Maintenance-energy-dependent dynamics of growth and poly(3-hydroxybutyrate) [P(3HB)] production by Azohydromonas lata MTCC 2311 using simple and renewable carbon substrates

BIOPROCESS ENGINEERING

Maintenance-energy-dependent dynamics of growth and poly(3-hydroxybutyrate) [P(3HB)] production by Azohydromonas lata MTCC 2311 using simple and renewable carbon substrates

M. Zafar^I,III,*; S. Kumar^II; S. Kumar^II; A. K. Dhiman^II; H.-S. Park^III,*

^IDepartment of Biological Engineering, Shobhit University, Meerut-250 110, U.P., India. Phone: [phone], Fax: [phone]. E-mail: [e-mail]

^IIDepartment of Chemical Engineering, Indian Institute of Technology Roorkee, Roorkee-247 667, Uttarakhand, India

^IIICenter for Clean Technology and Resource Recycling, University of Ulsan, Ulsan, Republic of Korea. Phone: [phone]; Fax: [phone], E-mail: [e-mail]

ABSTRACT

The dynamics of microbial growth and poly(3-hydroxybutyrate) [P(3HB)] production in growth/ non-growth phases of Azhohydromonas lata MTCC 2311 were studied using a maintenance-energy-dependent mathematical model. The values of calculated model kinetic parameters were: m_s1 = 0.0005 h^-1, k = 0.0965, µ_max = 0.25 h^-1 for glucose; m_s1 = 0.003 h^-1, k = 0.1229, µ_max = 0.27 h^-1 for fructose; and m_s1 = 0.0076 h^-1, k = 0.0694, µ_max = 0.25 h^-1 for sucrose. The experimental data of biomass growth, substrate consumption, and P(3HB) production on different carbon substrates were mathematically fitted using non-linear least square optimization technique and similar trends, but different levels were observed at varying initial carbon substrate concentration. Further, on the basis of substrate assimilation potential, cane molasses was used as an inexpensive and renewable carbon source for P(3HB) production. Besides, the physico-chemical, thermal, and material properties of synthesized P(3HB) were determined which reveal its suitability in various applications.

Keywords: Poly(3-hydroxybutyrate); Microbial dynamics; Cane molasses; Maintenance energy; Azohydromonas lata.

INTRODUCTION

In response to the problems associated with the application of synthetic oil-based polymers, the scientific communities are searching for alternative materials and their sustainable process development. The main concern of polymer industries and decision makers is to minimize the dependency on nondegradable polymers under the present scenario of environmental awareness (Akaraonye et al., 2010). It is of prime importance to assess the biodegradable polymer production potential of plants, microbes, and the archaea domain of life to reduce the environmental impact of petroleum-based polymers. The microbial production of polyhydroxyalkanoates (PHAs) is considered to be a 'green bio-refinery process', which protects the environment by reducing the CO₂ emission in the atmosphere (Chen, 2011). The microbial PHAs have a broader diversity in terms of monomer units, i.e., about 180 types of different monomers exist in nature. Poly(3-hydroxybutyrate) (P3HB) is a type of bacterial PHA that possesses almost similar thermo-plasticity and low O₂ permeability to that of petro-plastic. It is synthesized and accumulates intracellularly as carbon and energy granules up to 90% of the cellular dry weight and possesses unique thermal and material properties which make it suitable for various applications in processing industries, agriculture, and medical fields (Gouda et al., 2001; Chen, 2011).

Many bacterial species such as Alcaligenes sp., Pseudomonas sp., Aeromonas hydrophila, Rhodopseudomonas, Methylobacterium, Saccharophagus degradans, Bacillus sp., and Azotobacter sp., have been used extensively in the last decade for the production of PHAs (Castilho et al., 2009; Gouda et al., 2001; Annuar et al., 2006; Chen et al., 1991; Chen, 2011). The industrial production of biopolymer takes place under an excess of carbon source and a limited amount of certain nutrients such as nitrogen (A. latus, P. oleovorans, Ralstonia eutropha), carbon (Spirillum sp., Hyphomicrobium sp.), iron, magnesium (Pseudomonas sp.), oxygen (Azotobacter vinelandii, Rhizobium ORS 571), phosphate (Rhodobacter rubrum, Caulobacter crecentus), and potassium sulfate (Bacillus, Rhodospirillum sp.). The industrial production of PHAs is expensive due to the associated high cost of carbon sources, which can account for up to 29% of the overall production cost (Kim and Lenz, 2000). Therefore, it is essential to investigate the assimilation potential of bacterial strain towards simple carbon substrates such as glucose, fructose, sucrose, lactose, and xylose and subsequently renewable and inexpensive sources like cane molasses, cheese whey, and lignocellulosic materials.

It is essential to describe the dynamics of microbial growth and growth-associated production of PHAs in order to sustain the accumulation potential of microorganism. The suitable mathematical models have been used to describe the dynamic behavior of microorganisms using kinetic approaches based on growth parameters. Some of the researchers have incorporated 'maintenance expenditure' for painstaking explanations of microbial growth/product kinetics (Pirt, 1965, 1982; Bodegom, 2007; Nancib et al., 1993; Nielsen et al., 2005). The term physiological maintenance is used for endogenous metabolism which comprises the energy expenditure for osmo-regulation, cell-motility, active transport of bio-molecules, defense mechanisms, and proofreading/turnover of macromolecules (Bodegom, 2007; Nielsen et al., 2005). The physiological maintenance does not include the energy expenditures of metabolic pathways, storage of polymers (PHAs), and extracellular losses (Bodegom,2007). The maintenance expenditure is not constant throughout the fermentation process and varies with the substrate dependent growth with respect to specific growth rate (µ) and yield coefficient. Thus, in order to assess the economic feasibility of the use of carbon substrates, it is essential to evaluate the energy expenditures in terms of growth, maintenance, and P(3HB) formation.

In this study, the dynamics of microbial growth and P(3HB) production by A. lata MTCC 2311 using different carbon sources, namely glucose, fructose, and sucrose is assessed. The kinetics of growth and P(3HB) production are evaluated by including the growth-dependent-maintenance energy expenditure. A thorough discussion has been provided for a better insight of maintenance energy expenditure during growth and P(3HB) production. The experimental data derived for these carbon sources were fitted by mathematical models developed to represent the biomass growth, substrate consumption, and P(3HB) production profiles. Besides, the potential of A. lata for P(3HB) production using cane molasses as an inexpensive and renewable carbon source is also examined with its physico-chemical characterization.

MATERIALS AND METHODS

Microorganism and Seed Culture Preparation

The lyophilized culture of Alcaligenes latus MTCC 2311 was procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India, which is reclassified as Azohydromonas lata (Xie and Yokota, 2005). The culture was first revived in nutrient broth for 24 h followed by inoculum preparation in AL₂ medium (pH 7.0) which contained (g/L): Sucrose, 20.0; (NH₄)₂SO₄, 1.0; KH₂PO₄, 0.6; Na₂HPO₄.12H₂O, 3.6; MgSO₄.7H₂O, 1.0; CaCl₂.2H₂O, 0.1; citric acid, 0.1; and trace element solution 3 mL/L (Wang and Lee 1997). The inoculum was prepared by using 50 ml of AL₂ medium in 250 mL capacity conical flasks kept at 30 ºC and 180 rpm for 24 h in an orbital shaking incubator. The culture was maintained at 4 ºC on slants of AL₂ agar for further applications.

P(3HB) Production on Different Carbon Sources

In order to promote the P(3HB) production, 5% inoculum containing approximately 10⁶ cells/ml of medium was used to inoculate the P(3HB) production medium, which contained (g/L): carbon substrate (5 to 40), (NH₄)₂SO₄ 1.0, MgSO₄.7H₂O 1.0, citric acid 0.1, CaCl₂.2H₂O 0.1, Na₂HPO₄.12 H₂O 3.6, KH₂PO₄ 0.6, and Trace Element (TE) solution 3 mL/L, and pH 6.8. The different pure carbon substrates such as glucose, fructose, and sucrose were used. The P(3HB) production was carried out in AL₂ medium with varying C/N ratio at 30 ºC and agitation speed of 180 rpm for 72 h. The samples were collected at regular intervals for determination of biomass growth, P(3HB) concentration, and residual sugar concentration.

Analytical Procedures

The biomass concentration was estimated turbidimetrically at 600 nm using a UV-VIS Spectrophotometer (Lambda 35, Perkin Elmer, MA, USA). In addition, the bacterial cell pellet was collected by centrifugation at 8000×g for 10 min of 5 mL culture samples drawn at regular interval. The centrifuged cell pellet was washed twice with distilled water and dried at 80 ºC in a hot air oven to a constant weight. The quantification of P(3HB) was carried out by the propanolysis method with little modification using a gas chromatograph (Thermo, USA) (Riis and Mai 1988). The analysis of sugars was carried out on a high performance liquid chromatograph (HPLC) (Waters, USA), equipped with a sugar-pak column (6.5×250 mm length, Waters, USA) and a refractive index (RI) detector (model 24140, Waters). Deionized-water at 90 ºC was used as eluent with a flow rate of 0.5 ml/min. The cell-free supernatant was analyzed for residual (NH₄)⁺ ion concentration by the phenolhypochlorite method (Solozano 1969).

Physico-Chemical Characterization of Synthesized P(3HB)

Fourier transform infrared (FTIR) spectra of extracted P(3HB) were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo Scientific, USA) between 4000 and 500 cm^-1. The ¹³C Nuclear magnetic resonance (NMR) spectra of samples were recorded at 75.4 MHz on a model Av 500 MHz NMR spectrometer (Bruker Inc., USA) using deuterated chloroform (CDCl₃) as solvent. The thermal characteristics of P(3HB) samples were measured with a Pyris Diamond Thermogravimetric Analyser (Perkin Elmer Inc, Wellesley, MA, USA). The analysis was carried out in an inert gas atmosphere of nitrogen at a 100 mL/min flow rate in the temperature range from 25 to 250 ºC at a heating rate of 10 ºC/min. The molecular mass of the extracted P(3HB) sample was determined with a gel permeation chromatography (GPC) system (Waters Inc., MA, USA) at 40 ºC. Two columns in series (high resolution HSP gel HR 2.5 and HSP gel HR 3.0, 6.0×15 cm length, Waters) were used with a RI detector. A narrow dispersion polystyrene and tetrahydrofuran (THF) with a flow rate of 0.6 mL/min were used as the MW standard and mobile phase, respectively.

Maintenance-Energy-Dependent Kinetics and Development of Mathematical Models

Several batch studies were conducted to estimate the kinetic parameters of biological processes with particular attention to the substrate utilization for cell maintenance (Minkevich et al., 2000; Nielson et al., 2005; Nancib et al., 1993; Djavan and Jones, 1980). But little attention has been focused on the maintenance energy related kinetics during PHAs production (Liu et al., 2005; Wang et al., 2007). The microorganisms need energy for growth, intracellular/ extracellular product formation, and for maintenance functions such as transportation of cellular materials, osmotic regulation, defense against O₂ stress, cell motility, and for proofreading, synthesis, and turnover of cellular macromolecules, viz. enzyme, and RNA (Bodegom et al., 2007; Oliveira et al., 1992; Pirt, 1982). The kinetic approach to microbial dynamics requires the incorporation of maintenance energy expenditure with biomass growth under various types of bioprocesses such as primary/secondary metabolite production, bioremediation, bioaccumulation and biosorption (Bodegom et al., 2007).

During the fermentation process, substrates are utilized by microbial cells for biomass production, product formation, and for maintenance expenditure. The exponential growth of bacterial cell biomass is expressed as:

in which µ is the specific growth rate (h^-1) according to Monod:

where µ_max is the maximum specific growth rate (h^-1), K_sis the substrate saturation constant (g/L), and S is the substrate concentration (g/L).

Here, the intracellular P(3HB) accumulation is considered as a part of the biomass and both a growth and non-growth associated product (Wang and Lee, 1997). It is assumed that substrate inhibition of biomass growth is not observed. Thus, the microbial growth and growth-associated P(3HB) accumulation are associated with the types of substrate and their concentration.

During fermentative production of P(3HB), the initial microbial concentration (inoculum) and substrate are considered as reactants which, upon biocatalytic reaction, lead to an increase in the biomass growth as well as in P(3HB) accumulation. Thus, the microbial growth dynamics are determined by both the microbial specific growth rate and the substrate consumption rate.

The rate of substrate consumption, analogous to the biomass growth rate as a function of biomass concentration is given by:

where q_s is the specific substrate consumption rate and can be expressed in terms of the true growth yield and maintenance coefficients as:

where Y_{X /S} is maximum growth yield coefficient (g/g) and m_s is the maintenance energy coefficient (h^-1).

Upon substitution of Eq. (2) into Eq. (4)

When the product formation (P(3HB)) is related to carbon source consumption:

Upon substitution of Eq. (4) into Eq. (6)

where Y_P/S is the maximum P(3HB) yield coefficient (g/g).

In addition, the P(3HB) production by bacterial cells is described as:

where q_P(3HB) is the product formation rate, and K₁ and K₂ represent the growth and non-growth associated product constant, respectively.

The straight line for q_P(3HB) verses µ gives the slope K₁ and intercept K₂ and reveals the type of P(3HB) production. If the straight line passes through the origin, K₂is zero and hence the fermentation is growthassociated, otherwise it is of the mixed type, i.e., both growth and non-growth associated.

The free energy change during catabolic reaction is slightly coupled with the anabolic cellular biomass and biopolymer accumulation and the total energy flux is partitioned into biomass and the maintenance function (Wang et al., 2007).

The total maintenance energy expenditure is not constant throughout the bioprocess and can be divided into two components; one is a constant which is required throughout the cultivation process and the other component is growth dependent (Pirt, 1982). The second component of maintenance energy depends on the specific growth rate (µ) and decreases hyperbolically as a function of the specific growth rate. The maintenance is considered to be the consumption phenomenon, corresponding to energy wastage, which is increased under unfavorable environmental conditions. In general, a low maintenance value (m_s) is observed at high specific growth rate that favors biomass growth and metabolite production (Pirt, 1982).

According to Pirt, 1982:

m_s =m_s1 +m_s2 (10)

where, m_sis maintenance energy expenditure, ms₁ is the constant and ms₂ is the growth-dependent component of the maintenance coefficient.

where k is a positive quantity that depends on the substrate-microorganism system. The above relationship describes the cellular metabolic efficiency under specific environmental conditions. It is observed that the maintenance is high when the specific growth rate is low and vice versa.

In addition to the specific growth rate, the maintenance energy expenditure also depends on some physical factors such as temperature and salt concentration in the medium.

Upon substituting Eq. (11) into Eq. (4)

where k (1-µ µ_max )is growth dependent and m_s1 is the constant component of maintenance energy.

During the exponential growth of the microorganism m_s2 is required for the cell growth and cellular function in addition to m_s1. The amount of m_s2 decreases with the increase in growth rate and approaches zero at the maximum specific growth rate (µ_max). Eq. (12) can be rewritten as:

Eq. (14) is not exactly applicable in the case of cell dormancy resulting in a very slow specific growth rate (Pirt, 1982). In our study, Azohydromonas lata efficiently utilized the simple carbon substrate without any lag phase.

The percent of the total substrate (carbon source) consumption used for cell energy maintenance varies with the specific growth rate of microorganism and calculated as (Djavan and James, 1980):

where m_s (h^-1) is total maintenance expenditure (m_s1 +m_s2) and q_s is the specific substrate consumption rate (g/g).

RESULTS AND DISCUSSION

Maintenance-Energy-Dependent Kinetics and Mathematical Modeling

The dynamics of biomass growth and P(3HB) production by A. lata MTCC 2311 were studied in defined AL₂ medium supplemented with varying concentrations of glucose, fructose, and sucrose. About 65% of the initial concentration of these carbon sources (30 g/L) were metabolically assimilated and high values of biomass and P(3HB) concentrations of 8.35 and 3.60 g/L were observed with fructose followed by sucrose (7.92 and 3.50 g/L) and glucose (7.55 and 3.40 g/L), respectively. It is noteworthy that the simple carbohydrates such as glucose, fructose and sucrose are mostly processed through the EMP pathway to yield pyruvate and acetate as the main products. Acetate is partially converted into P(3HB) through synthesis of acetyl CoA and further consumed during the sporulation process of bacteria. It is also documented that the glucose in the medium represses the expression of the phospotransbutylase (Ptb) gene that is needed for PHB production (Shamala et al., 2009). This may be a possible reason due to which fructose gave a little higher yield than glucose and sucrose in the medium. Table 1 shows the calculated growth and P(3HB) kinetic parameters, along with the maintenance expenditure during microbial metabolism on various carbon substrates. Almost similar kinetic constants were observed for all tested sugars with slightly higher values for fructose: µ_max = 0.27 h^-1; Y_{X /S} = 0.45; Y_P/S = 0.20; q_s = 0.60; q_p = 0.12, and Y_c (carbon yield) = 0.24.

The maintenance expenditure is an important biological kinetic parameter, that is not constant throughout the bacterial metabolic processes. It may vary with the increase in substrate concentration and growth rate of the microorganism. The growth-ratedependent maintenance energy expenditure is represented by Eq. (11). In order to calculate both the constant and growth-dependent parts of the maintenance expenditure, the experimental data for biomass growth derived from varying sugar concentrations were fitted to Eq. (11) and can be represented as:

Eqs. (16) - (18) revealed that the maintenance energy requirement of microbial cells during the substrate assimilation and metabolic activities was associated with the growth and P(3HB) production. The maximum maintenance expenditure was observed with sucrose (0.01294 h^-1) followed by fructose (0.01218 h^-1) and glucose (0.00436 h^-1) in the medium (Table 1). This means that, in order to produce one gram of biomass by A. lata, about 12.94, 12.18, and 4.36 mg h^-1 of sucrose, fructose, and glucose, respectively, are needed just to meet the demands of cell energy maintenance. The assimilation of sucrose and fructose takes place through a branch metabolic pathway of glycolysis to convert them into pyruvate, which subsequently results in 3HB monomers through the β-oxidation pathway. Some additional maintenance energy is required during the expression of specific genes which lead to the synthesis of specific proteins, i.e., enzymes that participate in these metabolic pathways.

The maintenance energy was not constant throughout the fermentation process and changed with the specific growth rate of A. lata. Similarly, the percent of carbon used as maintenance energy decreased with the increase in specific growth rate (Fig. 1). It has been reported that the maintenance energy consumption corresponds to the energy wastage and increases under adverse environmental conditions (Nancib et al., 1993). At a low specific growth rate of 0.12 h^-1, about 22% of fructose was used as maintenance energy. However, only 2.14% of fructose expenditure was observed as maintenance energy at the high specific growth rate of 0.25 h^-1.

Similarly, about 16.85% and 15.93% of glucose and sucrose, respectively, were consumed as maintenance requirement at the low specific growth rate of 0.11 h^-1. These expenditures were decreased to 0.73% and 2.22% of glucose and sucrose, respectively, at the high specific growth rate of 0.24 h^-1. The high specific growth rate indicates a favorable environment for bacterial growth at which the maintenance requirement is comparatively low.

The varying concentration of carbon substrates such as glucose, fructose, and sucrose have significant effects on the observed specific microbial growth, specific substrate consumption, and P(3HB) production rates [Figs. 2(a)-(c)]. It can be seen that Eqs.(2), (5), and (8) have appropriately modeled the experimental data for biomass growth, substrate consumption, and P(3HB) formation, respectively. Fig. 2(a) shows the effect of fructose concentration on the specific growth rate, substrate consumption, and P(3HB) production rates, as well as satisfactory modeling of experimental data with determination of correlation coefficients (R²) of 0.98, 0.99, and 0.96, respectively. The maximum specific biomass growth rate of 0.27 h^-1 was reported at the concentration of 30 g/L. Similarly, the maximum substrate consumption ( q_p ) and P(3HB) production rates (q_P(3HB)) of 0.60 and 0.12 h^-1, respectively, were reported at the same concentration of fructose. Very similar trends were observed with glucose and sucrose in the medium, but to different extents (Fig. 2(b)-(c) and Table 1).

The calculated specific growth and specific P(3HB) production rates were fitted using Eq. (10) to predict the nature of P(3HB) synthesis. A straight line correlation was observed between µ and q_P(3HB)with a positive intercept on the y-axis (K₂) and slope (K₁) for all simple carbon sources, viz., glucose, fructose, and sucrose (Fig. 3). This indicates that the P(3HB) production is of the mixed type, i.e., both growth and non-growth associated. The higher value of K₁ than K₂ also indicates that the accumulation of P(3HB) inside the microbial cells is more growthassociated than non-growth associated (stationary phase).

P(3HB) Production Using Inexpensive Cane Molasses

Sugarcane molasses is an inexpensive and renewable carbon substrate derived from the massive sugar industry in India. The cane molasses obtained from a sugar industry (Uttam Sugar Mills Ltd., Roorkee, India) contains reducing sugars (46.8%) such as glucose (5.50%), fructose (9.54%), and sucrose (31.75%) in addition to some growth promoters, ions, minerals, and vitamins in abundance. The experiments were conducted at different concentrations (1.5 to 7.5%, w/v) of cane molasses to promote biomass growth and P(3HB) accumulation by A. lata. The course of dry cell weight, P(3HB) concentration, and the specific P(3HB) productivity is illustrated in Fig. 4. The optimum dry cell weight and P(3HB) of 7.85 and 3.75 g/L, respectively, were observed at 4.5% (w/v) of cane molasses and no significant further increase in biomass growth and P(3HB) was observed at high concentrations of cane molasses.

The maximum specific P(3HB) formation rate of 0.094 g/L.h was found at 4.5% (w/v) of cane molasses. The maximum specific growth rate (µ_max) of 0.29 h^-1 and substrate (cane molasses) saturation constant (Ks) of 1.67% (w/v) were estimated by the least square technique using the curve fitting toolbox of MATLAB 7.8.1 (The Mathworks Inc., MA, USA). The production of P(3HB) using cane molasses has been reported by some researchers (Beaulieu et al., 1995; Gouda et al., 2001). Beaulieu et al. (1995) reported a maximum accumulation of 26% (w/w) of dry weight by Alcaligenes eutropha DSM 545 on different concentrations of cane molasses. Similarly, Gouda et al. (2001) reported a maximum P(3HB) concentration of 46.20% per mg cell dry weight with 2% molasses, while best growth was obtained for Bacillus megaterium with 3% molasses.

Physico-Chemical, Thermal and Material Properties of Produced P(3HB)

The qualitative evaluation of the structural composition of P(3HB) produced by A.lata on cane molasses was analyzed by FTIR and NMR spectroscopy. The FTIR spectrum revealed the band at 1450 cm^-1 corresponding to the asymmetrical deformation of the C-H bond in the CH2 group, and the equivalent at 1380 cm^-1 for CH3 group (Fig. 5). The bands at 1728 and 1280 cm^-1 correspond to the stretching of the C=O bond, while a series of intense bands located between 1280 to 1000 cm^-1 represent the C-O bond stretch of the ester group and characterize the valence vibration of the carboxyl group. The ¹³C NMR spectrum represents the P(3HB) structural composition as shown in Fig. 6. The spectrum reveals the presence of different types of carbon atoms in the P(3HB) structure. The chemical shift signals at 19.75, 40.66, 67.45, and 169.10 ppm represent the different carbons such as CH3, CH2, CH and C=O, respectively. The FTIR absorption bands of extracted P(3HB) are in close agreement with those observed by Rozsa et al. (1996) and Oliveira et al. (2007), who characterized the PHB produced by Bacillus circulans in submerged fermentation and by Cupriavidus nector in solid state fermentation, respectively. The ¹³C NMR spectra of extracted P(3HB) illustrate the chemical shift signals and spectral signatures of the different functional groups of P(3HB) and are similar to the findings of other researchers (Doi et al., 1986; Rozsa et al., 1996; Oliveira et al., 2007). The thermal degradation of synthesized P(3HB) and standard P(3HB) were observed between 200 to 359 ºC and 200 to 246 ºC, respectively (Fig. 7). The DTA thermogram reveals the melting behavior (T_m) of the polymer; the Tm of P(3HB) is lower (163 ºC) than that of standard P(3HB) (169 ºC). The GPC analysis of extracted P(3HB) revealed a weight-average molecular weight (M_w), number-average molecular weight (M_n), and polydispersity index (D) of 2.384×10⁴ g/mol, 1.304×10⁴ g/mol, and 1.828, respectively. Sudesh et al. (2000) reported that the M_w of P(3HB) synthesized by wild bacteria ranges from 1×10⁴ to 3×10⁶ g/mol with a polydispersity of around two.

CONCLUSION

The dynamics of microbial growth and P(3HB) production on simple carbon substrates has been investigated with the incorporation of maintenanceenergy-dependent kinetics and mathematical models. The changes in maintenance energy expenditure has been successfully correlated with the varying specific growth rate and yields of biomass and P(3HB) during growth/P(3HB) production processes. Besides, the assimilation potential of cane molasses as an inexpensive and renewable carbon source for biomass growth and P(3HB) production has also been examined. The physico-chemical, thermal, and material properties of synthesized P(3HB) have been found to be similar to those of standard P(3HB).

NOMENCLATURE

k Constant that depends on the substrate-microorganism system

h^-1

K ₁ Growth-associated product constant (-) K ₂ Non-growth associated product constant h^-1 K _s Substrate saturation constant g/L M_n Number average molecular weight g/mol m_s Maintenance energy coefficient h^-1 m_s1 Constant component of the maintenance energy expenditure h^-1 m_s2 Growth dependent component of the maintenance energy expenditure h^-1 M_w Weight average molecular weight g/mol q_{P (3HB)} Specific P(3HB) formation rate h^-1 q_s Specific substrate consumption rate g.g^-1 S Concentration of substrates, glucose, fructose, and sucrose

g/L

T_m Melting Temperature C

Y_P/S P(3HB) yield coefficient, w.r.t. substrate consumption g/g Greek Letters μ Specific growth rate h^-1 μ_max Maximum specific growth rate h^-1 Abbreviations 3HB 3-hydroxybutyrate D

Polydispersity index

DTA

Differential Thermal Analysis

DTG Differential Thermogravimetry GPC

Gel Permeation Chromatography

HPLC

High performance liquid chromatography

MTCC

Microbial Type Culture Collection

P(3HB)

Poly(3-hydroxybutyrate)

PHA

Polyhydroxyalkanoate

PHAs Polyhydroxyalkanoates SEM Scanning Electron Microscopy TGA

Thermo-Gravimetric Analysis

(Submitted: December 6, 2012 ; Revised: July 20, 2013 ; Accepted: August 9, 2013)

• Akaraonye, E., Keshavarz, T. and Roy, I., Production of polyhydroxyalkanoates: The future green materials of choice. Journal of Chemical Technology and Biotechnology, 85, p. 732-743 (2010).
• Annuar, M. S. M., Tan, I. K. P., Ibrahim, S. and Ramachandran, K. B., Ammonium uptake and growth kinetics of Pseudomonas putida PGA1. Asia Pacific Journal of Molecular Biology and Biotechnology, 14(1), p. 1-10 (2006).
• Beaulieu, M., Beaulieu, Y., Melinard, J., Pandian, S., Goulet, J., Influence of ammonium salts and cane molasses on growth of Alcaligenes eutrophus and production of polyhydroxybutyrate. Applied and Environmental Microbiology, 61, p. 165-169 (1995).
• Bodegom, P. V., Microbial maintenance: A critical review on its quantification. Microbial Ecology, 53(4), p. 513-523 (2007).
• Castilho, L. R., Mitchell, D. A. and Freire, D. M. G., Production of polyhydroxyalkanoates (PHAs) from waste materials and byproducts by submerged and solid state fermentation. Bioresource Technology, 100, p. 5996-6009 (2009).
• Chen, G-Q., Industrial Production of PHA. In: Chen, G-Q., (Ed.), Plastics from Bacteria: Natural Functions and Applications. Microbiology Monographs, Vol., 14, Springer-Verlag, Germany, p. 121- 132 (2011).
• Chen, G-Q., Konig, K-H. and Lafferty, R. M., Production of poly-D(-)-3-hydroxybutyrate and polyD(-)-3-hydroxyvalerate by strains of Alcaligenes latus Antonie van Leeuwenhoek, 60, p. 61-66 (1991).
• Djavan, A. and James, A. M., Determination of the maintenance energy of Klebsiella aerogenes growing in continuous culture. Biotechnology Letters, 2(7), p.303-308 (1980).
• Gouda, M. K., Swellam, A. E., Omar, S. H., Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources. Microbiological Research, 156, p. 201-207 (2001).
• Kim, Y. B. and Lenz, R. W., Polyester from Microorganism, In: Scheper, T., (Ed.) Advances in Biochemical Engineering/Biotechnology: Biopolyester. Springer-Verlag, Germany, p. 52-77 (2000).
• Liu, Y., Liu, Q-S., Tay, J-H., Initial conditions-dependent growth kinetics in microbial batch culture. Process Biochemistry, 40, p. 155-160 (2005).
• Minkevich, I. G., Andreyev, S. V. and Eroshin, V. K., The effect of two inhibiting substrates on growth kinetics and cell maintenance of the yeast Candida valida Process Biochemistry, 36(3), p. 209-217 (2000).
• Nancib, N., Modelling of batch fermentation of a recombinant Escherichia coli producing glyceraldehyde-3-phosphate dehydrogenase on a complex selective medium. The Chemical Engineering Journal, 52(2), p. B35-B48 (1993).
• Nielsen, D. R., Daugulis, A. J. and McLellan, P. J., Quantifying maintenance requirements from the steady-state operation of a two-phase partitioning bioscubber. Biotechnology and Bioengineering, 90, p. 248-258 (2005).
• Oliveira, E. G., Morais, J. O. and Pereira, N., Determination of the energy maintenance coefficient of Zymomonas mobilis Biotechnology Letters, 14 (11), p. 1081-1084 (1992).
• Oliveira, F. C., Dias, M. L., Castilho, L. R. and Freire, D. M. G., Characterization of poly(3-hydroxybutyrate) produced by Cupriavidus necator in solidstate fermentation. Bioresource Technology, 98, p. 633-638 (2007).
• Pirt, S. J., The Maintenance energy of bacteria in growing cultures. Proceedings of the Royal Society of London. Series B, Biological Sciences, 163(991), p. 224-231 (1965).
• Pirt, S. J., Maintenance energy: A general model for energy-limited and energy-sufficient growth. Archives of Microbiology, 133, p. 300-302 (1982).
• Riis, V. and Mai, W., Gas chromatographic determination of poly-β-hydroxybutyric acid in microbial biomass after hydrochloric acid propanolysis. Journal of Chromatography, 445, p. 285-289 (1988).
• Rozsa, C., Gonzalez, M., Galego, N., Ortiz, P., Martinez, J., Martinez, R. and Gomez, M. R., Biosynthesis and characterization of poly(βhydroxybutyrate) produced by Bacillus circulans Polymer Bulletin, 435, p. 429-435 (1996).
• Shamala, T. R., Divyashree, M. S., Davis, R., Latha, K. K. S., Vijayendra, S. V. N. and Raj, B., Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by Fourier transform infrared spectroscopy and scanning electron microscopy. Indian Journal of Microbiology, 49, p. 251-258 (2009).
• Solozano, L., Determination of ammonia in natural waters by the phenol hypochlorite method. Limnology and Oceanography, 14, p. 799-801 (1969).
• Sudesh, K., Abe, H. and Doi, Y., Synthesis, structure and properties of Polyhydroxyalkanoates: Biological polyesters. Progress in Polymer Science, 25, p. 1503-1555 (2000).
• Wang, F. and Lee, S. Y., Poly (3-Hydroxybutyrate) production with high productivity and high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation. Applied and Environmental Microbiology, 63, p. 3703-3706 (1997).
• Wang, J., Fang, F. and Yu, H-Q., Substrate consumption and biomass growth of Ralstonia eutropha at various S0/X0 levels in batch cultures. Bioresource Technology, 98, p.2599-2604 (2007).
• Xie, C-H. and Yokota, A., Reclassification of Alcaligenes latus strains IAM 12599^T and IAM 12664 and Pseudomonas saccharophila as Azohydromonas lata gen. nov., comb. Nov., Azohydromonas australica sp. Nov. and Pelomonas saccharophila gen. nov., comb. Nov., respectively. International Journal of Systematic and Evolutionary Microbiology, 55, p. 2419-2425 (2005).

*

To whom correspondence should be addressed

Publication Dates

• Publication in this collection
  07 July 2014
• Date of issue
  June 2014

History

• Received
  06 Dec 2012
• Accepted
  09 Aug 2013
• Reviewed
  20 July 2013