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Substrate preference and oxygen requirement for cyanophycin synthesis by recombinant Escherichia coli
Biocatalysis and Agricultural Biotechnology 1 (2012) 9–14
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Substrate preference and oxygen requirement for cyanophycin synthesis by recombinant Escherichia coli$ Daniel K.Y. Solaiman n, Richard D. Ashby, Jonathan A. Zerkowski Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA
a r t i c l e i n f o
abstract
Article history: Received 12 April 2011 Received in revised form 10 August 2011 Accepted 17 August 2011 Available online 30 August 2011
Cyanophycin (CGP) is a bacterial bioproduct having a straight-chain poly(aspartic acid) as a backbone with arginine pendant groups attached to it. It has many potential industrial applications in the areas of water softening, hydrogel, metal–ion chelation, and nutriceuticals. Biotechnological production of CGP employs as producing strains the recombinant organisms that express heterologous cyanophycin synthase (cph) gene. A systematic study of fermentation parameters influencing CGP synthesis by a recombinant Escherichia coli expressing a cphA of Synechocystis sp. showed that high aeration conditions as provided by 400 rpm stirrer speed and 1.0 L/min air flow in a Sixfors vessel (450 mL culture workingvolume) resulted in high yields of cell biomass and crude CGP product. Glycerol substrate was found to yield 1.8-times higher crude CGP than glucose did under similar conditions. With glycerol as substrate, we found that a simplified fermentation scheme consisting of a straight 48-h fermentation at 37 1C (without a 30–37 1C temperature-shifting induction step) yielded compatible or higher amounts of crude CGP as those obtained under various temperature-shifting conditions. By studying the effects of glycerol concentration on CGP yields and analyzing glycerol consumption patterns, we demonstrated that substrate-to-product conversion could be increased by at least 15% and that costly leftover of unused substrate could be alleviated. The results yielded valuable information for optimization of fermentative production of CGP using glycerol that could be obtained as surplus coproduct from biodiesel production. Published by Elsevier Ltd.
Keywords: Bioglycerol Biopolymer Poly(amino acids)
1. Introduction Cyanophycin (CGP) is a microbial poly(amino acid) synthesized and sequestered as inclusion bodies by cyanobacteria (Oppermann¨ ¨ Sanio and Steinbuchel, 2002) and other microorganisms (Fuser ¨ and Steinbuchel, 2007; Norton et al., 2008). CGP is composed of a poly(aspartic acid) (poly(Asp)) backbone chain with arginine (Arg) residues attached as pendant groups (Fig. 1). CGP has been widely studied as renewable biobased polymers that can be used to replace petroleum-based materials in many industrial applications. In this respect, the use of CGP and its derivatives as water softeners to replace poly(acrylates) has found industrial acceptance (Joentgen et al., 1998; Schwamborn, 1998). Other potential industrial applications of CGP and derivatives are metal ion-exchange materials (Miller and Holcombe, 2001) and hydrogels (Yang et al., 2009). In biochemical and nutriceutical areas, CGP is studied as a precursor
$ Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. n Corresponding author. Tel.: þ1 215 233 6476; fax: þ 1 215 233 6795. E-mail address: [email protected] (D.K.Y. Solaiman).
1878-8181/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.bcab.2011.08.007
for the high value bioproducts such as aspartic acid and arginine ¨ (Van Beilen and Poirier, 2008; Konst et al., 2010) and the nutriceutic ¨ dipeptides (Sallam and Steinbuchel, 2010). Because of its potentially high commercial value, many studies have been directed to improve the efficiency and economics of fermentation processes to produce ¨ ¨ CGP (Obst and Steinbuchel, 2004; Reinecke and Steinbuchel, 2009). The development of recombinant bacteria expressing gene(s) responsible for CGP biosynthesis (i.e., the cyanophycin synthase genes, cph) is an important undertaking in advancing bioprocess technologies towards future commercialization of CGP. A recombinant Escherichia coli clone expressing the cphA gene of Synechocystis sp. strain PCC6803 (i.e., E. coli [pMa/c5-914::cphA]) is widely used in studies aimed to develop efficient fermentation process for CGP production (Frey et al., 2002; Mooibroek et al., 2007; Elbahloul et al., 2005a,b; Solaiman et al., in press). Although technical scale fermentation for CGP synthesis had been evaluated using E. coli [pMa/c5914::cphA] (Frey et al., 2002), a systematic study to examine growth parameters important for production of CGP is still lacking and much needed. This is especially imperative in view of the low yields CGP obtained in a recent study using meat-and-bone meal as a source of nitrogen and amino acids (Solaiman et al., in press). We report in this paper our in-depth study on substrate preference and fermentation conditions to improve the efficiency of substrate-to-CGP conversion.
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NH2
+NH2
NH
O
NH
O OH O
O NH
NH OH
NH2
OH
O
O OH
n
O Fig. 1. Cyanophycin (CGP) structure.
Two substrates were compared in this study: Glucose was examined because not only is it a commonly used fermentation substrate in various embodiments (e.g., corn and sugar-cane syrups) but is also the primary constituent of cellulosic biomass hydrolysates; and glycerol was studied because it is the coproduct of biodiesel production, and creating value-added products from glycerol is paramount to the commercial viability of biodiesel industry. The information resulted from this study is invaluable for future development of sustainable fermentation processes that use low-cost or surplus feedstock to produce CGP, which is needed to ensure commercial viability of biotechnological production processes (Koutinas et al., 2007; Sanders et al., 2007).
2. Materials and methods 2.1. Microorganism and culture maintenance Genetically modified E. coli DH1 containing a recombinant plasmid pMa/c5-914::cphA (Frey et al., 2002) was used in this study. A long-term storage stock culture of E. coli [pMa/c5914::cphA] in growth medium supplemented with 15% glycerol as a cryoprotectant was stored in a 80 1C freezer. LB medium (1% w/v tryptone, 0.5% w/v yeast extract, 0.5% NaCl) containing carbenicillin (Cb, 50 mg/mL) was used for routine culture growth at 37 1C. Solid media were prepared by adding 1.0–1.2% (w/v) of agar to LB broth before autoclaving. 2.2. Bench-scale bioreactor fermentation A Sixfors Multi-Fermenter system (Infors AG, Bottmingen, Switzerland) was used in this study. The system consists of six fermentation vessels (500-mL capacity), and the fermentation conditions (i.e., stirrer speed, air-flow rate, pH) of each vessel can be independently set by a microprocessor-controlled console. The rate of oxygenation of the medium was determined using an oxygen electrode (OXYPROBE D140, Broadley-James, Irvine, CA, USA). The vessel content was first deaerated by bubbling nitrogen gas through it for at least 2 h, and the pO2 (partial O2 pressure) reading on the instrument panel was then set at zero. The vessel content was then fully aerated by bubbling in-house air at 1.0 L/min with 400 rpm stirrer speed for at least 18 h (i.e., overnight), and the pO2 reading was then set to 100. To obtain the initial rates of pO2 increase under high- (400 rpm stirrer speed, 1.0 L/min air flow) or low- (150 rpm, 0.3 L/min air flow) aeration conditions, the vessel content was first deaerated again as described earlier. Aeration was then commenced under either the high- or the low-aeration conditions. The increase of pO2 as a function of time was recorded and
used to calculate the initial rates of aeration. The results showed that the rate of oxygenation under the low-aeration conditions were only (2071) % of that occurred under high-aeration conditions. The growth medium was a casamino acids (CA)-supplemented (5 g/L) mineral salts medium (collectively termed MM; Frey et al., 2002; Nakano et al., 1997) containing per L (in grams): citric acid trisodium salt dihydrate (3), KH2PO4 (5.55), K2HPO4 (4.27), NaH2PO4 H2O (2.25), (NH4)2SO4 (1.00), NH4Cl (0.10), MgCl2 6H2O (1.25), CaCl2 2H2O (0.02), thiamine–HCl (0.005), and 1 mL of a trace element solution. Carbenicillin (50 mg/mL) was used in all fermentation medium to prevent the loss of the recombinant plasmid in E. coli [pMa/c5-914::cphA]. Thiamine–HCl (a 50 mg/mL stock solution which was sterilized with a 0.45-mm membrane filter) and trace element solution were added to MM (pH 7) after the medium had been autoclaved and cooled to ambient temperature. The composition of the trace element solution was (mg per 100 mL 0.1N HCl): FeSO4 7H2O (2.0), MnSO4 H2O (1289.7), ZnSO4 7H2O (871.4), CoCl2 6H2O (644.4), CuCl2 2H2O (320.8), Na2MoO4 2H2O (270.3), AlCl3 6H2O (147.1) and H3BO3 (51.2). We evaluated glucose and glycerol as fermentative substrates for the synthesis of CGP. As appropriate, stock glucose solution (40% w/v; sterilized using a 0.45-mm membrane filter) was added to the culture medium to achieve the desired test concentrations (see Table 1). When glycerol was tested, we directly added 100% glycerol stock solution to MM to achieve the desired concentrations (see Tables 1–3) before autoclaving.
Table 1 Glucose and glycerol as carbon source under different aeration conditionsa. Substrate Stirrer Air flow speed (rpm) (L/min)
Wet cell yield (g/L)
Dry cell yield (g/L)
Crude CGP yield (mg/L)
Glucose Glucose Glycerol Glycerol
10.7 6.5 11.3 6.9
2.70 1.38 2.78 1.53
14.0 3.8 6.4 3.8
400 150 400 150
1.0 0.3 1.0 0.3
a Cells were grown in mineral salts medium (MM) (450 mL) containing carbenicillin (50 mg/mL) and a carbon source (2% w/v glucose or glycerol) in 500 mL-capacity vessels of a Sixfors Multi-Fermenter system. Cells were first grown at 30 1C for 22 h, then the temperature was increased to 37 1C for the duration of the experiment. NH4Cl (final concentration [Cf] ¼4 g/L) and the carbon source ([Cf] glucose¼ 1.25% w/v; [Cf] glycerol ¼1% v/v) were added to the appropriate culture at 46 h after initial inoculation. Cells were harvested at 70 h after the start of the experiment.
Table 2 Comparative CGP yields at different aeration rates and induction times with glycerol as substratea. Temperatures
Stirrer speed Air flow (L/ Dry cell (rpm) min) yield (g/L)
Crude CGP yield (mg/L)
30 1C, 6 h; then 37 1C, 42 h 30 1C, 24 h; then 37 1C, 24 h 37 1C, 48 h
400 150 400 150 400 150
133.2 7 12.7 17.9 70.2 7.07 0.2 15.2 71.7 149.5 7 39.1 17.6b
1.0 0.3 1.0 0.3 1.0 0.3
2.87 0.2 1.67 0.1 3.67 0.6 2.07 0.1 3.77 0.2 2.07 0.3
a Experimental set-up was basically as described in footnote of Table 1. Glycerol (2% w/v) was the starting carbon source in all experiments. NH4Cl ([Cf]¼ 4 g/L) and glycerol ([Cf] glycerol ¼ 1% v/v) were added to the cultures at 24 h after initial inoculation. Growth temperatures for the duration of the fermentation were as shown in the Table. Cells were harvested at 48 h after the initial inoculation. Duplicate experiments were performed for each set of fermentation conditions (i.e., n¼ 2). b The duplicate experiments yielded widely different values of crude CGP yields of 17.6 and 99.6 mg/L. Consistent with the results of the other low-aeration experiments, only the lower value is tabulated.
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Table 3 Concentration dependence and consumption of glycerol in CGP productiona. Initial glycerol concentration (v/v) (%)
2.0 1.0 0.5
Dry cell yield (g/L)
3.3 70.1 3.3 70.3 3.9 70.1
Crude CGP yield (mg/L)
82.2 7 11.0 63.2 7 2.0 50.7 7 2.2
Glycerol concentration (%, v/v)b Initial
Immediately before feeding
Immediately before feeding
At harvest
1.99 7 0.01 0.94 7 0.01 0.68 7 0.01
1.16 7 0.02 *b **b
2.68 70.06 0.857 0.02 *b
1.96 7 0.08 0.69 70.01 **b
a Experimental set-up was essentially as described in footnote of Table 1. Glycerol (0.5–2% v/v) was the starting carbon source in all experiments. NH4Cl ([Cf] ¼ 4 g/L) and glycerol ([Cf] glycerol ¼half of the initial concentration) were added to the cultures at 24 h after initial inoculation. Cells were grown for the entire 48-h fermentation at 37 1C under high aeration conditions (i.e., 400 rpm stirrer speed and 1.0 L/min air flow rate). Duplicate experiments were performed for each glycerol initial concentration tested (i.e., n¼2). b Glycerol concentration was determined using an HPLC as described in Section 2. *HPLC peak for glycerol was present but was too low to be picked up by the integration program of the instrument due to a high- and noisy-background/baseline. **HPLC peak for glycerol was not present in chromatogram.
Seed cultures were prepared in Erlenmeyer flasks (with a capacity Z5 culture volume) by growing E. coli [pMa/c5914::cphA] in MM containing the appropriate substrate (i.e., glucose or glycerol) for 18–24 h at 30 1C and 250 rpm shaking. We used 10 mL of the seed culture to inoculate 450-mL growth medium in each fermenter vessel (500-mL capacity). The air-flow rate and stirrer speed were set at values specified for each experiment (see Tables 1–3). We also studied the effect of upshifting growth temperature on the yields of cell mass and crude CGP. In all experiments, cells were fed at 24 h time-point after the initial inoculation with NH4Cl (by adding a 0.45-mm filter-sterilized 0.2 g/L stock solution to a final concentration of 4 g/L) and glucose (by adding a 0.45-mm filter-sterilized 40% w/v stock solution to a final concentration of 1.25% w/v) or glycerol (by adding 100% glycerol to a final concentration of 10% v/v). The amounts of NH4Cl and carbon source added approximated the total amounts added in Frey et al. (2002)’s large-scale multiplefed batch study. Aliquots of cultures were removed for glucose or glycerol determination at the following points: the start of the experiment, immediately before and after the addition of NH4Cl and carbon source at 24 h after the initial inoculation, and the end of experiment at 48 h after the initial inoculation. Cells were harvested and weighed (to obtain wet cell mass, WCM) at the end of experiment by centrifugation (Sorvall RC5B Plus, GS-3 rotor, 6500 rpm, 30 min, 4 1C), and were then lyophilized to determine the dry cell weight (DCW). Crude CGP was extracted using a method adopted from Frey et al. (2002). Briefly, the dried cells were resuspended in deionized water (1 mL per 0.1 g of cells). The suspension was acidified to pH 1 by adding concentrated HCl, which was then stirred for 6 h at ambient temperature. The mixture was centrifuged (Sorvall RC5B Plus, GS-3 or SS-34 rotor depending on volume, 6500–7500 rpm, 30–60 min, 4–10 1C) and the supernatant was collected in a clean flask. The cell pellet was subjected to a second acid extraction by resuspending it in 0.1 N HCl (1 mL per 0.1 g of original cell mass) and stirring for 1 h. Following a centrifugation step (see above for conditions), the supernatant was combined with the previously saved first-round acid extract. We isolated the crude CGP from the combined acidic cell extracts by precipitation at pH 7 through the addition of 10 N NaOH aqueous solution. The precipitates were collected by centrifugation (see above for conditions) and lyophilized to yield the crude CGP preparation. Alternatively, differential filtration technique was used to isolate crude CGP fraction in solution form. The combined acid extract was first filtered through a 0.45-mm MCE membrane filter (MILLEX HA, Millipore). The filtrate was loaded on a 0.1N NaOH-prewashed Amicon Ultracel 10k NMWL filter unit and centrifuged in a swinging-bucket rotor at 4000 rpm (3345 g) for 10 min at 4 1C in an AccuSpin 1R centrifuge (Fisher Scientific). The retentate (100–200 mL) constituted crude CGP fraction in liquid form.
2.3. Analysis of glucose and glycerol Glucose and glycerol concentrations were determined using a Shimadzu Prominence HPLC system equipped with an evaporative light scattering detector (ELSD-LTII) and a SIL-20A/C autosampler. Culture samples were filtered using 0.2-mm Acrodisc PVDF membrane filters (PALL Life Sciences) and the filtrates were used in the analysis. A guard column and a Chromegabond Carbohydrate column (5 mm, 100 A, 25 cm 4.6 mm; ES Industries, West Berlin, NJ) were employed for sample analysis. Column temperature was maintained at 40 1C using a CTO 20A chromatography column oven. Elution was effected using an isocratic mobile phase (acetonitrile/water mixture, 75/25, v/v) at 1 mL/min for 15 min. Glucose and glycerol solutions ranging from 0–4% (w/v) were prepared and used as standards. We used also an enzymatic assay to determine glycerol concentration prior to the development of the HPLC method described earlier. A Free Glycerol Determination Kit (SigmaAldrich) was used for this purpose. Culture samples were filtered through 0.2-mm Acrodisc PVDF membrane filters prior to use in the enzymatic assay. The absorbance at 540 nm was measured on either a Shimadzu BioSpec-1601 UV/visible spectrophotometer or a microplate reader. 2.4. SDS-PAGE analysis Denaturing discontinuous gel electrophoresis was performed on a Mini PROTEAN 3 Cell apparatus (BioRad Laboratories, Hercules, CA), using Ready Gel Precast 10% polyacrylamide gels (BioRad Laboratories). Lyophilized crude CGP was added to a 2X Sample Loading Buffer solution (18), heated to 95 1C for 4 min, and centrifuged (in an Eppendorf microcentrifuge at 14,000 g, 15 min, ambient temperature) to remove insoluble substance. An aliquot of the supernatant was loaded on the polyacrylamide gel, and electrophoresis was performed in TRIS–glycine–SDS buffer system (Laemmli, 1970). Polypeptides in the gel were visualized with Coomassie Blue dye (Ausubel et al., 1987) or using a SimplyBlue SafeStain Kit (Invitrogen) according to the supplier’s instructions. SDS-PAGE Markers (Bio-Rad Laboratories) were used as molecular-weight standards.
3. Results and discussion 3.1. Substrate preference and the effect of aeration conditions We first compared the yields of cell mass and crude CGP obtained under high- and low-aeration conditions using two different substrates, i.e., glucose and glycerol, as carbon source. While
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glucose in various forms (e.g., corn syrup and beet molasses) is a commonly used substrate in microbial fermentation, we are interested in glycerol as a carbon source because of its potential as a lowcost surplus coproduct from the biodiesel industry. Results in Table 1 showed that as expected with E. coli, high-aeration conditions (400 rpm stirrer speed, 1.0 L/min air flow) led to a significantly higher yield of cell biomass (measured as wet- and dry-cell weight) than the low-aeration conditions (150 rpm, 0.3 L/min air flow) did. The type of carbon source, however, did not affect the biomass yields under our experimental conditions. The crude CGP yields obtained by neutralization (pH 7) of acid extracts of the harvested cells also showed significant increase at high aeration rates (Table 1). The higher yield of crude CGP obtained with high-aeration conditions paralleled the observed increase in cell biomass under these conditions. We performed an SDS–PAGE analysis on the crude CGPs obtained from the glucose- and glycerol-fed cells to assess the CGP contents in the isolated crude products. The positive effect of aeration on the yield of CGP is most prominently shown by the results obtained with the glucose samples, and to a lesser extent also with those from glycerol-fed fermentation (Fig. 2). A comparison of the relative intensities of the ca. 27 kDa-bands attributed to CGP showed that high-aeration conditions (lanes 1 and 3, Fig. 2) led to a higher level of CGP synthesis than the low-aeration conditions (lanes 2 and 4, Fig. 2), suggesting that high oxygen content favors CGP production. This oxygen dependency is an important finding for CGP synthesis by genetically engineered E. coli that has not been previously reported. It is interesting to note that the production of surfactin – a microbial product similar to CGP synthesized via nonribosomal assembly of amino acids into polypeptide – is also favored by high aeration conditions (Yeh et al., 2006). Elbahloul et al. (2005a,b) had showed that CGP content of wild-type Acinetobacter calcoaceticus ADP1 could be increased by culturing the organism in baffled Erlenmeyer flasks to increase aeration. Aeration rate or oxygen content of a culture is an important fermentation parameter
that influences the biosynthesis of microbial products including the secondary metabolites (Frykman et al., 2002), and our results further add to the list of industrially useful microbial products whose production is closely tied to oxygen content of the medium. Notwithstanding that the mechanistic basis for the increased CGP synthesis as a result of high aeration rate is not determined, ours and other researchers’ results clearly point to the importance of providing enough dissolved oxygen in culture medium in order to obtain high CGP production. More importantly, our results further show that glycerol is preferred as a substrate than glucose in supporting CGP synthesis. By comparing the intensities of the CGP bands in lanes 1 and 3 of Fig. 2 using an image integration program (ChemiImager 5500, Alpha Innotech Corp., San Leandro, CA), we estimated that glycerol afforded 1.8-times higher yield of CGP than glucose did under similar experimental conditions. This result agrees with the observation of Frey et al. (2002) in their large-scale fermentation runs. In their study, glycerol at the initial feed concentration of 2% afforded the synthesis of 1.64 g CGP/L culture (i.e., CGP content of 12% w/w CDW at 13.7 g/L cell density). Glucose at 2.5% initial feeding, on the other hand, only produced 0.23 g CGP/L culture (i.e., CGP content of 3% w/w CDW at 7.8 g/L cell density). The metabolic basis for the different ability of glucose and glycerol to support CGP production is not presently understood. Nevertheless, our confirmatory results to those of Frey et al. (2002) reinforce the conclusion that glycerol is a preferred substrate over glucose for CGP production. In fact, many studies on CGP synthesis have been performed using glycerol in place of glucose as a substrate with the consideration that glycerol is preferable (Steinle et al., 2010; Aboulmagd et al., 2001). A practical implication of this observation is that the glycerol coproduct stream from biodiesel production could in fact be a desired substrate for CGP production, which in turn would improve the economics of the biofuel production by making available a value-added coproduct to the overall biorefinery scheme. 3.2. Effects of temperature on CGP production
Fig. 2. SDS-PAGE of crude cyanophycin preparations. Lyophilized CGP (0.5–2.2 mg) was solvated in a Sample Loading Buffer solution (Laemmli, 1970) at a concentration of 10 mg/mL. The mixture was heated at 95 1C for 4 min, and centrifuged to remove insoluble materials. Electrophoresis was performed on a 10% polyacrylamide gel with 10 mL of the supernatant samples. Crude CGP preparations from cells grown on glucose (lanes 1 and 2) and glycerol (lanes 3 and 4) at 400 rpm stirrer speed and 1 L/min air flow (lanes 1 and 3) or 150 rpm stirrer speed and 0.3 L/min air flow (lanes 2 and 4) are shown. CGP at 27 kDa is indicated with an arrow head (c). M, molecular-weight protein markers.
While performing the experiments reported in Table 1, we observed that the cell density (as monitored by absorbance at 600 nm) of E. coli [pMa/c5-914::cphA] culture at the end of the 6 h initial cultivation at 30 1C was low and inconsistent. We decided to perform a separate set of experiments to examine the effects of shifting the incubation temperature on the yields of dry cell mass and CGP. Results from this set of experiments show that a rather consistent crude CGP yield of 15–18 mg/L was observed under low aeration conditions (150 rpm stirrer speed; 0.3 L/min air flow rate) regardless of the temperature regiments used (Table 2). Moreover, we could only reach a low cell density of o2 g/L when fermentation was carried out under the low aeration conditions, again regardless of the temperature settings (Table 2). These results show that under low aeration conditions, incubation temperature between 30 and 37 1C does not affect the already low yields of cell mass and CGP. The results obtained under high aeration conditions (400 rpm stirrer speed; 1.0 L/min air flow rate), on the other hand, show a clear dependence of CGP production on the variation of incubation temperature. We noted from the results that there is a direct correlation between the length of incubation at 37 1C and the crude CGP yield. When the entire 48 h of fermentation was performed at 37 1C, a crude CGP yield of 150 mg/L was observed (Table 2). Conversely, when half (i.e., 24 h) of the time of fermentation was performed at 30 1C followed by the other half (i.e., 24 h) at 37 1C, the crude CGP yield dramatically dropped to 7 mg/L (Table 2). The CGP yield obtained with the first 6 h of fermentation performed at 30 1C and the rest (i.e., 42 h) at 37 1C
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was 133 mg/L (Table 2), which was not statistically different from that observed with the entire fermentation performed at 37 1C. These results suggest that CGP production occurs mainly when the culture temperature is at 37 1C. The cell mass yield, on the other hand, was not significantly affected by the different temperature regiments used here under high aeration conditions. Data in Table 2 show that regardless of temperature-shifting pattern, dry cell mass yields were only slightly varied between 2.8 and 3.7 g/L and were not considered statistically significant. We had initially adapted the 30–37 1C temperature-shift pattern based on the large-scale experimental set-up of Frey et al. (2002). The rationale was that the expression of the cphA gene in the recombinant plasmid (i.e., pMa/c5-914::cphA) is under the regulation of a temperature-sensitive repressor (i.e., cI857; Frey et al., 2002). Therefore, Frey et al. (2002) first grew the cells under cphA-uninduced state at 30 1C to achieve as high a cell density as possible before implementing a temperature up-shift to 37 1C to de-repress cphA gene expression for CGP synthesis. Our results, however, indicated that an optimal CGP production could occur without the need for the initial growth of cells under cphA-repressed state at 30 1C, thus alleviating the inconvenience of monitoring cell growth and temperature shift during initial stage of fermentation. These results underscored the importance of a systematic study of experimental conditions at bench-scale fermentation to identify parameters crucial to CGP production, despite of earlier study at large-scale technical production Frey et al. (2002). 3.3. Effect of glycerol concentration and its conversion efficiency to CGP An efficient bioprocess requires that substrate conversion or utilization and the product yield are optimal. We thus conducted a series of experiments to study the effect of glycerol concentration on crude CGP yields under high aeration conditions. The results (Table 3) show that crude CGP yield is positively correlated with the total glycerol concentration fed to the culture. At initial glycerol concentration of 2% followed by 1% feeding midway through the fermentation, we obtained a crude CGP yield of 82.2 mg/L (Table 3). When 0.5% initial followed by 0.25% midcourse feedings of glycerol were tested, only 50.7 mg/L of crude CGP yield was produced at the end of the fermentation (Table 3). The patterns of glycerol consumption are valuable to ensure efficient substrate utilization. At 2% initial and 1% mid-course feedings of glycerol, close to 2/3 of the total substrate added still remained at the end of fermentation (Table 3, 1st row data). Conversely, it appeared that all of the added glycerol was consumed in the experiments in which the initial and midway feedings were 0.5% and 0.25%, respectively (Table 3, 3rd row data). A conversion rate of 50.7 mg CGP/7.5 g glycerol was obtained under this complete consumption condition. When we started the fermentation with 1% glycerol concentration, we observed that nearly all of it was consumed midway through the fermentation (Table 3, 2nd row data). Further addition of 0.5% glycerol resulted in a leftover of substrate at a concentration of 0.69%, which amounted to an overall substrate consumption rate of 54%. Under this substrate feeding scheme, a substrate-toproduct conversion rate of 63.2 mg CGP/8.1 g glycerol was observed. Taken together, these data suggest that an optimal conversion rate could be achieved by providing enough glycerol for CGP biosynthesis to complete (as in Table 3, 2nd row experiments), without an excessively wasteful addition of the substrate (as in Table 3, 1st row experiments). The effect of the concentration of carbon-source substrate on CGP synthesis had only been reported by Frey et al. (2002) in a technical-scale fermentation study. Contrary to our findings, however, Frey et al. (2002) found that lowering the initial concentration of glycerol
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from 4% to 2% resulted in a nearly 6-fold increase of CGP production. The different results observed between our benchscale and Frey et al.’s technical-scale fermentations again underscore the necessity of systematic optimization of bioprocessing systems to further the commercial prospects of CGP production.
Acknowledgment The authors thank Nicole Crocker and Bun-Hong Lai for ¨ technical assistance, and Professor Alexander Steinbuchel ¨ ¨ Munster, ¨ (Westfalische Wilhelms-Universitat, Germany) for the kind gift of Escherichia coli [pMa/c5-914::cphA]. References ¨ Aboulmagd, E., Voss, I., Oppermann-Sanio, F.B., Steinbuchel, A., 2001. Heterologous expression of cyanophycin synthetase and cyanophycin synthesis in the industrial relevant bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida. Biomacromolecules 2, 1338–1342. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., et al., 1987. Analysis of proteins. In: Ausubel, et al. (Eds.), Current Protocols in Molecular Biology. John Wiley & Sons, New York (Chapter 10). ¨ Elbahloul, Y., Krehenbrink, M., Reichelt, R., Steinbuchel, A., 2005a. Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1. Applied Environmental Microbiology 71, 858–866. ¨ Elbahloul, Y., Frey, K., Sanders, J., Protamylasse, Steinbuchel A., 2005b. A residual compound of industrial starch production, provides a suitable medium for large-scale cyanophycin production. Applied Environmental Microbiology 71, 7759–7767. ¨ Frey, K.M., Oppermann-Sanio, F.B., Schmidt, H., Steinbuchel, A., 2002. Technicalscale production of cyanophycin with recombinant strains of Escherichia coli. Applied Environmental Microbiology 68, 3377–3384. Frykman, S.A., Tsuruta, H., Starks, C.M., Regentin, R., Carney, J.R., Licari, P.J., 2002. Control of secondary metabolite congener distributions via modulation of the dissolved oxygen tension. Biotechnology Progress 18, 913–920. ¨ ¨ Fuser, G., Steinbuchel, A., 2007. Analysis of genome sequences for genes of cyanophycin metabolism: identifying putative cyanophycin metabolizing prokaryotes. Macromolecular Bioscience 7, 278–296. ¨ Joentgen, W., Groth, T., Steinbuchel, A., Hai, T., Oppermann, F.B., 1998. Polyasparaginic Acid Homopolymers and Copolymers, Biotechnical Production and Use Thereof. International patent application, WO 98/39090. ¨ Konst, P.M., PMCCD, Turras, Franssen, M.C.R., Scott, E.L., Sanders, J.P.M., 2010. Stabilized and immobilized Bacillus subtilis arginase for the biobased production of nitrogen-containing chemicals. Advanced Synthesis and Catalysis 352, 1493–1502. Koutinas, A.A., Wang, R.-H., Webb, C., 2007. The biochemurgist—bioconversion of agricultural raw materials for chemical production. Biofuels Bioproducts and Biorefining 1, 24–38. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Miller, T.C., Holcombe, J.A., 2001. Comparison and evaluation of the synthetic biopolymer poly-L-aspartic acid and the synthetic plastic polymer poly-acrylic acid for use in metal ion-exchange systems. Journal of Hazardous Materials 83, 219–236. Mooibroek, H., Oosterhuis, N., Giuseppin, M., Toonen, M., Franssen, H., Scott, E., et al., 2007. Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied Microbiology and Biotechnology 77, 257–267. ¨ Nakano, K., Rischke, M., Sato, S., Markl, H., 1997. Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Applied Microbiology and Biotechnology 48, 597–601. Norton, J.M., Klotz, M.G., Stein, L.Y., Arp, D.J., Bottomley, P.J., Chain, P.S.G., et al., 2008. Complete genome sequence of Nitrosospira multiformis, an ammoniaoxidizing bacterium from the soil environment. Applied Environmental Microbiology 74, 3559–3572. ¨ Obst, M., Steinbuchel, A., 2004. Microbial degradation of poly(amino acid)s. Biomacromolecules 5, 1166–1176. ¨ Oppermann-Sanio, F., Steinbuchel, A., 2002. Occurrence, functions and biosynthesis of polyamides in microorganisms and biotechnological production. Naturwissenschaften 89, 11–22. ¨ Reinecke, F., Steinbuchel, A., 2009. Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. Journal of Molecular Microbiology and Biotechnology 16, 91–108. ¨ Sallam, A., Steinbuchel, A., 2010. Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production. Applied Microbiology Biotechnology 87, 815–828. Sanders, J., Scott, E., Weusthuis, R., Mooibroek, H., 2007. Bio-refinery as the bioinspired process to bulk chemicals. Macromolecular Bioscience 7, 105–117.
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Schwamborn, M., 1998. Chemical synthesis of polyaspartates: a biodegradable alternative to currently used polycarboxylate homo- and copolymers. Polymer Degradation and Stability 59, 39–45. Solaiman Daniel, K.Y., Garcia Rafael, A., Ashby Richard, D., Piazza George, J., ¨ Steinbuchel Alexander, Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer. New Biotechnology, in press. doi:10.1016/j.nbt. 2011.03.025. ¨ Steinle, A., Witthoff, S., Krause, J.P., Steinbuchel, A., 2010. Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases. Applied Environmental Microbiology 76, 1062–1070.
Van Beilen, J.B., Poirier, Y., 2008. Production of renewable polymers from crop plants. Plant Journal 54, 684–701. Yang, J., Wang, F., Tan, T., 2009. Controlling degradation and physical properties of chemical sand fixing agent-poly(aspartic acid) by crosslinking density and composites. Journal of Applied Polymer Science 111, 1557–1563. Yeh, M.-S., Wei, Y.-H., Chang, J.-S., 2006. Bioreactor design for enhanced carrierassisted surfactin production with Bacillus subtilis. Process Biochemistry 41, 1799–1805.
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Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab
Substrate preference and oxygen requirement for cyanophycin synthesis by recombinant Escherichia coli$ Daniel K.Y. Solaiman n, Richard D. Ashby, Jonathan A. Zerkowski Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA
a r t i c l e i n f o
abstract
Article history: Received 12 April 2011 Received in revised form 10 August 2011 Accepted 17 August 2011 Available online 30 August 2011
Cyanophycin (CGP) is a bacterial bioproduct having a straight-chain poly(aspartic acid) as a backbone with arginine pendant groups attached to it. It has many potential industrial applications in the areas of water softening, hydrogel, metal–ion chelation, and nutriceuticals. Biotechnological production of CGP employs as producing strains the recombinant organisms that express heterologous cyanophycin synthase (cph) gene. A systematic study of fermentation parameters influencing CGP synthesis by a recombinant Escherichia coli expressing a cphA of Synechocystis sp. showed that high aeration conditions as provided by 400 rpm stirrer speed and 1.0 L/min air flow in a Sixfors vessel (450 mL culture workingvolume) resulted in high yields of cell biomass and crude CGP product. Glycerol substrate was found to yield 1.8-times higher crude CGP than glucose did under similar conditions. With glycerol as substrate, we found that a simplified fermentation scheme consisting of a straight 48-h fermentation at 37 1C (without a 30–37 1C temperature-shifting induction step) yielded compatible or higher amounts of crude CGP as those obtained under various temperature-shifting conditions. By studying the effects of glycerol concentration on CGP yields and analyzing glycerol consumption patterns, we demonstrated that substrate-to-product conversion could be increased by at least 15% and that costly leftover of unused substrate could be alleviated. The results yielded valuable information for optimization of fermentative production of CGP using glycerol that could be obtained as surplus coproduct from biodiesel production. Published by Elsevier Ltd.
Keywords: Bioglycerol Biopolymer Poly(amino acids)
1. Introduction Cyanophycin (CGP) is a microbial poly(amino acid) synthesized and sequestered as inclusion bodies by cyanobacteria (Oppermann¨ ¨ Sanio and Steinbuchel, 2002) and other microorganisms (Fuser ¨ and Steinbuchel, 2007; Norton et al., 2008). CGP is composed of a poly(aspartic acid) (poly(Asp)) backbone chain with arginine (Arg) residues attached as pendant groups (Fig. 1). CGP has been widely studied as renewable biobased polymers that can be used to replace petroleum-based materials in many industrial applications. In this respect, the use of CGP and its derivatives as water softeners to replace poly(acrylates) has found industrial acceptance (Joentgen et al., 1998; Schwamborn, 1998). Other potential industrial applications of CGP and derivatives are metal ion-exchange materials (Miller and Holcombe, 2001) and hydrogels (Yang et al., 2009). In biochemical and nutriceutical areas, CGP is studied as a precursor
$ Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer. n Corresponding author. Tel.: þ1 215 233 6476; fax: þ 1 215 233 6795. E-mail address: [email protected] (D.K.Y. Solaiman).
1878-8181/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.bcab.2011.08.007
for the high value bioproducts such as aspartic acid and arginine ¨ (Van Beilen and Poirier, 2008; Konst et al., 2010) and the nutriceutic ¨ dipeptides (Sallam and Steinbuchel, 2010). Because of its potentially high commercial value, many studies have been directed to improve the efficiency and economics of fermentation processes to produce ¨ ¨ CGP (Obst and Steinbuchel, 2004; Reinecke and Steinbuchel, 2009). The development of recombinant bacteria expressing gene(s) responsible for CGP biosynthesis (i.e., the cyanophycin synthase genes, cph) is an important undertaking in advancing bioprocess technologies towards future commercialization of CGP. A recombinant Escherichia coli clone expressing the cphA gene of Synechocystis sp. strain PCC6803 (i.e., E. coli [pMa/c5-914::cphA]) is widely used in studies aimed to develop efficient fermentation process for CGP production (Frey et al., 2002; Mooibroek et al., 2007; Elbahloul et al., 2005a,b; Solaiman et al., in press). Although technical scale fermentation for CGP synthesis had been evaluated using E. coli [pMa/c5914::cphA] (Frey et al., 2002), a systematic study to examine growth parameters important for production of CGP is still lacking and much needed. This is especially imperative in view of the low yields CGP obtained in a recent study using meat-and-bone meal as a source of nitrogen and amino acids (Solaiman et al., in press). We report in this paper our in-depth study on substrate preference and fermentation conditions to improve the efficiency of substrate-to-CGP conversion.
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NH2
+NH2
NH
O
NH
O OH O
O NH
NH OH
NH2
OH
O
O OH
n
O Fig. 1. Cyanophycin (CGP) structure.
Two substrates were compared in this study: Glucose was examined because not only is it a commonly used fermentation substrate in various embodiments (e.g., corn and sugar-cane syrups) but is also the primary constituent of cellulosic biomass hydrolysates; and glycerol was studied because it is the coproduct of biodiesel production, and creating value-added products from glycerol is paramount to the commercial viability of biodiesel industry. The information resulted from this study is invaluable for future development of sustainable fermentation processes that use low-cost or surplus feedstock to produce CGP, which is needed to ensure commercial viability of biotechnological production processes (Koutinas et al., 2007; Sanders et al., 2007).
2. Materials and methods 2.1. Microorganism and culture maintenance Genetically modified E. coli DH1 containing a recombinant plasmid pMa/c5-914::cphA (Frey et al., 2002) was used in this study. A long-term storage stock culture of E. coli [pMa/c5914::cphA] in growth medium supplemented with 15% glycerol as a cryoprotectant was stored in a 80 1C freezer. LB medium (1% w/v tryptone, 0.5% w/v yeast extract, 0.5% NaCl) containing carbenicillin (Cb, 50 mg/mL) was used for routine culture growth at 37 1C. Solid media were prepared by adding 1.0–1.2% (w/v) of agar to LB broth before autoclaving. 2.2. Bench-scale bioreactor fermentation A Sixfors Multi-Fermenter system (Infors AG, Bottmingen, Switzerland) was used in this study. The system consists of six fermentation vessels (500-mL capacity), and the fermentation conditions (i.e., stirrer speed, air-flow rate, pH) of each vessel can be independently set by a microprocessor-controlled console. The rate of oxygenation of the medium was determined using an oxygen electrode (OXYPROBE D140, Broadley-James, Irvine, CA, USA). The vessel content was first deaerated by bubbling nitrogen gas through it for at least 2 h, and the pO2 (partial O2 pressure) reading on the instrument panel was then set at zero. The vessel content was then fully aerated by bubbling in-house air at 1.0 L/min with 400 rpm stirrer speed for at least 18 h (i.e., overnight), and the pO2 reading was then set to 100. To obtain the initial rates of pO2 increase under high- (400 rpm stirrer speed, 1.0 L/min air flow) or low- (150 rpm, 0.3 L/min air flow) aeration conditions, the vessel content was first deaerated again as described earlier. Aeration was then commenced under either the high- or the low-aeration conditions. The increase of pO2 as a function of time was recorded and
used to calculate the initial rates of aeration. The results showed that the rate of oxygenation under the low-aeration conditions were only (2071) % of that occurred under high-aeration conditions. The growth medium was a casamino acids (CA)-supplemented (5 g/L) mineral salts medium (collectively termed MM; Frey et al., 2002; Nakano et al., 1997) containing per L (in grams): citric acid trisodium salt dihydrate (3), KH2PO4 (5.55), K2HPO4 (4.27), NaH2PO4 H2O (2.25), (NH4)2SO4 (1.00), NH4Cl (0.10), MgCl2 6H2O (1.25), CaCl2 2H2O (0.02), thiamine–HCl (0.005), and 1 mL of a trace element solution. Carbenicillin (50 mg/mL) was used in all fermentation medium to prevent the loss of the recombinant plasmid in E. coli [pMa/c5-914::cphA]. Thiamine–HCl (a 50 mg/mL stock solution which was sterilized with a 0.45-mm membrane filter) and trace element solution were added to MM (pH 7) after the medium had been autoclaved and cooled to ambient temperature. The composition of the trace element solution was (mg per 100 mL 0.1N HCl): FeSO4 7H2O (2.0), MnSO4 H2O (1289.7), ZnSO4 7H2O (871.4), CoCl2 6H2O (644.4), CuCl2 2H2O (320.8), Na2MoO4 2H2O (270.3), AlCl3 6H2O (147.1) and H3BO3 (51.2). We evaluated glucose and glycerol as fermentative substrates for the synthesis of CGP. As appropriate, stock glucose solution (40% w/v; sterilized using a 0.45-mm membrane filter) was added to the culture medium to achieve the desired test concentrations (see Table 1). When glycerol was tested, we directly added 100% glycerol stock solution to MM to achieve the desired concentrations (see Tables 1–3) before autoclaving.
Table 1 Glucose and glycerol as carbon source under different aeration conditionsa. Substrate Stirrer Air flow speed (rpm) (L/min)
Wet cell yield (g/L)
Dry cell yield (g/L)
Crude CGP yield (mg/L)
Glucose Glucose Glycerol Glycerol
10.7 6.5 11.3 6.9
2.70 1.38 2.78 1.53
14.0 3.8 6.4 3.8
400 150 400 150
1.0 0.3 1.0 0.3
a Cells were grown in mineral salts medium (MM) (450 mL) containing carbenicillin (50 mg/mL) and a carbon source (2% w/v glucose or glycerol) in 500 mL-capacity vessels of a Sixfors Multi-Fermenter system. Cells were first grown at 30 1C for 22 h, then the temperature was increased to 37 1C for the duration of the experiment. NH4Cl (final concentration [Cf] ¼4 g/L) and the carbon source ([Cf] glucose¼ 1.25% w/v; [Cf] glycerol ¼1% v/v) were added to the appropriate culture at 46 h after initial inoculation. Cells were harvested at 70 h after the start of the experiment.
Table 2 Comparative CGP yields at different aeration rates and induction times with glycerol as substratea. Temperatures
Stirrer speed Air flow (L/ Dry cell (rpm) min) yield (g/L)
Crude CGP yield (mg/L)
30 1C, 6 h; then 37 1C, 42 h 30 1C, 24 h; then 37 1C, 24 h 37 1C, 48 h
400 150 400 150 400 150
133.2 7 12.7 17.9 70.2 7.07 0.2 15.2 71.7 149.5 7 39.1 17.6b
1.0 0.3 1.0 0.3 1.0 0.3
2.87 0.2 1.67 0.1 3.67 0.6 2.07 0.1 3.77 0.2 2.07 0.3
a Experimental set-up was basically as described in footnote of Table 1. Glycerol (2% w/v) was the starting carbon source in all experiments. NH4Cl ([Cf]¼ 4 g/L) and glycerol ([Cf] glycerol ¼ 1% v/v) were added to the cultures at 24 h after initial inoculation. Growth temperatures for the duration of the fermentation were as shown in the Table. Cells were harvested at 48 h after the initial inoculation. Duplicate experiments were performed for each set of fermentation conditions (i.e., n¼ 2). b The duplicate experiments yielded widely different values of crude CGP yields of 17.6 and 99.6 mg/L. Consistent with the results of the other low-aeration experiments, only the lower value is tabulated.
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Table 3 Concentration dependence and consumption of glycerol in CGP productiona. Initial glycerol concentration (v/v) (%)
2.0 1.0 0.5
Dry cell yield (g/L)
3.3 70.1 3.3 70.3 3.9 70.1
Crude CGP yield (mg/L)
82.2 7 11.0 63.2 7 2.0 50.7 7 2.2
Glycerol concentration (%, v/v)b Initial
Immediately before feeding
Immediately before feeding
At harvest
1.99 7 0.01 0.94 7 0.01 0.68 7 0.01
1.16 7 0.02 *b **b
2.68 70.06 0.857 0.02 *b
1.96 7 0.08 0.69 70.01 **b
a Experimental set-up was essentially as described in footnote of Table 1. Glycerol (0.5–2% v/v) was the starting carbon source in all experiments. NH4Cl ([Cf] ¼ 4 g/L) and glycerol ([Cf] glycerol ¼half of the initial concentration) were added to the cultures at 24 h after initial inoculation. Cells were grown for the entire 48-h fermentation at 37 1C under high aeration conditions (i.e., 400 rpm stirrer speed and 1.0 L/min air flow rate). Duplicate experiments were performed for each glycerol initial concentration tested (i.e., n¼2). b Glycerol concentration was determined using an HPLC as described in Section 2. *HPLC peak for glycerol was present but was too low to be picked up by the integration program of the instrument due to a high- and noisy-background/baseline. **HPLC peak for glycerol was not present in chromatogram.
Seed cultures were prepared in Erlenmeyer flasks (with a capacity Z5 culture volume) by growing E. coli [pMa/c5914::cphA] in MM containing the appropriate substrate (i.e., glucose or glycerol) for 18–24 h at 30 1C and 250 rpm shaking. We used 10 mL of the seed culture to inoculate 450-mL growth medium in each fermenter vessel (500-mL capacity). The air-flow rate and stirrer speed were set at values specified for each experiment (see Tables 1–3). We also studied the effect of upshifting growth temperature on the yields of cell mass and crude CGP. In all experiments, cells were fed at 24 h time-point after the initial inoculation with NH4Cl (by adding a 0.45-mm filter-sterilized 0.2 g/L stock solution to a final concentration of 4 g/L) and glucose (by adding a 0.45-mm filter-sterilized 40% w/v stock solution to a final concentration of 1.25% w/v) or glycerol (by adding 100% glycerol to a final concentration of 10% v/v). The amounts of NH4Cl and carbon source added approximated the total amounts added in Frey et al. (2002)’s large-scale multiplefed batch study. Aliquots of cultures were removed for glucose or glycerol determination at the following points: the start of the experiment, immediately before and after the addition of NH4Cl and carbon source at 24 h after the initial inoculation, and the end of experiment at 48 h after the initial inoculation. Cells were harvested and weighed (to obtain wet cell mass, WCM) at the end of experiment by centrifugation (Sorvall RC5B Plus, GS-3 rotor, 6500 rpm, 30 min, 4 1C), and were then lyophilized to determine the dry cell weight (DCW). Crude CGP was extracted using a method adopted from Frey et al. (2002). Briefly, the dried cells were resuspended in deionized water (1 mL per 0.1 g of cells). The suspension was acidified to pH 1 by adding concentrated HCl, which was then stirred for 6 h at ambient temperature. The mixture was centrifuged (Sorvall RC5B Plus, GS-3 or SS-34 rotor depending on volume, 6500–7500 rpm, 30–60 min, 4–10 1C) and the supernatant was collected in a clean flask. The cell pellet was subjected to a second acid extraction by resuspending it in 0.1 N HCl (1 mL per 0.1 g of original cell mass) and stirring for 1 h. Following a centrifugation step (see above for conditions), the supernatant was combined with the previously saved first-round acid extract. We isolated the crude CGP from the combined acidic cell extracts by precipitation at pH 7 through the addition of 10 N NaOH aqueous solution. The precipitates were collected by centrifugation (see above for conditions) and lyophilized to yield the crude CGP preparation. Alternatively, differential filtration technique was used to isolate crude CGP fraction in solution form. The combined acid extract was first filtered through a 0.45-mm MCE membrane filter (MILLEX HA, Millipore). The filtrate was loaded on a 0.1N NaOH-prewashed Amicon Ultracel 10k NMWL filter unit and centrifuged in a swinging-bucket rotor at 4000 rpm (3345 g) for 10 min at 4 1C in an AccuSpin 1R centrifuge (Fisher Scientific). The retentate (100–200 mL) constituted crude CGP fraction in liquid form.
2.3. Analysis of glucose and glycerol Glucose and glycerol concentrations were determined using a Shimadzu Prominence HPLC system equipped with an evaporative light scattering detector (ELSD-LTII) and a SIL-20A/C autosampler. Culture samples were filtered using 0.2-mm Acrodisc PVDF membrane filters (PALL Life Sciences) and the filtrates were used in the analysis. A guard column and a Chromegabond Carbohydrate column (5 mm, 100 A, 25 cm 4.6 mm; ES Industries, West Berlin, NJ) were employed for sample analysis. Column temperature was maintained at 40 1C using a CTO 20A chromatography column oven. Elution was effected using an isocratic mobile phase (acetonitrile/water mixture, 75/25, v/v) at 1 mL/min for 15 min. Glucose and glycerol solutions ranging from 0–4% (w/v) were prepared and used as standards. We used also an enzymatic assay to determine glycerol concentration prior to the development of the HPLC method described earlier. A Free Glycerol Determination Kit (SigmaAldrich) was used for this purpose. Culture samples were filtered through 0.2-mm Acrodisc PVDF membrane filters prior to use in the enzymatic assay. The absorbance at 540 nm was measured on either a Shimadzu BioSpec-1601 UV/visible spectrophotometer or a microplate reader. 2.4. SDS-PAGE analysis Denaturing discontinuous gel electrophoresis was performed on a Mini PROTEAN 3 Cell apparatus (BioRad Laboratories, Hercules, CA), using Ready Gel Precast 10% polyacrylamide gels (BioRad Laboratories). Lyophilized crude CGP was added to a 2X Sample Loading Buffer solution (18), heated to 95 1C for 4 min, and centrifuged (in an Eppendorf microcentrifuge at 14,000 g, 15 min, ambient temperature) to remove insoluble substance. An aliquot of the supernatant was loaded on the polyacrylamide gel, and electrophoresis was performed in TRIS–glycine–SDS buffer system (Laemmli, 1970). Polypeptides in the gel were visualized with Coomassie Blue dye (Ausubel et al., 1987) or using a SimplyBlue SafeStain Kit (Invitrogen) according to the supplier’s instructions. SDS-PAGE Markers (Bio-Rad Laboratories) were used as molecular-weight standards.
3. Results and discussion 3.1. Substrate preference and the effect of aeration conditions We first compared the yields of cell mass and crude CGP obtained under high- and low-aeration conditions using two different substrates, i.e., glucose and glycerol, as carbon source. While
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glucose in various forms (e.g., corn syrup and beet molasses) is a commonly used substrate in microbial fermentation, we are interested in glycerol as a carbon source because of its potential as a lowcost surplus coproduct from the biodiesel industry. Results in Table 1 showed that as expected with E. coli, high-aeration conditions (400 rpm stirrer speed, 1.0 L/min air flow) led to a significantly higher yield of cell biomass (measured as wet- and dry-cell weight) than the low-aeration conditions (150 rpm, 0.3 L/min air flow) did. The type of carbon source, however, did not affect the biomass yields under our experimental conditions. The crude CGP yields obtained by neutralization (pH 7) of acid extracts of the harvested cells also showed significant increase at high aeration rates (Table 1). The higher yield of crude CGP obtained with high-aeration conditions paralleled the observed increase in cell biomass under these conditions. We performed an SDS–PAGE analysis on the crude CGPs obtained from the glucose- and glycerol-fed cells to assess the CGP contents in the isolated crude products. The positive effect of aeration on the yield of CGP is most prominently shown by the results obtained with the glucose samples, and to a lesser extent also with those from glycerol-fed fermentation (Fig. 2). A comparison of the relative intensities of the ca. 27 kDa-bands attributed to CGP showed that high-aeration conditions (lanes 1 and 3, Fig. 2) led to a higher level of CGP synthesis than the low-aeration conditions (lanes 2 and 4, Fig. 2), suggesting that high oxygen content favors CGP production. This oxygen dependency is an important finding for CGP synthesis by genetically engineered E. coli that has not been previously reported. It is interesting to note that the production of surfactin – a microbial product similar to CGP synthesized via nonribosomal assembly of amino acids into polypeptide – is also favored by high aeration conditions (Yeh et al., 2006). Elbahloul et al. (2005a,b) had showed that CGP content of wild-type Acinetobacter calcoaceticus ADP1 could be increased by culturing the organism in baffled Erlenmeyer flasks to increase aeration. Aeration rate or oxygen content of a culture is an important fermentation parameter
that influences the biosynthesis of microbial products including the secondary metabolites (Frykman et al., 2002), and our results further add to the list of industrially useful microbial products whose production is closely tied to oxygen content of the medium. Notwithstanding that the mechanistic basis for the increased CGP synthesis as a result of high aeration rate is not determined, ours and other researchers’ results clearly point to the importance of providing enough dissolved oxygen in culture medium in order to obtain high CGP production. More importantly, our results further show that glycerol is preferred as a substrate than glucose in supporting CGP synthesis. By comparing the intensities of the CGP bands in lanes 1 and 3 of Fig. 2 using an image integration program (ChemiImager 5500, Alpha Innotech Corp., San Leandro, CA), we estimated that glycerol afforded 1.8-times higher yield of CGP than glucose did under similar experimental conditions. This result agrees with the observation of Frey et al. (2002) in their large-scale fermentation runs. In their study, glycerol at the initial feed concentration of 2% afforded the synthesis of 1.64 g CGP/L culture (i.e., CGP content of 12% w/w CDW at 13.7 g/L cell density). Glucose at 2.5% initial feeding, on the other hand, only produced 0.23 g CGP/L culture (i.e., CGP content of 3% w/w CDW at 7.8 g/L cell density). The metabolic basis for the different ability of glucose and glycerol to support CGP production is not presently understood. Nevertheless, our confirmatory results to those of Frey et al. (2002) reinforce the conclusion that glycerol is a preferred substrate over glucose for CGP production. In fact, many studies on CGP synthesis have been performed using glycerol in place of glucose as a substrate with the consideration that glycerol is preferable (Steinle et al., 2010; Aboulmagd et al., 2001). A practical implication of this observation is that the glycerol coproduct stream from biodiesel production could in fact be a desired substrate for CGP production, which in turn would improve the economics of the biofuel production by making available a value-added coproduct to the overall biorefinery scheme. 3.2. Effects of temperature on CGP production
Fig. 2. SDS-PAGE of crude cyanophycin preparations. Lyophilized CGP (0.5–2.2 mg) was solvated in a Sample Loading Buffer solution (Laemmli, 1970) at a concentration of 10 mg/mL. The mixture was heated at 95 1C for 4 min, and centrifuged to remove insoluble materials. Electrophoresis was performed on a 10% polyacrylamide gel with 10 mL of the supernatant samples. Crude CGP preparations from cells grown on glucose (lanes 1 and 2) and glycerol (lanes 3 and 4) at 400 rpm stirrer speed and 1 L/min air flow (lanes 1 and 3) or 150 rpm stirrer speed and 0.3 L/min air flow (lanes 2 and 4) are shown. CGP at 27 kDa is indicated with an arrow head (c). M, molecular-weight protein markers.
While performing the experiments reported in Table 1, we observed that the cell density (as monitored by absorbance at 600 nm) of E. coli [pMa/c5-914::cphA] culture at the end of the 6 h initial cultivation at 30 1C was low and inconsistent. We decided to perform a separate set of experiments to examine the effects of shifting the incubation temperature on the yields of dry cell mass and CGP. Results from this set of experiments show that a rather consistent crude CGP yield of 15–18 mg/L was observed under low aeration conditions (150 rpm stirrer speed; 0.3 L/min air flow rate) regardless of the temperature regiments used (Table 2). Moreover, we could only reach a low cell density of o2 g/L when fermentation was carried out under the low aeration conditions, again regardless of the temperature settings (Table 2). These results show that under low aeration conditions, incubation temperature between 30 and 37 1C does not affect the already low yields of cell mass and CGP. The results obtained under high aeration conditions (400 rpm stirrer speed; 1.0 L/min air flow rate), on the other hand, show a clear dependence of CGP production on the variation of incubation temperature. We noted from the results that there is a direct correlation between the length of incubation at 37 1C and the crude CGP yield. When the entire 48 h of fermentation was performed at 37 1C, a crude CGP yield of 150 mg/L was observed (Table 2). Conversely, when half (i.e., 24 h) of the time of fermentation was performed at 30 1C followed by the other half (i.e., 24 h) at 37 1C, the crude CGP yield dramatically dropped to 7 mg/L (Table 2). The CGP yield obtained with the first 6 h of fermentation performed at 30 1C and the rest (i.e., 42 h) at 37 1C
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was 133 mg/L (Table 2), which was not statistically different from that observed with the entire fermentation performed at 37 1C. These results suggest that CGP production occurs mainly when the culture temperature is at 37 1C. The cell mass yield, on the other hand, was not significantly affected by the different temperature regiments used here under high aeration conditions. Data in Table 2 show that regardless of temperature-shifting pattern, dry cell mass yields were only slightly varied between 2.8 and 3.7 g/L and were not considered statistically significant. We had initially adapted the 30–37 1C temperature-shift pattern based on the large-scale experimental set-up of Frey et al. (2002). The rationale was that the expression of the cphA gene in the recombinant plasmid (i.e., pMa/c5-914::cphA) is under the regulation of a temperature-sensitive repressor (i.e., cI857; Frey et al., 2002). Therefore, Frey et al. (2002) first grew the cells under cphA-uninduced state at 30 1C to achieve as high a cell density as possible before implementing a temperature up-shift to 37 1C to de-repress cphA gene expression for CGP synthesis. Our results, however, indicated that an optimal CGP production could occur without the need for the initial growth of cells under cphA-repressed state at 30 1C, thus alleviating the inconvenience of monitoring cell growth and temperature shift during initial stage of fermentation. These results underscored the importance of a systematic study of experimental conditions at bench-scale fermentation to identify parameters crucial to CGP production, despite of earlier study at large-scale technical production Frey et al. (2002). 3.3. Effect of glycerol concentration and its conversion efficiency to CGP An efficient bioprocess requires that substrate conversion or utilization and the product yield are optimal. We thus conducted a series of experiments to study the effect of glycerol concentration on crude CGP yields under high aeration conditions. The results (Table 3) show that crude CGP yield is positively correlated with the total glycerol concentration fed to the culture. At initial glycerol concentration of 2% followed by 1% feeding midway through the fermentation, we obtained a crude CGP yield of 82.2 mg/L (Table 3). When 0.5% initial followed by 0.25% midcourse feedings of glycerol were tested, only 50.7 mg/L of crude CGP yield was produced at the end of the fermentation (Table 3). The patterns of glycerol consumption are valuable to ensure efficient substrate utilization. At 2% initial and 1% mid-course feedings of glycerol, close to 2/3 of the total substrate added still remained at the end of fermentation (Table 3, 1st row data). Conversely, it appeared that all of the added glycerol was consumed in the experiments in which the initial and midway feedings were 0.5% and 0.25%, respectively (Table 3, 3rd row data). A conversion rate of 50.7 mg CGP/7.5 g glycerol was obtained under this complete consumption condition. When we started the fermentation with 1% glycerol concentration, we observed that nearly all of it was consumed midway through the fermentation (Table 3, 2nd row data). Further addition of 0.5% glycerol resulted in a leftover of substrate at a concentration of 0.69%, which amounted to an overall substrate consumption rate of 54%. Under this substrate feeding scheme, a substrate-toproduct conversion rate of 63.2 mg CGP/8.1 g glycerol was observed. Taken together, these data suggest that an optimal conversion rate could be achieved by providing enough glycerol for CGP biosynthesis to complete (as in Table 3, 2nd row experiments), without an excessively wasteful addition of the substrate (as in Table 3, 1st row experiments). The effect of the concentration of carbon-source substrate on CGP synthesis had only been reported by Frey et al. (2002) in a technical-scale fermentation study. Contrary to our findings, however, Frey et al. (2002) found that lowering the initial concentration of glycerol
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from 4% to 2% resulted in a nearly 6-fold increase of CGP production. The different results observed between our benchscale and Frey et al.’s technical-scale fermentations again underscore the necessity of systematic optimization of bioprocessing systems to further the commercial prospects of CGP production.
Acknowledgment The authors thank Nicole Crocker and Bun-Hong Lai for ¨ technical assistance, and Professor Alexander Steinbuchel ¨ ¨ Munster, ¨ (Westfalische Wilhelms-Universitat, Germany) for the kind gift of Escherichia coli [pMa/c5-914::cphA]. References ¨ Aboulmagd, E., Voss, I., Oppermann-Sanio, F.B., Steinbuchel, A., 2001. Heterologous expression of cyanophycin synthetase and cyanophycin synthesis in the industrial relevant bacteria Corynebacterium glutamicum and Ralstonia eutropha and in Pseudomonas putida. Biomacromolecules 2, 1338–1342. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., et al., 1987. Analysis of proteins. In: Ausubel, et al. (Eds.), Current Protocols in Molecular Biology. John Wiley & Sons, New York (Chapter 10). ¨ Elbahloul, Y., Krehenbrink, M., Reichelt, R., Steinbuchel, A., 2005a. Physiological conditions conducive to high cyanophycin content in biomass of Acinetobacter calcoaceticus strain ADP1. Applied Environmental Microbiology 71, 858–866. ¨ Elbahloul, Y., Frey, K., Sanders, J., Protamylasse, Steinbuchel A., 2005b. A residual compound of industrial starch production, provides a suitable medium for large-scale cyanophycin production. Applied Environmental Microbiology 71, 7759–7767. ¨ Frey, K.M., Oppermann-Sanio, F.B., Schmidt, H., Steinbuchel, A., 2002. Technicalscale production of cyanophycin with recombinant strains of Escherichia coli. Applied Environmental Microbiology 68, 3377–3384. Frykman, S.A., Tsuruta, H., Starks, C.M., Regentin, R., Carney, J.R., Licari, P.J., 2002. Control of secondary metabolite congener distributions via modulation of the dissolved oxygen tension. Biotechnology Progress 18, 913–920. ¨ ¨ Fuser, G., Steinbuchel, A., 2007. Analysis of genome sequences for genes of cyanophycin metabolism: identifying putative cyanophycin metabolizing prokaryotes. Macromolecular Bioscience 7, 278–296. ¨ Joentgen, W., Groth, T., Steinbuchel, A., Hai, T., Oppermann, F.B., 1998. Polyasparaginic Acid Homopolymers and Copolymers, Biotechnical Production and Use Thereof. International patent application, WO 98/39090. ¨ Konst, P.M., PMCCD, Turras, Franssen, M.C.R., Scott, E.L., Sanders, J.P.M., 2010. Stabilized and immobilized Bacillus subtilis arginase for the biobased production of nitrogen-containing chemicals. Advanced Synthesis and Catalysis 352, 1493–1502. Koutinas, A.A., Wang, R.-H., Webb, C., 2007. The biochemurgist—bioconversion of agricultural raw materials for chemical production. Biofuels Bioproducts and Biorefining 1, 24–38. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Miller, T.C., Holcombe, J.A., 2001. Comparison and evaluation of the synthetic biopolymer poly-L-aspartic acid and the synthetic plastic polymer poly-acrylic acid for use in metal ion-exchange systems. Journal of Hazardous Materials 83, 219–236. Mooibroek, H., Oosterhuis, N., Giuseppin, M., Toonen, M., Franssen, H., Scott, E., et al., 2007. Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied Microbiology and Biotechnology 77, 257–267. ¨ Nakano, K., Rischke, M., Sato, S., Markl, H., 1997. Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Applied Microbiology and Biotechnology 48, 597–601. Norton, J.M., Klotz, M.G., Stein, L.Y., Arp, D.J., Bottomley, P.J., Chain, P.S.G., et al., 2008. Complete genome sequence of Nitrosospira multiformis, an ammoniaoxidizing bacterium from the soil environment. Applied Environmental Microbiology 74, 3559–3572. ¨ Obst, M., Steinbuchel, A., 2004. Microbial degradation of poly(amino acid)s. Biomacromolecules 5, 1166–1176. ¨ Oppermann-Sanio, F., Steinbuchel, A., 2002. Occurrence, functions and biosynthesis of polyamides in microorganisms and biotechnological production. Naturwissenschaften 89, 11–22. ¨ Reinecke, F., Steinbuchel, A., 2009. Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers. Journal of Molecular Microbiology and Biotechnology 16, 91–108. ¨ Sallam, A., Steinbuchel, A., 2010. Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production. Applied Microbiology Biotechnology 87, 815–828. Sanders, J., Scott, E., Weusthuis, R., Mooibroek, H., 2007. Bio-refinery as the bioinspired process to bulk chemicals. Macromolecular Bioscience 7, 105–117.
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D.K.Y. Solaiman et al. / Biocatalysis and Agricultural Biotechnology 1 (2012) 9–14
Schwamborn, M., 1998. Chemical synthesis of polyaspartates: a biodegradable alternative to currently used polycarboxylate homo- and copolymers. Polymer Degradation and Stability 59, 39–45. Solaiman Daniel, K.Y., Garcia Rafael, A., Ashby Richard, D., Piazza George, J., ¨ Steinbuchel Alexander, Rendered-protein hydrolysates for microbial synthesis of cyanophycin biopolymer. New Biotechnology, in press. doi:10.1016/j.nbt. 2011.03.025. ¨ Steinle, A., Witthoff, S., Krause, J.P., Steinbuchel, A., 2010. Establishment of cyanophycin biosynthesis in Pichia pastoris and optimization by use of engineered cyanophycin synthetases. Applied Environmental Microbiology 76, 1062–1070.
Van Beilen, J.B., Poirier, Y., 2008. Production of renewable polymers from crop plants. Plant Journal 54, 684–701. Yang, J., Wang, F., Tan, T., 2009. Controlling degradation and physical properties of chemical sand fixing agent-poly(aspartic acid) by crosslinking density and composites. Journal of Applied Polymer Science 111, 1557–1563. Yeh, M.-S., Wei, Y.-H., Chang, J.-S., 2006. Bioreactor design for enhanced carrierassisted surfactin production with Bacillus subtilis. Process Biochemistry 41, 1799–1805.