Potassium deficiency results in accumulation of ultra-high molecular weight poly-β-hydroxybutyrate in a methane-utilizing mixed culture
Article first published online: 16 APR 2008
© 2008 The Authors. Journal compilation © 2008 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 105, Issue 4, pages 1054–1061, October 2008
How to Cite
Helm, J., Wendlandt, K.-D., Jechorek, M. and Stottmeister, U. (2008), Potassium deficiency results in accumulation of ultra-high molecular weight poly-β-hydroxybutyrate in a methane-utilizing mixed culture. Journal of Applied Microbiology, 105: 1054–1061. doi: 10.1111/j.1365-2672.2008.03831.x
- Issue published online: 16 SEP 2008
- Article first published online: 16 APR 2008
- 2007/2038: received 19 December 2007, revised 20 February 2008 and accepted 3 March 2008
- methanotrophic bacteria;
- Methylocystis sp. GB25;
- potassium deficiency;
- ultra-high molecular weight PHB
Aims: To investigate the effect of various single nutrient deficiencies on poly-β-hydroxybutyrate (PHB) biosynthesis in a methane-utilizing mixed culture (dominant species Methylocystis sp. GB 25 DSM 7674).
Methods and Results: Poly-β-hydroxybutyrate accumulation experiments were performed in 7 and 70 l bioreactors and initiated by potassium, sulfur or iron deficiency. After 24 h the PHB content reached levels of 33·6%, 32·6% and 10·4% respectively. Interestingly a polymer with an ultra-high average-weight molecular weight (Mw) of 3·1 MDa was accumulated under potassium-limited conditions. When sulfur and iron were lacking Mw were lower by 20·6 and 41·6%. Potassium-deficiency experiments were furthermore characterized by a maximum specific PHB formation rate 0·08 g g−1residual biomass (R) h−1 and a yield coefficient of 0·45 g PHB g−1 CH4.
Conclusions: Biosynthesis of an ultra-high Mw PHB in a methane-utilizing mixed culture can be induced by potassium deficiency.
Significance and Impact of the Study: This study greatly extends the knowledge in the field of bacterial biopolymer formation with gaseous substrates. The special system used here combines the use of methane a low-cost substrate available from natural and renewable sources with the possibility of employing a mixed-culture in an open system for the synthesis of a high-value product.
Recently the growing interest in and the great potential of biopolymers in general and polyhydroxyalkanoates (PHAs) in particular has been highlighted by several reviews (Hazer and Steinbuchel 2007; Philip et al. 2007; Suriyamongkol et al. 2007; Verlinden et al. 2007). Special attention was given to the use of mixed cultures and the advantages to work under nonsterile conditions (Dias et al. 2006; Gurieff and Lant 2007).
Previously we investigated poly-β-hydroxybutyrate (PHB) formation in a methane-utilizing mixed culture (dominant species Methylocystis sp. GB25) under nitrogen, phosphorus and magnesium deficiency in an open system. Maximum values for PHB content of 50% and Mw of 2·5 MDa were determined under those conditions (Wendlandt et al. 2001). Among possible strategies for the production of PHA the use of methane as a cheap and renewable carbon source can be regarded as a very attractive approach for biopolymer production (Yamane 1993; Rehm and Steinbüchel 1999).
Generally speaking PHA synthesis is induced when the supply of nutrients essential for growth is imbalanced. Such nutrients can be macro elements like nitrogen or phosphorus but also microelements like magnesium, sulfur, iron, potassium, manganese, copper, sodium, cobalt, tin or calcium (Kim and Lenz 2001). Even though all elements play different roles within the bacterial metabolism a universal overflow metabolism is triggered under conditions of carbon excess and nutrient limitation. This leads to a more stable energy state by using the surplus carbon to form intracellular storage substances like PHA (Steinbüchel 1996). In this way bacteria are able to adjust to unfavourable environmental conditions by securing carbon and energy resources which can be used once nutrient limitations have been overcome (Kadouri et al. 2005).
The majority of studies in the field of microbial PHA synthesis were carried out under nitrogen deficiency as nitrogen demand for growth is high and limiting conditions can be achieved within a very short period of time (minutes) after interrupting the supply. For other nutrients like phosphorus intracellular storage systems can delay the induction of polymer accumulation (Kornberg et al. 1999). Even though nitrogen deficiency is most commonly used comparative studies revealed that key parameters like maximum content, specific formation rates and molecular weights (Mw) of PHA depend on the nutrient-limiting conditions for a particular species (Suzuki et al. 1986; Kim et al. 1997). Interestingly, a methanol-utilizing bacterium (Methylobacterium organophilum) accumulates a maximum level of PHB under potassium deficiency (Kim et al. 1999a) whereas sulfur deficiency is most effective for a mutant strain of R. eutropha (Steinbüchel and Schlegel 1989).
The objectives of this study were to characterize PHB accumulation and polymer properties in a methane-utilizing mixed culture under potassium, sulfur and iron deficiency. Those elements where chosen because of their central role in bacterial metabolism and because previous results suggested unusual effects of limited supply of these elements on biopolymer formation (see above).
Materials and methods
All chemicals were at least analytical grade and supplied by Merck, Darmstadt (Germany). Methane (purity 99·5%) was obtained from Linde AG (Germany).
Culture conditions and bioreactors
All cultivations were performed in specially equipped 7 l and 70 l pressure bioreactors (P ≤ 0·6 MPa, UD5, UD50, B. Braun Biotech International, Germany) in two stages (a) a continuous growth phase (t = 24 h, dilution rate D = 0·17 h−1) and (b) discontinuous PHB accumulation phase (t = 24 h, D = 0). During the first stage a mineral salt medium designed for the growth of up to 60 g dry mass per litre was used which contained per litre water: 1·68 ml H3PO4 (80%), 2100 mg KH2PO4, 1500 mg MgSO4·7H2O, 47·1 mg CuSO4·5H2O, 83·34 mg MnSO4H2O, 100·68 mg FeSO4·7H2O, 19·32 mg ZnCl2, 2·16 mg CoSO4·7H2O, 11·16 mg Al2(SO4)3·18H2O, 52·98 mg Ca(NO3)2·4H2O, 2·46 mg Na2MoO4·2H2O, 77·16 mg H3BO3, 4·62 mg CrCl3·6H2O and 6·54 mg NiSO4·7H2O.
During PHB accumulation concentrated versions (33×) of the above medium depleted of potassium, sulfur and iron, respectively, were supplied manually (in aliquots not exceeding 0·1% of reactor volumes) to establish a single nutrient deficiency.
Individual supply systems for gases (methane, air, nitrogen) enabled continuous feeding of both the carbon source and oxygen. Under standard fermentation conditions the inlet gas consisted of 25% methane and 75% air. In order to maintain the dissolved oxygen level at 15% air saturation, agitation speed, total gas flow rates and pressure were varied using a digital measurement and control system connected to a process computer. More details can be found elsewhere (Wendlandt et al. 2001). For safety reasons the laboratory was equipped with continuous methane monitoring system which would automatically interrupt methane supply in case its concentration exceeded a threshold value of 0·1%.
The composition of inlet and exhaust gas was determined by using a gas analysis system based on paramagnetic gas analysis (Magnos 6G, Hartmann and Braun, Frankfurt, Germany) for oxygen, and infrared absorption gas analysis (Uras 10P, Hartmann and Braun) for methane and carbon dioxide. In addition, the bioreactor was connected online to a quadrupole process mass spectrometer (QMG 421, Balzers, Switzerland).
Bacterial dry weight was determined gravimetrically. Fifty microlitre volumes of cell suspension were centrifuged at 19 195 g for 20 min. The pellet was washed twice with demineralized water and dried in a preweight container at 105°C for 36–48 h. Total biomass concentrations (g l−1) were calculated based on the weight differences before and after drying.
Ion concentrations were determined in the supernatant after centrifugation (19 195 g, 20 min) and filtration (0·2 μm syringe filter). Sulfate and potassium concentrations were measured by ion chromatography (DX 100, Dionex, Sunnyvale, CA). Iron concentrations were determined with commercially available cuvette test systems LCK321 (0·2–6 Fe mg l−1) and LCKW021 (0·05–2 Fe mg l−1) using a spectrophotometer (CADAS 100, LPG210, Dr Lange, Berlin, Germany).
PHB content of biomass
Biomass was harvested by centrifugation (19 195 g, 20 min) washed twice with demineralized water and freeze-dried. Lipids were removed by pre-extraction with 80% methanol (20 ml per gram dry weight) for 1 h at 50°C in stirred glass flasks. PHB was extracted from pretreated biomass for 1 h at 80°C using 1,2-dichloroethane (16 ml per gram dry weight). Non-PHB containing material was removed by filtration. PHB was precipitated from solution in a fourfold volume of 80% methanol, separated by filtration, washed twice with pure methanol and dried at 60°C for 2 h.
Average molecular weight and distribution
The average Mw and the Mw distribution of the polymer was determined by gel permeation chromatography (GPC) (Knauer, Berlin, Germany) using refractive index detection and polystyrene standards (solvent: 1,2,4-trichlorobenzene, concentration: 0·2–0·4 mg ml−1).
PHB content and time course of PHB formation
After 24 h there was no difference in PHB content under sulfur (32·6 ± 2·0%) and potassium (33·6 ± 0·3%) deficiency. In contrast iron deficiency resulted in the formation of only 10·4 ± 1·2% PHB.
Figures 1–3 show time courses of PHB formation for representative experiments under sulfur, potassium and iron deficiency. At the start of the experiments (t = 0 h) all investigated ions were present at concentrations adequate for growth. Within the first 3 h 99·7%, 66·8% and 23·5% of the sulfate, potassium and iron ions respectively, were utilized to form additional (PHB free) biomass (8·0, 6·7 and 3·8 g l−1). Under sulfur and potassium deficiency its concentration remained constant after the initial rise (Figs 1 and 2) whereas under iron deficiency a further noticeable increase was observed (Fig. 3). In all cases PHB synthesis was initiated 2–3 h after the start as the respective nutrient concentration either approached zero (sulfate) or fell below a critical level (17 mg l−1 for potassium and 0·26 mg l−1 for iron). Interestingly the increase in potassium concentration (11·8 mg l−1 after 10 h to 43 mg l−1 after 24 h, Fig. 2) did not affect growth or PHB synthesis.
Weight-average molecular weight
Potassium deficiency resulted in the formation of PHB with a Mw of 3·1 ± 0·02 MDa. This, to our knowledge, is the highest value ever reported for methanotrophic bacteria. It exceeds the Mw obtained under sulfur deficiency (Table 1) and previous results using ammonium, phosphorus or magnesium limitation by at least 20% (Wendlandt et al. 2001). The lowest value was observed when iron was lacking (1·7 MDa). The Mw distribution, characterized by homogeneity (Mw/Mn − 1), was similar for all nutrient-limiting conditions with values varying between 1·7 and 2·4 (data not shown).
|Deficiency||S (2)||K (3)||Fe (2)|
|PHB (%)||32·6 ± 2·0||33·6 ± 0·3||10·4 ± 1·2|
|qPHB,ov (g g−1 R h−1)||0·024 ± 0·002||0·021 ± 0·001||0·005 ± 0·001|
|qPHB,max (g g−1 R h−1)||0·048 ± 0·004||0·081 ± 0·007||0·024 ± 0·003|
|YPHB/CH4 (g g−1)||0·40 ± 0·038||0·45 ± 0·027||0·22 ± 0·019|
|Mw (MDa)||2·46 ± 0·07||3·1 ± 0·01||1·81 ± 0·03|
PHB formation rates
Specific PHB formation rates varied with nutrient limitation and time (Fig. 4). Maximum values were achieved 4–9 h after the start of the experiments. The highest value (0·08 g PHB g−1 R h−1) was observed under potassium deficiency whereas sulfur and iron deficiency resulted in lower rates of 0·05 and 0·02 respectively (Table 1). Overall formation rates based on the whole 24 h duration of the experiment were similar for sulfur and potassium deficiency (0·021 and 0·024 g PHB g−1 R h−1, Table 1).
Yield of PHB formation
In this study, the yields of PHB formation YPHB/CH4 describing the amount of methane consumed for the formation of 1 g polymer were determined to be 0·45 for potassium, 0·40 for sulfur and 0·22 for iron deficiency (Table 1). These values amount to 82%, 73% and 40%, respectively, of the maximum value achieved under phosphorus limitation (0·55 g PHB g−1 CH4) (Wendlandt et al. 2001) which is comparable to the theoretical yields of 0·54 and 0·73 which have been calculated based on two different co-enzyme dependence and regeneration pathways (Yamane 1993; Babel et al. 2001).
PHB content and time course of PHB formation
The PHB content accumulated under sulfur or potassium deficiency corresponds to only two thirds of the maximum value of 51·3% obtained for the methane-utilizing mixed culture under ammonium deficiency (Wendlandt et al. 2001). PHB formation was induced at near to zero concentrations under sulfur depletion experiments but an unusually high threshold of 17 mg l−1 was found for potassium. This phenomenon was also observed with M. organophilum where a potassium threshold value of 25 mg l−1 was published (Kim et al. 1996). Furthermore potassium was never completely exhausted at any time during the experiments. Lowest levels of 12 mg l−1 were reached after 8 h. Thereafter potassium concentrations steadily increased to about 40 mg l−1 after 24 h (Fig. 2). It has been reported that a significant decrease in external potassium level disturbs the osmotic equilibrium within bacterial cells and results in the release of potassium ions into the medium (Tempest and Wouters 1981). To establish if this was the reason for the observed increase in potassium concentrations in the liquid phase, a potassium analysis of dried biomass was performed. Indeed a 50% decrease during the 24 h period was detected (data not shown). The methane-utilizing mixed culture seems unable to recover from the perturbations resulting from potassium deficiency as neither bacterial growth nor PHB synthesis did resume after potassium levels returned to normal external concentrations (t > 12 h, Fig. 2).
Under iron deficiency PHB was accumulated only to a very low extent (10·4%). This can possibly be explained by the central role of iron as a cofactor in methanotrophic metabolisms. The initial step in the oxidation of methane is catalysed by methane monoxygenase (MMO) which, depending on the copper concentration in the medium, can be expressed in either a soluble MMO or a membrane-associated particulate form (pMMO) (Murrell et al. 2000a). Under the conditions used here (copper concentrations of ≥0·2 mg l−1) pMMO is formed (Cook and Shiemke 1996; Hanson and Hanson 1996; Murrell et al. 2000b). It has been suggested that copper and iron are present in the three active centers of pMMO and that both are required for its full activity (Zahn and DiSpirito 1996; Takeguchi et al. 1999; Basu et al. 2003). Thus, it appears plausible that PHB synthesis is hampered under iron-deficient conditions.
As for Methylocystis sp. GB25 the maximum achievable polymer content depends on the nature of the limiting nutrient for several PHB producers for example Pseudomonas putida, M. organophilum or Pseudomonas sp. K (Suzuki et al. 1986; Kim et al. 1997, 1999b). Metabolic analysis using M. organophilum demonstrated that under potassium limitation intracellular energy levels, measured as ATP, were more balanced compared with sulfur and nitrogen limited conditions and the highest PHB content was accumulated (Kim et al. 1999a). Similar studies using the methane-utilizing mixed culture could help to find an explanation for our experimental results.
Weight-average molecular weight
The formation of PHB with an unusually high Mw≥3 MDa under potassium limitation is an exciting finding. This might open up new applications for the polymer. Films made from ‘ultra-high-molecular-weight’ PHB by a two-step drawing and annealing procedure have greatly improved mechanical properties while retaining a similar biodegradability compared with lower Mw PHB (Kusaka et al. 1998, 1999; Aoyagi et al. 2003; Kahar et al. 2005; Murakami et al. 2007). However, the cause for this unexpected outcome is not clear at this stage. Very little is known about the mechanisms by which different nutrient deficiencies affect the Mw of PHA. Daniel et al. showed that the Mw of PHB accumulated by Pseudomonas sp. 135 (carbon source methanol) was different under ammonium deficiency (0·37 MDa) and magnesium deficiency (0·26 MDa) (Daniel et al. 1992). Like for the Methylocystis sp. GB25-dominated mixed culture there was no relationship between PHB content and Mw e.g. highest Mw were not achieved under conditions where PHB accumulation was maximal.
A negative correlation between the carbon source concentration and the average Mw has been reported on several occasions (Suzuki et al. 1988; Hori et al. 1994; Taidi et al. 1994; Kusaka et al. 1997) and might offer an explanation for the generally high Mw (>1·7 MDa) of PHB from Methylocystis sp. GB25. Methane is hardly soluble in aqueous solutions [25 mg l−1 at 20°C (Geventman 1999)] and therefore its concentration in the liquid medium is considerably lower compared with highly soluble substrates like glucose or methanol.
It has also been shown that the specific combination of substrate, organism and nutrient deficiency as well as the fermentation conditions (pH, temperature) have an effect on Mw and weight distribution of PHAs (Suzuki et al. 1988; Daniel et al. 1992; Chen and Page 1994; Hori et al. 1994; Asenjo et al. 1995; Yeom and Yoo 1995; Yoon et al. 1996; Quagliano et al. 2001).
Except for results published by our group no data on Mw for PHB produced by methanotrophic bacteria are available. For methanol utilizing bacteria those values are lower at around 1·8 MDa [Methylobacterium extorquens (Bourque et al. 1995)]. Compared with other native PHB producers the Mw of PHB accumulated under potassium deficiency by Methylocystis sp. GB25-dominated mixed culture is one of the highest ever reported. Only for Azotobacter vinelandii UWD a higher Mw of 4 MDa has been published (Chen and Page 1994). It also has been shown that PHB of exceptionally high Mw of up to 22 MDa can be synthesized in recombinant Escherichia coli XL1-Blue (Kusaka et al. 1997; Choi and Lee 2004). As PHA metabolism consists of two main routes, namely PHA synthesis (key enzyme: PHA synthase) and intracellular PHA degradation (key enzyme: PHA depolymerase), their balance affects the length of the polymer chain (Sudesh et al. 2000). Systematic studies need to be conducted to investigate how the availability of nutrients influences the activity of these opposing pathways. Enzymes responsible for intracellular degradation of PHB are absent in E. coli XL-1. This might offer an explanation for the formation of such ‘ultra-long’ polymer chains. Thus it could be speculated that the activity of intracellular PHB degradation enzymes might be affected by the availability of potassium in our methane-utilizing mixed culture.
PHB formation rates
Maximum specific PHB formation rates under sulfur, potassium and iron deficiency were 36·8%, 44·7% and 86·8% lower than the highest value previously reported under ammonium deficiency (0·123 g PHB g−1 R h−1) (Wendlandt et al. 2001). The same trend was seen for overall PHB formation rates (based on 24 h period) which were only 61%, 34·1% and 80·5%, respectively, of those determined under phosphor-deficient conditions (0·038 g PHB g−1 R h−1) (Wendlandt et al. 2001).
Varying specific PHB formation rates with different single nutrient deficiencies were also determined for Ralstonia eutropha. With gluconate as carbon source maxima of 0·28 and 0·23 g PHB g−1 R h−1 for sulfur and ammonium limitation, respectively, have been reported (Steinbüchel and Schlegel 1989) whereas with glucose under ammonium deficiency a rate of 0·21 g PHB g−1 R h−1 was determined (Henderson and Jones 1997). It was shown that the intracellular level of NAD(P)H as well as the NAD(P)H/NAD(P) ratio varied with different nutrient-limiting conditions, suggesting that enzymes involved in PHB synthesis (and therefore specific PHB formation rates) are directly regulated by tnicotinamide cofactors (Lee et al. 1995). It is generally accepted that the NAD(P)H/NAD(P) ratio regulates the flux of acetyl-CoA either towards the TCA cycle or PHB synthesis by affecting the two key enzymes citrate synthase (TCA) and β-ketothiolase (PHB) (Anderson and Dawes 1990; Mothes et al. 1996; Henderson and Jones 1997). Again, to fully explain our findings the above mentioned enzymes and nicotinamide concentrations for methanotrophic bacteria need to be studied.
Yield of PHB formation
The yields for bacterial PHB synthesis (based on methane consumption) under all conditions studied here were lower compared with the theoretical values, which have been estimated to range between 0·54 and 0·73 g PHB g−1 CH4 (Yamane 1993; Babel et al. 2001). Provided NADPH is used as cofactor for acetoacetyl-CoA reductase in the second step of PHB synthesis the lower value of 0·54 is valid. Higher yields can only be achieved assuming NADH is the cofactor as its regeneration is also possible via the oxidation of formaldehyde to CO2 as part of the main oxidation cascade for methane (Asenjo and Suk 1986). Even though enzymes involved in PHB synthesis have not yet been elucidated for Methylocystis sp. GB25 the maximum yield achieved under phosphorus limitation (Wendlandt et al. 2001) might indicate that both possible regeneration pathways are being used. This combined mechanism was also reported for the methylotrophic bacterium Methylobacterium rhodesianum MB126 which assimilates carbon via the serine pathway (as does Methylocystis) (Mothes 1997; Breuer and Babel 1999). To our knowledge there are no other published experimental yields for methanotrophic bacteria.
Yields determined using a methane-utilizing mixed culture are similar to values published for R. eutropha 0·5 g PHB g−1 glucose (Gostomski and Bungay 1996) or recombinant E. coli 0·43 g PHB g−1 glucose (Yu et al. 2000) but higher compared with most other PHB-producing bacteria and substrates such as Alcaligenes latus (0·37 g PHB g−1 sucrose) (Yamane et al. 1996), Methylobacterium extorquens (0·22 g PHB g−1 methanol) (Bourque et al. 1995) or Pseudomonas cepecia (0·147 g PHB g−1 lactose) (Young et al. 1994). This fact emphasizes the great potential of methane as a carbon source for biopolymer production.
In summary, the results presented here demonstrate that PHB of an ultra-high Mw is accumulated under potassium deficiency in a methane-utilizing mixed culture. These findings might contribute to the development of a methane-based PHB production process which yields a high-quality biopolymer at competitive costs (Listewnik et al. 2007).
This paper is dedicated to the memory of Dr Karin-Dagmar Wendlandt who sadly passed away in 2007. The authors would like to thank E. Knaak for technical assistance and acknowledge the support of G. Rogge and I. Mäusezahl in routine microbiological analysis.
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