netherlands

A N D H . G . D. N I E K U S 2

(Received 29 July 1985)

~~~

Product excretion by Methylophilus sp. NCIB 12047, Pseudomonas extorquens NCIB 9399 and Pichia pastoris during growth on methanol was examined. These organisms possess the ribulose monophosphate pathway. the serine pathway and the dihydroxyacetone pathway of C, assimilation, respectively. Only Methyluphilus sp. NCIB 12047 produced significant amounts of extracellular product from methanol under conditions of nitrogen limitation in chemostat culture. This was a low-viscosity extracellular polysaccharide containing glucose and mannose in the ratio 3 : 1. Maximum polysaccharide production occurred under nitrogen limitation at a methanol/ammonium sulphate ratio > 10. The other two organisms responded to nitrogen limitation by increasing the rate of methanol oxidation to C 0 2 . The maximum yield for polysaccharide production by Methjllophilus sp. was 0-34 g (g oxygen)-' and 0.30 g (g methanol)-'. The maximum specific rate of polysaccharide production was 0.18 g (g protein)-' h-I. Methylophilus sp. grew readily under oxygen limitation and excreted an extracellular polysaccharide under these conditions. Examination of the biochemical pathways for polysaccharide production via the various C, fixation routes indicates that the ribulose monophosphate pathway is energetically the most favourable. Polymer production by Methylophilus sp. is energetically neutral in terms of net ATP demand; however, the rate of ATP utilization for polymer production is equivalent to 65 to 80% of that required for cell production at the same growth rate. The results reported suggest that the energetic constraints imposed by the various pathways of C, assimilation strongly influence both the rate of synthesis and the composition of exopolysaccharides produced by methylotrophs.

INTRODUCTION

Methanol is used as a chemical intermediate in the production of formaldehyde, acetic acid and solvents, and its world production is currently approximately 12 x lo6 t per year. The traditional outlets for methanol are expected to show only a modest growth (4-5 % per year) over the next 10-20 years (Marshall, 1982). However, methanol is available in large quantities, it is a very pure and relatively cheap chemical and therefore it could be an attractive alternative feedstock for the fermentation industry in the future.

The production of single-cell protein from methanol has been the driving force for a large number of publications on the metabolism of C, compounds (Anthony, 1982). However, very few reports concerning the microbial conversion of methanol into products have been published (Anthony, 1982; Crawford & Hanson, 1984; Morinaga & Hirose, 1984; Hou, 1984). There are four main pathways of C, assimilation; each one is associated with a different efficiency of

Abbreviations: DHAP, dihydroxyacetone phosphate; FAB, fast-atom bombardment; FP, flavoprotein; ICL, isocitrate lyase; KDPGA, 2-keto-3-deoxy-6-phosphogluconate aldolase; MDH, methanol dehydrogenase ; PQQ, pyrroloquinoline quinone; RMP, ribulose monophosphate ; TA, transaldolase.

0001-2854 0 1986 SGM

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

780 J . D . LINTON A N D OTHERS

carbon fixation and therefore ultimately determines the final yield of any bioconversion of methanol. 'This paper reports a study of metabolite formation from methanol by three methylotrophs and the results allow some general conclusions to be drawn regarding both the type and efficiency of metabolite excretion by methylotrophs possessing different pathways of C assimilation.

METHODS

Micro-organisms. Three rriethylotrophs were studied. (i) A type 1 methylotroph identified as a Methylophilus sp. (NCIB 12047) by the National Collection <3f Industrial Bacteria, Torry, Aberdeen. It possesses hexulose phosphate synthetase, glucose-6-phosphate dehydrogenase and transaldolase but no detectable activity of fructose-l,6-bisphosphate aldolase or sedoheptulose- 1,7-bisphosphatase, which implies the operation of the Entner-Doudoroff variant of the ribulose monophosphate (RMP) cycle. (ii) A pink-pigmented strain, Pseudotnonas extorquens NCIB 9399, which possesses the serine pathway. (iii) A yeast strain, donated by Dr A. E. Humphrey, University of Pennsylvania, which has recently been characterized by the National Collection of Yeast Cultures, Norwich, as a strain of Pichi? pastoris.

Growth medium jbr bacterial cultures. The niedium was made in five separate portions which were sterilized separately and then combined. Portion 1 (g 1-I): H3P0,, 2.6; MgS0,.7H20, 0.80; CaC12.2H20, 0.08; nitrilotriacetic acid, 0.12; trace element solution 100 ml 1 - I . Portion 11: FeSO,, 0-01 56. Portion 111: antifoam, 0.1 ml 1- ' . Portions IV and V contained (NH.,),SO, and methanol respectively; the concentrations used were varied as required. The complete medium, coniaining 26.5 mM-phosphoric acid, was neutralized in the fermenter with an equimolar mixture of KOH/NaOH. The former, approximately 13.5 mM, served as the source of K t . The trace elements solution contained (g 1-I): ZnS0,.7HZ0, 0.29; CuSO,. 5 H 2 0 , 0.24; MnS0,.4H20, 0-225; CoC12.6H20, 0-27; H3B03, 0.15; NazMo0,.2H20, 0.45.

Growth mediunijor theyrast strain. The tung.11 medium described previously (Linton et al., 1984) was used for continuous culture studies which were done a'. pH 4.5.

Continuous culture. A Biotech fermenter of w Drking volume approximately 3.5 I was operated with virtually no head space as described previously (Linton et al.. 1984).

Measurements of'.@ow rate. carbon content, Oz and C O , . The flow rate of medium into the fermenter was measured over a period of at least 1 h by mems of a graduated cylinder (500ml) in the medium input line. Similarly, culture output from the fermenter was measured by collecting culture effluent in a graduated cylinder. For routine analysis of cultures, cells were removed by centrifugation at 40000 g for 20 min.

The carbon contents of total culture and culture supernatant were measured using a Beckrnan 915-B total organic carbon analyser.

Oxygen consumption and C 0 2 production try chemostat cultures was continuously monitored as described previously (Linton et al., 1984). Gas flow rates were measured using a column bubble meter.

Methanol, ammonia, total carbohydrate und protein determinations. Methanol concentrations in the input and effluent medium were determined by gas liquid chromatography, using a Varian Aerograph series 1400 fitted with a Poropak Q column at 160 "C. The ammonia concentrations in the input and effluent medium were determined using a Technicon Autoanalyser 11 (Technicon Industrial Methods NO 108-7OW). The concentration of extracellular carbohydrate in culture supernatants was determined by the phenol/sulphuric acid method (Herbert et al., 1971). Culture supernatant protein was determined by the Lowry method. The content of intracellular stored glucose was determined as described by Linton 6: Cripps (1978). Analysis of bacterial carbon, nitrogen, hydrogen and phosphorus content was as described previously (Linton & Buckee, 1977).

Composition oj'estraccllulur poljwccharidr. Isolated polysaccharide (freeze-dried, 10 mg) was heated to 95 "C for 16 h with 0.24 M-H2S0, in a sealed vial. The acidic solution was neutralized with Dowex A62-X8 (HC0,-) anion- exchange resin, filtered through glass wool to remove resin and made up to standard volumes with distilled water. The component sugars were determined by coilversion to their corresponding peracetylated aldononitriles as described previously (Linton & Cripps. 1978).

Fast-atom bontburdniertt (FAB) muss ~ p t ' ~ ~ t r o s ~ o p y . This was done on a VCi Analytical 7070E mediurn-resolution mass spectrometer. FAB spectra were obtained i i , both positive and negative ion modes for all samples examined. The samples were loaded onto the FAB target as aqueous solutions at about 1 pg pl-I as 1 p1 films. Glycerol was used as the matrix. The atom gun was operated at 8 kV and xenon was employed as the bombarding gas.

RESULTS

Product formation by the three methqlotrophic micro-organisms studied will be discussed separately.

Product Jorrnution by Methylophilus sp. NCIB 12047

The medium used for chemostat studizs supported up to 14 g bacterial dry wt I - ' . Under methanol limitation (D = 0.05 h- l ) the yield from methanol, YC'H,Ok-l, was 0.43 g (g

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

Exopolysaccharide production from methanol 78 I

Table 1. Eject of the C : N ratio on biomass and product yields oJ' Methylophilus sp. NCIB 12047 grown in continuous culture under standard conditions ( D = 0.05 h-' , 30 "C, p H 6-8)

CH,OH :(NH,)?SO, ratio .

CH30H (g 1-I) Input output

output

(NH,),SO, (g I - ' ) Input

Bacterial dry weight (g I - ' ) Bacterial protein content (%dry wt N x 6.25)

Bacterial intracellular polyglucose content (% dry wt)

Extracellular polysaccharide (g 1-' ) Ypolysac [g polysaccharide (g CH,OH)-*] YCH,OH [g bacterial dry wt (g CH,OH)-' ] Yo? [g bacterial dry wt (g 02)-'] Supernatant protein/

Carbon balance (:d): Input methanol carbon Bacterial carbon Soluble product carbon C 0 2 carbon Recovery

bacterial protein (%)

2.0 2.9 3.0 3.2 6,4 9.2 12.6 14-4

6.55 19.33 37.5 10-05 40.0 38.5 43-25 37.75 0 0 0 0 0 0 0 6.5

3.3 6.62 12.75 3.15 6.25 4.15 3.43 2.62 1.13 1.21 2.07 0.27 0.07 0.04 0.04 0.03 2.92 7.56 13.91 4.23 10.48 6.55 6.46 4.6

75.9 77.1 76 77.1 70.6 65.0 62.5 63.4

0 - - 0 5.6 17 16.4 15 0.38 1.42 2.43 0.36 6-88 12 14.37 9-67 0.06 0.07 0.06 0.04 0.17 0.31 0.33 0.31 0.44 0.39 0.37 0.42 0.26 0.17 0.14 0.15 - -- 0.40 - - 0.17 0.15 0.15

8.8 12 15 1 1 11.5 12 12 13

- 100 - - 100 100 100 - 43.8 - - 22.5 18.4 19.7

- - 13.9 - - 35.4 39.5 46.3 - - 36.3 - - 53.5 49.1 48.2 - - 94.0 - - 111.3 107.0 114.2

-, Not determined.

methanol)-' at methanol concentrations < 10 g 1-l but at higher methanol concentrations there was a marginal decrease in yield (Table 1).

Efect of the CH3 OH : (NH&so4 ratio on product excretion. Methylophilus sp. NCIB 12047 was initially grown under methanol limitation at a constant dilution rate of 0.05 h-l and at a fixed methanol concentration of 40 g 1-l in the feed medium. Steady states were maintained at various C : N ratios and metabolite excretion was examined (Table 1).

The yield of bacteria from methanol and oxygen (g dry wt g-l) decreased as the CH30H:(NH4)2S04 ratio increased from 2 to 12.6; however, further increase in the ratio (ratios up to 17 were measured although the data are not given) did not result in any further decrease in the yield from methanol or oxygen (Table 1). Conversely, the yield of metabolites excreted increased with increasing CH30H : (NH4)2S04 ratio but no further increase in metabolite yield was observed above a ratio of 12-6 (Table 1). Virtually 100% of the product carbon excreted could be accounted for as a low-viscosity extracellular polysaccharide which was composed of glucose and mannose in the ratio 3 : 1 .

Under carbon limitation in the chemostat the culture supernatant contained soluble carbon which was composed of protein and carbohydrate and amounted to approximately 9 to 15 % of the steady-state cellular carbon (Table 1). This material probably originated from bacterial lysis as a number of methylotrophs undergo a growth-rate-dependent lysis which results in the accumulation of high levels of organic carbon in the culture supernatant at low growth rates (Dostalek & Molin, 1975; Linton & Buckee, 1977; Drozd et al., 1978). With Methylophilus sp. the amount of supernatant protein when expressed as a percentage of the steady-state cellular protein was independent of the C : N ratio even when excess methanol was present in the culture, indicating that there was no change in the lysis rate under these conditions (Table 1).

As the bacterial protein content decreased with increasing C : N ratio, all rates have Been expressed in terms of mmol (g cell protein)-' h-l . This decrease in the cellular protein content was caused by the accumulation of intracellular glucose-containing storage material (Table 1). The effect of increasing the C : N ratio on the rate of methanol (qCH,OH) and oxygen (qo2) utilization and C 0 2 production (qco,) is shown in Fig. 1. All three parameters reached a maximum rate at a CN,OH : (N&)2S04 ratio of approximately 10. As the pmax of Methylophilus

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

782 J . D . L I N T O N A N D O T H E R S

C 0

3 7 ‘s 24

2 4 6 8 1 0 1 2 1 CH,OH : (NH,),SO, ratio

Fig. 1

.u 4 16

I

2 4 6 8 10 12 14 16 CH,OH : (NH,),SO, ratio

Fig. 2 Fig. 1 . Effect of the CH,OH :(NH,)2S0, ratio in the medium feed on the rate of methanol (0) and oxygen (0) consumption and CO1 produ;tion (A) by a chemostat culture of Methylophilus sp. grown at 30 “C, pH 6.8 and a dilution rate of 0.05 h-I.

Fig. 2. Effect of the CH,0H:(NH, )2S0 , ratio on (a) the rate of extracellular polysaccharide production (e) and (b) the rate of ATP utilization for cell production (-) and polysaccharide production (0) by Methylophilus sp. (conditions as for Fig. 1) .

sp. is approximately 0.46 h-l, in the absence of any change in the growth efficiency from oxygen this organism would be expected to express a 40, of approximately 49 mmol g-l h-’ at pmax. Therefore the apparent saturation of methanol metabolism (qo2 = 18.8) is probably due to regulation rather than saturation of the respiratory or methanol-uptake system. The rate of polysaccharide production (qpolysac) increased with increasing C : N ratio up to a maximum of 0.18 g polymer (g bacterial protein)-* h-l, beyond which further increase in the C : N ratio did not result in an increase in the qpolysac (Fig. 2a). The maximum observed extracellular polysaccharide yield was 0.30 g (g methanol)-’ and 0.34 g (g oxygen)-’.

Energetics of polysaccharide produclion. Methylophilus sp. possesses the 2-keto-3-deoxy-6- phosphogluconate aldolase and transaldolase (KDPGA/TA) variant of the RMP pathway of formaldehyde assimilation. Thus the net production of 2 moles of phosphoglycerate from methanol occurs as follows :

6CH30H + 2NAD+ + 4ATP + 6l’QQ = 2phosphoglycerate + 2NADH2 + 4ADP + 2P, + 6PQQH2

The production of glucose 1-phosphate from 2 moles of phosphoglycerate requires 2ATP + 2NADH2. Thus the overall conversion of methanol into glucose 1-phosphate requires 6ATP + 2NADH2. As the production of phosphoglycerate results in the formation of 2NADH2 and 6 ATP (via PQQH,), the overall production of glucose 1-phosphate is energetically neutral. However, a further UTP (ATP) is required per hexose incorporated into polysaccharide but this can be supplied by the combustion of I /7 moles of methanol to CO, via the dissimilatory RMP pathway. The overall stoichiometry of polysaccharide production from methanol via the RMP pathway is therefore :

6.142CH30H + 6.142PQQ + 0.285NAD + 7ATP = [CgHloOs] + 6‘142PQQHz + 7ADF’ + 7P, + 0*285NAD(P)H2 + O.142CO2 + 0.858H20

6’142PQQH2 + 0’285NAD(P)H2 + 3.21302 + 7ADP + 7P, = 6.142PQQ + 0*285NAD(P) + 7ATP + 6.427H20

Net: 6.142CH30H + 3.2130, = [C6H1005] + 0.142c02 + 7-285H20

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

Exopolysaccharide production from methanol 783

Table 2 . Efect of increasing the severity of oxygen limitation on a chemostat culture of Methylophilus sp. NCIB 12047 maintained at D = 0.05 h-l, 30 "C, pH 6.8 on the yield of cells

and metabolic products (cellular yields from oxygen and formaldehyde during carbon-limited growth on jormaldehyde are also shown)

Nutrient limitation . . . C

HCHO (g 1-I) Input output

output

CHJOH (g 1-') Input

Bacterial dry weight (g 1-I) Bacterial protein (% dry wt N x 6.25) Supernatant protein/cell protein (%) Soluble product carbon (g 1-I) YCH,OH [g bacterial dry wt (g CH,OH)-' 1

Yo? [g bacterial dry wt (g O,)-l] Yproduct Is product c (g CH30H C)-lI qpol>srrc [g (g protein)-' h-' 1 qCH,OH [mmol (g protein)-' h-'1 qo2 [mmol (g protein)-' h-'1 qco2 [mmol (g protein)-' h-'1 Extracellular polysaccharide (g I-' ) Carbon balance (%) :

Bacterial carbon Soluble product carbon COz carbon Recovery

0 0

9-5 0.0 3.96

77.5 16 0.66

0.42 0.56 0.16 0-0 10 4.8 3.60 1.65 0.62

45 16 31 92

0

0 0

19.2 1.5 7.27

76.8 11 1.14

0.4 1 0.35 0-15 0.01 1 4.96 5.8 1 2.47 1 -29

45 15 43

103

0

0 0

36-8 18.1 5.93

73.7 7.3 1.71

0.32 0-32 0.24 0.0 I 3 6.83 6-63 2.70 1 062

37 24 40

101

0

0 0

41.4 28.1

1.4 73.1 16.5

1.35

0.10 0.1 1 0.27 0.056

2 1 a45 19.68 7.6 1.15

1 1 27 32 70

P

0 0

40.2 5.5 6-24

65 25 4.16

0.18 0.15 0.3 1 0.10

13.36 16 5 *O 8.44

21 31 41 93

C

24 0

-

-

3-05 72.5 12.2 0-76

0*13* 0.17 0.095 * 0

18*85* 13.19 10.3 0

16 9

63 88

* Expressed in terms of HCHO rather than CH,OH.

Therefore the production of a glucose : mannose (3 : 1) polysaccharide from methanol is virtually energetically neutral in terms of ATP and NAD(P)H2. Nevertheless, the rate of ATP consumption (7 ATP per hexose incorporated) for extracellular polysaccharide production from methanol is large in comparison to that required for cell production (Fig. 2b), and under conditions optimal for polysaccharide production (CH30H : (NH&SO4 > 10 the q A T p ) for polymer production is equivalent to 65 to 80% of that required for cell production, assuming a Y,,, for cells of between 8.3 and 10.6gmol-' (Anthony, 1982). The overall q A T p value for polymer production by Methylophilus sp. is 4 mmol ATP (g dry wt)-l h-l, which is remarkably close to that calculated for the production of xanthan and alginate by Xanthomonas carnpestris (3.8 mmol g-l h-l) and Pseudomonasaeruginosa (4.1 mmol g-' h-l) (Jarman & Pace, 1984). It is not clear whether the rate of polymer production is a means of controlling the rate of ATP turnover under conditions of methanol excess or the q A T p imposes the overall rate of polymer product ion.

Product formation by Methylophilus sp. under other nutrient limitations. In order to examine the possibility that methylotrophs may excrete fermentation products, Methylophilus sp. was grown under oxygen limitation in a chemostat. The severity of oxygen limitation was progressively increased and this resulted in an increase in the steady-state concentration of unmetabolized methanol in the culture (Table 2). Under these conditions the yield of cells from methanol and oxygen decreased while the amount of methanol carbon excreted as soluble products increased. Polysaccharide was produced under oxygen limitation but in very low concentrations and it accounted for all the carbon other than lysis products. No other products could be detected by HPLC or FAB mass spectrographic analysis, apart from traces of acetate. Nevertheless, the rate of extracellular polysaccharide production increased fivefold when the severity of oxygen limitation was increased (Table 2). The maximum rates of methanol and oxygen consumption and of C 0 2 excretion were very similar to the maximum values observed under nitrogen limitation (Table 2, Fig. 1).

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

784 I . D. L I N T O N AND OTHERS

Phosphate limitation. Under phosphate limitation polysaccharide accounted for 90 % of the product carbon excreted (Table 2). The remaining 10% was accounted for as protein. The high levels of supernatant protein under phosphate limitation caused foaming and it was difficult to maintain a constant volume in the fermenter.

Metabolite production fromformafduhyde. As oxygen-limited growth on methanol did not lead to the excretion of fermentation products it could be argued that regeneration of the oxidized form of methanol dehydrogenase may restrict the metabolism of methanol under these conditions. It was therefore interesting to examine the possibility of formaldehyde utilization under these conditions, particularly as this C1 compound is at the same oxidation/reduction level as a carbohydrate.

Carbon limitation was demonstrated by growing the culture on four different concentrations of formaldehyde from 14 to 36 g 1-I (D = 0.05 h-*) which all gave a cell yield between 0.12 and 0.15 g dry wt (g formaldehyde)-'. The low yield from formaldehyde was accompanied by a low yield from oxygen (Table 2). It was not possible to grow Methyfophilus sp. under oxygen limitation with formaldehyde because as soon as the air supply was restricted, formaldehyde accumulated and the cells were killed. As methanol dehydrogenase (MDH), a PQQ-containing enzyme, is located on the periplasmic surface of the membrane (Duine & Frank, 1981 ; Jones et a f . , 1982) it is possible that formaldehyde is metabolized via MDH to formate and then to C 0 2 via a NAD-linked formate dehydrogenase. The oxidation of methanol via MDH has been shown to be linked to at most one mole ol' ATP per mole methanol oxidized (Dawson & Jones, 1981 ; Drozd & Wren, 1980). Consequently the oxidation of formaldehyde to C 0 2 via the above linear pathway would result in production of a maximum of 4ATP per formaldehyde oxidized compared to 7ATP per mole methanol oxidized via MDH and the dissimilatory RMP pathway. Thus the yield from methanol would be expected to be approximately 57% of that from methanol, i.e. 0.23 g (g formaldehyde)-', which is higher than the experimental values measured (0.12 to 0.15 g g-l) during carbon-limited growth of Methyfophilus sp. on .formaldehyde, suggesting that the formate oxidation step is associated with the production of less than 3ATP per mole formate converted to C 0 2 .

Product fortnation by Pichia pastoris Pichiapastoris was grown in continuous culture at a dilution rate of 0.05 h-' under both carbon

and nitrogen limitation; repeated attempts were made to grow the yeast under oxygen limitation, but it proved impossible to attain a steady state,

One reason why this organism was difficult to grow, and in particular did not readily reach a steady state under oxygen limitation, may be that it is very sensitive to restriction of the oxygen supply, In one experiment with a nitrogen-limited culture, the air supply was stopped for 2 min and then resumed. This resulted in an immediate wash-out of the culture and formaldehyde (44 mg 1-*) was detected in the culture medium. The cell yield under methanol limitation (0.41 g dry wt g-l) was high compared to published yield values (Sahm, 1977; Tani et af . , 1978) although well within the range theoretically possible. Under nitrogen limitation the yield on a dry weight basis appeared to decrease only marginally. However, the nitrogen content of nitrogen-limited cells was considerably lower than that of carbon-limited cells and the yield decreased markedly when expressed as g protein (g methanol)-' (Table 3). The low nitrogen content of nitrogen-limited cultures was partially due to the accumulation of intracellular glucose-containing storage material (Table 3). Under conditions of nitrogen limitation no soluble metabolites were excreted and a greater proportion of methanol carbon was oxidized to Cot compared to that for carbon-limited growth (Table 3).

Product formation by P.reudomonas extorquens NCIB 9399 Pseudomonas extorquens when grown under methanol limitation exhibited yield values typical

of methylotrophs possessing the serine pathway of formaldehyde assimilation. Under nitrogen limitation this organism failed to produce measurable levels of soluble metabolites but as with

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

Exopolysaccharide production from methanol 785

Table 3. Eflect of growth under methanol and nitrogen limitation on the yield of cells and products and the respiratory activity of' Pseudomonas extorquens NCIB 9399 and Pichia pastoris

Pichia pastoris Pseudomonus extorquens

Dilution rate (h-I) Nutrient limitation CH,OH (g I - ' )

Input output

output

(NH4)2SO, (g I - ' 1 Input

Cellular N content (% dry wt) Cellular dry weight (g 1-' ) Cellular protein (% dry wt N x 6.25) Intracellular polyglucose (% dry wt) YCHJOH [g protein (g C H 0 - W ' 1 yo, [g protein (g O,)-'I qCH30H [mmol (g protein)-' h-'1 qo2 [mmol (g protein)-' h-'1 qcol [mmol (g protein)-' h-'1 Yproduct [g product C (g CH@H C>-' I Supernatant carbon (g 1-I) Carbon balance (%) :

Cellular carbon Soluble product carbon CO, carbon Recovery

f

0.05 C

15.5 0

5.12 2.64

10.1 7.23

63 4.6 0.29 0.3 1 5.3 5.0 2.6 0.0 17 0.1 1

49 2

45 96

3

0.047 N

37.3 0

3.55 < 0.02

5.90 13.96 36.8 14.3 0.138 0.1 28

10.68 11.4 6.5 0.017 0.24

42 2

61 105

0.1 C

9.62 0

-

-

10.69 4.05

66.8 3.2 0.28 0.33

1 1 *42 10 5.0 0.05 0.17

53.7 4.9

43.7 102-3

0.1 N

9.67 0.08

1.8 <0.1

8.2 2.6

51.2 17.6 0.139 0.147

22.8 21.2 13.2 0.05 0.20

35 5

60 100

the yeast, accumulation of intracellular glucose-containing storage material occurred, and the rate of C 0 2 production was considerably higher than that observed during carbon-limited growth (Table 3).

DISCUSSION

In this study Methylophilus sp., NCIB 12047, the strain possessing the RMP pathway of C, fixation, was the only organism that excreted significant amounts of extracellular metabolites during carbon-sufficient growth on methanol. Metabolite excretion was not detected with Pseudornonas extorquens, which possesses the serine pathway, nor with Pichia pastoris, which is presumed to possess the dihydroxyacetone pathway of C , assimilation. In both organisms over- metabolism of methanol occurred but this was confined to oxidation to C 0 2 . Methylophilus sp. grew readily and produced extracellular polysaccharide under oxygen limitation whereas the other strains, which were unable to produce polymer, grew poorly. Methanol, a small uncharged molecule, probably diffuses into the cell at an appreciable rate. When the respiratory capacity is limited by the supply of oxygen, polymer production is a considerably (three times) faster and more efficient means of methanol (formaldehyde) removal than is oxidation, provided that its production does not lead to a net over-production of reducing equivalents.

An examination of the overall stoichiometry of extracellular polysaccharide production via the various pathways of C1 fixation (Table 4) affords an explanation of the experimental results. In the production of an extracellular polysaccharide containing glucose and mannose (3 : 1) from methanol via the RMP pathway the formation of 6 moles of formaldehyde generates 85% of the ATP requirement for the synthesis of a hexose unit of a polysaccharide. The remaining ATP requirement is met by oxidation of 0.142 moles of methanol to C 0 2 via the dissimilatory RMP pathway. Alternatively the incorporation of 1 mole of pyruvate per 2 hexose units of polysaccharide would supply this additional ATP demand. In both cases polymer production is balanced in terms of ATP and reducing equivalents. It is interesting that Methylophilus sp. does not produce exopolysaccharide from formaldehyde but instead oxidizes it to C 0 2 . Unlike polymer production from methanol, production from formaldehyde is ATP limited.

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

Table 4. Theoretical stoichiometry of po lysaccharide formation from methanol via various pathways of C1 Jixation (the stoichiometry for the production of these products from glucose is included for comparison)

Carbon Pathway of sQllrCP c; fiw3tion

Energy balance in the production of 1 mole of product (no. of moles) Overall stoichiometry

oxidi7ed to water via respiration) A (assuming that excess reducing equivalents are 3 Ysubstrate yo

bQQH1 YH, FPH, NAD(P)H1 ATP (g g-I) (g g")

0 0.82 1.57 Methanol ICL + Serine 10CH30H + 902 = [ C ~ H I O O ~ ] 4CO2 + 15HZO 1 6 2 0 0 0.50 0-56

Methanol DHAP 7.166CH3OH + 4.74902 = [ C ~ H I O O ~ ] + 1.166C02 + 9.33HzO 0 0 0 0 0 0.71 1.06 Methane RMP(KDPGA/TA) 12CH, + 180, = [C6Hlo05] + 6C02 + 19H20 5 0 0 0434 0.28 0 0 Glucose 1.052C6H1206 + 0.31202 = [C~HIOO,] + 0.312CO2 + 1'312HzO 0 0 0 0 0 0.85 16.22

* Containing an NAD-linked formaldehyde dehydrogenase.

Methanol RMP (KDPGA/TA) 6.142CH30H + 3.2130, = [C6Hlo0,] + 0-142C02 + 7-285H20 0 0 0 0

Methanol *ICL + Serine 7CH3OH + 4-50, = [ C ~ H ~ O O ~ ] + COz + 9H2O 0 0 1 0 0 0.72 1.125

U

t: Z

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

Exopo fysaccharide production from methanol 787

Polysaccharide production from methane via the RMP pathway is NADH limited (Anthony, 1978, 1982) because of the NADH requirement of the methane mono-oxygenase reaction. The severity of the NADH limitation is increased in the serine pathway. As a consequence, synthesis of the shortfall of NADH for polysaccharide production results in a concomitant overproduction of PQQH, . Similarly, polysaccharide production via the serine pathway is NADH limited, but the extent of this limitation will depend on whether the formaldehyde dehydrogenase present is NAD linked or not (Table 4). Here again the extent to which other reducing equivalents are concomitantly over-produced will depend on the extent of the NADH limitation (Table 4). As a consequence of the conversion of methanol to formaldehyde via an oxidase in the dihydroxyacetone pathway a higher proportion of additional methanol has to be oxidized to CO, in order to satisfy the biosynthetic demand for ATP via this pathway in comparison to the RMP pathway (Table 4).

In agreement with our findings, a recent survey of 77 obligate and facultative methylotrophs (Grinberg et al., 1984) reported that the best extracellular polysaccharide producers (in terms of rates and yields) were those possessing the RMP pathway of C1 assimilation. Nevertheless polysaccharide production from methanol by methylotrophs possessing the serine pathway and from methane by those possessing the RMP and serine pathways has been reported. A close examination of the component sugars of these extracellular polysaccharides suggests that their oxidation/reduction state, i.e. the sugar composition, is related to the energetic constraints imposed by the various pathways of C1 fixation as outlined above. For example, extracellular polysaccharides produced by micro-organisms possessing pathways of C fixation that are NADH limited with respect to polysaccharide synthesis contain reduced sugars like rhamnose and fucose, and in most cases uronic acids. The production of uronic acids presumably leads to a reduction in the demand for NADH and to a smaller overproduction of other reducing equivalents. The presence of these reduced sugars under conditions of NADH limitation suggests that their production involves the participation of reducing equivalents other than N AD(P)H.

Examples of polysaccharide produced from methane include Methyfornonas sp. (RMP pathway), glucose : fucose :mannose : galactose : uronic acid (38-48 : 1 1-20 : 7-21 : 1 1-1 8 : 10-20; Meyers & Westlake, 1977) and bacterium H-2, glucose : mannose : galactosamine : glucuronic acid (2.1 : 10.5 : 23 : 19.6; Chida et af., 1983). Polysaccharides produced from methanol by organ- isms possessing the serine pathway include Methyfocystisparvus OBBP, glucose : rhamnose : man- nose : galactose : fucose (8 1.9 : 14- 1 : 1 a 9 : 1 ~5 : 0.7 ; Hou et af., 1978) ; Pseudomonas viscogena Ts, galactose : mannose : allose :glucose :glucuronic acid (54-5 : 13.3 : 9.8 : 10.7 : 1 1 ; Misaki et af., 1977, 1979) and Hyphomicrobium sp., glucose : mannose : 2-0-methylmannose : pyruvate (2 : 1 : 1 : 1 ; Kanamara et af., 1982). It is interesting that Methyfornonas mucosa (RMP pathway) produced an extracellular polysacc haride from methanol containing glucose : mannose : galac- tose :pyruvate (10-30 : 3-15 : 3-15 : 5-35; Finn et af., 1977) whereas an organism from the same genus produced a polysaccharide from methane containing fucose and uronic acids (Meyers & Westlake, 1977). A mixed culture utilizing methane produced an extracellular polysaccharide containing glucose : galactose : mannose : fucose : rhamnose (1 : 0.36 : 0.19 : 0.3 1 : 0.16) from meth- ane but a polymer containing only glucose : galactose : mannose (1 : 0.67 : 0.42) from methanol (Huq et af., 1978). Similarly the obligate methane-utilizer Methyfococcus therrnophifus 1 10 (RMP pathway) produced an extracellular polysaccharide containing glucose, mannose, rhamnose, xylose and glucosamine from methane whereas Pseudornonas sp. IT (RMP pathway) produced a polysaccharide containing only glucose, mannose, galactose and pyruvic acid from methanol (Grinberg et af., 1984).

The bioconversion of methanol into chemicals suffers from one major constraint : whereas in general the yield of a particular product from methanol is likely to be similar to that from glucose on a carbon-carbon basis, the yield from oxygen is likely to be considerably lower (Table 4). The oxygen demand for extracellular polysaccharide formation from methanol is at least 1 0-fold greater than that for production from glucose (Table 4).

Part of this work was carried out as part of a joint-venture research programme between Shell Research Ltd and Gist-Brocades NV, Delft. We would like to thank Dr J. A. Page for carrying out mass-spectrcgraphic analysis and M. R. Platten for gas-liquid chromatographic analysis of polysaccharides.

Downloaded from www.microbiologyresearch.org by

IP: 54.39.17.49

On: Sun, 11 Feb 2018 18:30:47

788 J . D . L I N T O N A N D OTHERS

REFERENCES

ANTHONY, C. (1978). The prediction of growth yields of methylotrophs. Journal of’ General Microbiology 104,

ANTHONY, C. (1982). The Biochemistry of’ Methylo- trophs. London : Academic Press.

CHIDA, K . , SHEN. G., KODAMA, T. & MINODA, Y. (1983). Acidic polysaccharide production from methane by a new methane-oxidising bactei,ium H2. Agricultural und Biologicd Chemistry 47,

CRAWFORD, R. L. & HANSON, R . S . (editors) (1‘44). Microhiul Growth on C, Compounds (Procertlinps qf ’ 4t h Int ernut ionul Spipo.sium). Wash i ng ton, IX : American Society for Microbiology.

DAWSON, M. J . & JONES, C. W. (1981). Respiration- linked proton-translocation in the obligate methylo- troph Methylophilus methylotrophus. Biochemical Journal 194, 9 15-924.

DOSTALEK, M. & MOLIN, N. (1975). Studies of biomass production of methanol oxidising bacteria. In Single Cell Protein, vol. 2, pp. 3955401. Edited by S R. Tannenbaum & D. I . C. Wang. Cambridge, Mitss. : MIT Press.

SON, R. J. (1978). An in situ assessment of the specific lysis rate in continuous cultures of Methylococcuy sp: ( N C I B 11083) grown on methane. F E M S Micro- biology Lottors 4, 3 I 1 ~ 3 14.

DROZD, J. W . &WREN, S. J. (1980). Growthenergetics in the production of bacterial single cell protein from methanol. Biotechnology and Bioengineering 22, 353- 362.

DUINE, J . A. & FRANK, J . (1981). Methanol dehydro- genase: a quinoprotein. In Microbial Growth on C , Compounds, pp. 31-41. Edited by H. Dahon. London : Heyden.

FINN, R. K., TANNAHILL, A. L. & LAPTEWICZ, J . E. (1977). Production of heteropolysaccharide by fer- mentation of methanol. United States Patent 40 16 085.

GRINBERG, T. A., KOSENKO, L. V. & MALASHENKO. R. Yu. (1984). Formation of exopolysaccharide by methylotrophic microorganisms. Mikrobiologicherkii Zhurnal46, 22-26 (in Russian, English summary).

HERBERT, D., PHIPPS, P. J . & STRANGE, R. E. (19*’1). Chemical analysis of microbial cells. Method.\ in Microbiology 5B, 2 10-344.

Hou, C. T., LASKIN, A. L. & PATEL, R. N. (19:’s). Growth and polysaccharide production by Methylo- cystis parvus OBBP on methanol. Applied (2nd Environmental Microbiology 37, 800-804.

Hou, C. T. (1984). Other applied aspects of methylo- trophs. In Methylotrophs: Microbiology, Biochemhtry and Genetics, pp. 146-167. Edited by C . T. Hou. Florida: CRC Press,

9 1-1 04.

275-280.

DROZD, J . w . , LINTON, J . D., DOWNS, J . &STEPHEN-

HUQ, M. N., RALPH, B. J. & RICKARD, P. A. D. (1978). The extracellular polysaccharide of a methylotrophic culture. Australian Journal of Biological Science 31,

JARMAN, T. R. & PACE, G. W. (1984). Energy requirements for microbial exopolysaccharide syn- thesis. Archives of Microbiology 137, 231-235.

JONES, C. W., KINGSBURY, S. A. & DAWSON, M. J . (1982). The partial resolution and dye-mediated reconstitution of methanol oxidase activity in Methylophilus methylotrophus. FEMS Microbiology Letters 13, 195-200.

KANAMARA, K., IWAMURO, Y., MIKAMI, Y., OBI, Y. & KISAKI, T. (1982). 0-2-Methyl-~-mannose in an extracellular polysaccharide from Hyphomicrobium sp. Agricultural and Biological Chemistry 46, 24 19- 2424.

LINTON, J. D. & BUCKEE, J . C. (1977). Interaction in a methane-utilizing mixed bacterial culture in a chemostat. Journal of’General Microbiology 101,2 19- 225.

LINTON, J . D. & CRIPPS, R. E. (1978). The occurrence and identification of intracellular polyglucose stor- age granules in Methylococcus NCIB 11083 grown in a chemostat on methane. Archives of’ Microbiology

LINTON, J. D., AUSTIN, R. M. & HAUGH, D. E. (1984). The kinetics and physiology of stipitatic acid and gluconate production by carbon sufficient cultures of Penicillium stipitatum growing in continuous culture. Biotechnology and Bioengineering 26, 1455- 1464.

MARSHALL, E. F. (1982). Methanol: emerging uses, new syntheses. Chemical Technology 6, 358-362.

MEYERS, A. P. & WESTLAKE, D. J. C . (1977). Fermentation process for the production of micro- bial biomass and a heteropolysaccharide bio- polymer. GB Patent 1580439.

MISAKI, A., TSUBURAYA, Y. & KAKUTA, M. (1979). D- Allose-containing polysaccharide synthesized from methanol by Pseudomonas sp. Carbohydrate Research

MISAKI, A., TSUBURAYA, Y., KAKUTA, M., TAKEMOTA, H., IGARASHI, T . & HASHIMOTA, T. (1977). Charac- terization of a polysaccharide produced by the methanol-assimilating bacterium. Tokyo Soda Ken- kya Hokoku 23, 89-96.

MORINAGA, Y. & HIROSE, Y . (1984). Production of metabolites by methylotrophs. In Meth.v/orroph.s: Microbiology, Biotc~chnologv untl Genrtics, pp. I 46 167. Edited by C. T. Hou. Florida: C‘RC Press.

SAHM, H. (1977). Metabolism of methanol by yeasts. Advances in Biochemical Engineering 6 , 77-1 03.

TANI, Y., KATO, N . & YAMADA, H. (1978). Utilization of methanol by yeasts. Advances in Applied Micro- biology 24, 1 6 5 1 86.

31 1-316.

117, 41-48.

75, C8-C10.