MICROBIAL PRODUCTION OF POLYHYDROXYALKANOATES USING INEXPENSIVE AND RENEWABLE MATERIALS

Abstract

The application of biotechnological process for the industrial production of value-added products is promising under the sustainable development. It is essential to develop the suitable bio-conversion processes in which a broad range of industrial byproducts and surplus materials can be utilized to reduce their impact on environment. The microbial based biopolymers, polyhydroxyalkanoates (PHAs) are considered as an alternative to the conventional petroleum derived xenobiotic plastics as they possess similar physico-chemical, thermal and material properties and, hence, have comparatively good processing abilities. PHAs are also getting major attention due to their biodegradability and biocompatible nature and their production comes• under `green bio-refining processes' as they protect the environment by reducing the CO2 emission in atmosphere. In this study the assimilation potentials of procured and sludge isolated bacterial strains towards pure carbon substrates (glucose, fructose, sucrose, and glycerol) and subsequently on their renewable and inexpensive sources (cane molasses and glycerol based substrate) were studied extensively for the production of PHAs. The dynamics of microbial growth and P(3HB) production of Azohydromonas lata MTCC 2311 using glucose, fructose, and sucrose was examined and the kinetic parameters were evaluated with the incorporation of maintenance energy expenditure. The experimental growth data derived on various carbon sources were fitted to the proposed maintenance energy based mathematical models and kinetic parameters were calculated as follows: msl = 0.0005 h-', k = 0.0965, μmat 0.25 h-1 for glucose; msl = 0.003 h-1, k = 0.1229, μmat 0.27 h-1 for fructose; and msl = 0.0076 h-1, k = 0.0694, μma, 0.25 h-1 for sucrose. The maintenance energy expenditure was found to change throughout the fermentation process as a function of the specific growth rate. The per cent of carbon used as maintenance energy expenditure was found to decrease with the increase in specific growth rate. At low specific growth rate of 0.12 h-1, about 22% of fructose used by bacterial cells as a maintenance energy which was decreased to 2.14% at high specific growth rate of 0.25 h-1. Similarly, with the glucose and sucrose in the medium, about 16.85% and 15.93%, respectively, were noticed as maintenance energy requirement at low specific growth rate of 0.11 h-I. These expenditures were decreased to 0.73% and 2.22% at high specific growth rate of 0.24 h-1 of glucose and sucrose, respectively. The high specific growth rate indicates a favourable environment in which the maintenance requirement is low. The maximum maintenance expenditure of 0.01294 h-' was observed with ._ _; sucrose in the medium followed by fructose (0.01218 h"1) and glucose (0.00436 h-1) in the medium. It means that about 12.94, 12.18, and 4.36 mg of sucrose, fructose, and glucose, respectively, were needed just to meet the demand of cell maintenance energy during the production of one gram of biomass per h. A linear correlation was observed between g and gp(3HB) with positive values of intercept K2 and slope K1 for fructose, glucose, and sucrose which indicated that the P(3HB) production by A. lata was mixed type, i.e. both growth and non-growth associated in nature. Furthermore, the high value of Kl and comparatively low value of K2 indicated that the accumulation of P(3HB) inside the microbial cells was more growth associated than non-growth associated (stationary phase). The effect of varying concentration of (NH4)2SO4 on the specific growth rate (p) of A. lata was also examined and fitted well with the Aiba growth model that incorporated the substrate inhibition parameter (K; = 2.24 g/L) with R2 values of 0.9827. It was also concluded that the NH4+ concentration in the medium should be kept around 0.8 g/L to get maximum biomass growth and favorable conditions for P(3HB) accumulation. The combination of sucrose with urea (among combination of three carbon sources namely, sucrose, fructose, and glucose and four nitrogen sources namely, (NH4)2SO4, NH4C1, urea, and NH4NO3) was identified as best carbon and nitrogen source, respectively, for maximum biomass (8.92 g/L) and P(3HB) (4.24 g/L) concentration in AL2 medium. Further, sucrose and urea along with the trace-element (TE) solution, essential for biomass growth, were chosen for their level optimization by using statistical / artificial intelligence based multivariate optimization technique to get maximum biomass and P(3HB) concentrations. Response surface methodology (RSM) and artificial neural network models (ANN) were successfully navigated the experimental data, obtained in accordance with the central composite design. The effects of sucrose (3.2 — 36.82 g/L), urea (0.16- 1.84 g/L), and TE solution (0.32- 3.68 ml/L) on biomass and P(3HB) concentrations were investigated. The modeling and optimization ability of hybrid ANN-GA with higher ii accuracy had been shown in finding optimum concentrations of medium variables than hybrid RSM-GA. Hybrid ANN-GA predicted the maximum biomass concentration (12.25 g/L) at the optimum level of medium variables: sucrose, 35.27 g/L; urea, 1.55 g/L; and TE solution, 0.42 ml/L. Whereas, the maximum predicted P(3HB) concentration (5.95 g/L) was reported at: sucrose, 35.20 g/L; urea, 1.58 g/L; and TE solution, 0.36 ml/L. The validation with additional set of data shows that the predictive errors (%) in biomass and PHB concentrations were 3.67 and 2.52, respectively for shake flask experiments, whereas, the predictive errors (%) were 13.80 and 14.28, respectively for bioreactor experiments. Subsequently, the P(3HB) produced on cane molasses in medium was subjected for its physico-chemical, thermal, and material characterization. This has revealed that the produced P(3HB) possesses the similar properties as that of standard P(3HB) and is suitable for processing and blending for various applications. On the basis of sucrose assimilation potential of A. lata, further attempts were made to produce the copolymer P(3HB-co-3HV) using cane molasses supplemented with the volatile fatty acids (VFAs), namely propionic acid and valeric acid. In the first step, the effect of supplementation of VFAs in cane molasses solution was examined. The maximum biomass concentration, P(3HB-co-3HV) concentration, and 3HV content (mol%) were obtained at a concentration of 30 mM/L for both the propionic and valeric acids. The maximum biomass concentration of 9.6 g/L, P(3HB-co-3HV) concentration of 5.6 g/L, and 3HV content of 16.5 mol% were observed with propionic acid in medium, however, a little lower values of 8.2 g/L of biomass concentration, 5.4 g/L of (P(3HB-co-3HV) concentration, and 14.5 mol% of 3HV content was obtained with valeric acid in medium. In addition, the effect of varying concentration of molasses (1.5-9.0%, w/v) on biomass concentration, P(3HB-co-3HV) concentration, and 3HV content (mol%) was studied. Maximum biomass and P(3HB-co-3HV) concentrations of 10.25 and 6.25 g/L, respectively, were observed at 4.5% (w/v) cane molasses concentration corresponding to 20.15 g/L of total sugar in the medium. Further increase in the concentration of cane molasses leads to the decrease in both biomass and P(3HB-co-3HV) concentrations. The maximum specific growth rate (μmax) of 0.29 h-1 and substrate (molasses) saturation constant (KS) of 1.67% (w/v) were estimated by the least square curve fitting of experimental data to Monod kinetic model. iii The cane molasses solution supplemented with propionic acid and urea were optimized for the production of copolymer P(3HB-co-3HV) by Azohydromonas lata MTCC 2311 using artificial intelligence based multivariate optimization technique. Genetic algorithm (GA) was used for the optimization of P(3HB-co-3HV) production through the simulation of artificial neural network (ANN) and response surface methodology (RSM) models. The predictions of hybrid ANN-GA were found better than those of hybrid RSM-GA and in good agreement with experimental findings. The maximum P(3HB-co-3HV) concentration of 7.35 g/L was predicted at optimum levels of medium variables: cane molasses, 4.29% (w/v); urea, 0.53 g/L; and propionic acid, 12.60 mmol/L by hybrid ANN-GA approach. Similarly, 16.84 mol% of 3HV content was predicted at 3.89% (w/v) of cane molasses, 0.50 g/L of urea, and 20.40 mmol/L of propionic acid by hybrid ANN-GA approach. Upon validation, 7.20 g/L and 16.30 mol% of P(3HB-co-3HV) concentration and 3HV content were found in the shake flask, whereas 6.70 g/L and 16.35 mol%, were observed in a 3-L bioreactor, respectively. Further, the effects of agitation and aeration rates on copolymer P(3HB-co-3HV) production by Azohydromonas lata MTCC 2311 using optimized cane molasses solution in a 3-L bioreactor were investigated. The experiments were conducted in a three level factorial design by varying the impeller (150-500 rev/min) and aeration (0.5-1.5 vvm) rates. The experimental data were fitted to the mathematical models [quadratic polynomial equation and artificial neural network (ANN)] and process variables were optimized by GA-coupled models. ANN and hybrid ANN-GA were found superior for modeling and optimization of process variables, respectively. The maximum P(3HB-co-3HV) concentration of 7.45 g/L with 21.50 mol% of 3HV was predicted at process variables: agitation speed, 287 rev/min; and aeration rate, 0.85 vvm, which upon validation gave 7.20 g/L of P(3HB-co-3HV) with 21 mol% of 3HV with the prediction error (%) of 3.38 and 2.32, respectively. Agitation speed established a relative high importance of 72.19% than of aeration rate (27.80%) for copolymer accumulation. The volumetric gas-liquid mass transfer coefficient (kLa) was strongly affected by agitation and aeration rates. The highest P(3HB-co-3HV) productivity of 0.163 g/L.h was achieved at 0.17 s1 of kLa value. During the early phase of copolymer production process, 3HB monomers were accumulated which were shifted to 3HV units (9 to 21 %) during cultivation period of iv 24 to 42 h. An enhancement of 7.5% and 34% were reported for P(3HB-co-3HV) production and 3HV content, respectively, by hybrid ANN-GA paradigm. The carbon yield of 0.58 and 0.48 were calculated on optimized solution given by hybrid ANN-GA and RSM-GA, respectively. These calculated values were found comparable to the theoretical carbon yield of 0.48 and 0.50 on pure substrates glucose and sucrose. This increase in 3HV content (mol%) of copolymer P(3HB-co-3HV) has enhanced the thermal and material properties such as Tm, degradation temperature profile, and M of produced P(3HB-co-3HV) significantly. The Tm of P(3HB-co-3HV) was found significantly lower (150 °C) than that of standard PHB (169 °C). Similarly, the difference between Tm and decomposition temperature (200 °C) was high enough to increase their processing window for various applications. The GPC analysis of copolymer P(3HB-co-3HV) extracted from A. lata cells has revealed that the weight average molecular weight (Mw), number average molecular weight (M„), and polydispersity index (D) are 2.0616x104 g/mol, 1.2117x104 g/mol, and 1.701, respectively. Besides, the acidogenic-fermentation of cane molasses solution of 3.0 and 4.5% was carried out at initial pH of 5, 6, and 7 by using mixed microbial consortium of activated sludge. With 3.0% cane molasses solution, the maximum 93 Cmmol/L of VFAs concentration was observed at acidic pH of 5.0, followed by a decrease in VFAs concentration at higher pH values. Similarly, with 4.5% cane molasses solution, a little higher concentrations of VFAs were observed as 96, 86, and 66 Cmmol/L at initial pH of 5.0, 6.0, and 7.0, respectively. The clarified cane molasses solution (fermented) was used for the production of P(3HB-co-3HV) by A. lata and maximum biomass concentration of 6.5 g/L, P(3HB-co-3HV) concentration of 2.1 g/L, and 3HV content of 14 mol% were observed with 4.5% cane molasses. In addition, a pure microbial strain was isolated from the activated sludge sample enriched in glycerol based E2 medium. The isolated strain was subjected to molecular characterization based on 16S rDNA technique and was identified as Bacillus sp. RER002. The potential of sludge isolated Bacillus sp. RER002 to accumulate the value-added PHAs of various short and medium chain lengths using glycerol based medium was examined in a 3L bioreactor. The mathematical models based on logistic, Luedeking-Piret and Luedeking-Piret-like equations were v developed to describe and navigate the experimental data of active biomass growth, P(3HB) accumulation, and consumption of glycerol, respectively in the range of 10-40 g/L. The model predictions were attested by some statistical/mathematical constants and found satisfactory with the experimental data. The accumulation of P(3HB) was found to be growth associated with; comparatively high values of Kl (0.2435-0.5477) than K2 values (2.2x 10-6-9.1 x 10-3) within the concentration range of 10-40 g/L of glycerol in the medium. In addition, some free fatty acids such as octanoic, palmitic, stearic, and oleic acids were used to supplement the glycerol based medium to incorporate the 3HHx-monomer in copolymer at the concentration 6.53, 4.5, 4.0, and 3.5 mol%, respectively. Besides, the concentration of 3.2 g/L of P(3HB-co-3HHx) was also found at 30 g/L of glycerol in simulated crude glycerol (CG1) medium with Yp,s of 0.16 g/g. Subsequently, the improvement in physico-chemical and thermal properties of isolated copolymer was found which revealed its suitability in various processing. Hence, the suitable batch process for the production of PHAs of different monomers, viz. 3HB, 3HV, and 3HHx using sugar cane molasses and glycerol based substrate as renewable carbon substrates have been developed in a 3L bioreactor. The various physico-chemical, thermal, and material properties of produced PHAs have suggested their applications in various fields like, biomedical, biomaterial and tissue engineering and biodegradable packaging materials.