Advanced approaches to produce polyhydroxyalkanoate (PHA) biopolyesters in a sustainable and economic fashion

Impact of different by-products from the biodiesel industry and bacterial strains on the production, composition, and properties of novel polyhydroxyalkanoates containing achiral building blocks

Waste streams containing volatile fatty acids (VFAs) can be used for polyhydroxyalkanoate (PHA) production by mixed microbial cultures (MMCs) and most of the operating strategy for MMCs PHA production includes moderate organic loading and nitrogen limitation. However, waste streams in reality commonly contain different concentration of carbonand nitrogen (for example COD=24.0 g/L, Ammonia N=6.58 mg/L for fermented paper mill wastewater and COD=5978.17 mg/L, Ammonia N=398.18 mg/L for sludge fermentation liquid). This paper aims to investigate whether there is an optimal strategy for MMCs PHA production that appeal to a wide carbon and nutrient level spectrum. Three typical sequence batch reactors (SBRs) submitted to aerobic dynamic feeding (ADF) mode were operated under the same C/N ratio, VFAs composition and hydraulic retention time (HRT) but different combination of sludge retention time (SRT), organic load rate (OLR) and cycle length (CL) to enrich PHA accumulating MMCs from municipal activated sludge. The PHA production capacity of SBRs under nutrient excess, limitation and starvation conditions (Cmol/Nmol ratio equals to 8, 40 and ∞, respectively) was evaluated in batch assays. The succession of microbial communities in SBRs and batch assays was analyzed by the method of terminal restriction fragment length polymorphism (T-RFLP). Batch assays of SBR#1 (long SRT, low OLR, long CL), SBR2# (short SRT, high OLR, long CL) and SBR#3 (short SRT, high OLR, short CL) showed similar results under nutrient starvation condition, with PHA content of 46.60 wt% (g PHA/g VSS), 46.46 wt% and 47.12 wt% achieved respectively after 7.5 h reaction, while batch assay of SBR#3 reached the maximum PHA content (54.85 wt%) under nutrient excess condition, compared to that of SBR#1 and SBR#2 of 49.99 wt% and 50.04 wt%, respectively. Regarding active biomass growth, batch assays of SBR#2 and SBR#3 showed an increase of 20.61% (g Biomass/g Initial Biomass) and 38.92% under nutrient limitation, 19.80% and 24.79% under nutrient excess, respectively, while no apparent growth occurred in batch assay of SBR#1 (8.92% and 4.15% under nutrient limitation and excess respectively). Negative growth were observed under nutrient starvation of all SBRs because of sampling loss. Due to the inhibition of free ammonia under nutrient excess condition, biomass growth was less compared to that under nitrogen limited condition. The results showed that SBR#3 had the best overall PHA production performance considering its relatively high PHA content and productivity in all nutrient conditions, which will guarantee the production performance with adaptability for a wide range of VFA-rich waste streams. Nitrogen has great impact on the biomass yield especially when OLR is high, the presence of nitrogen results in the increase of biomass consequently increases the final PHA productivity that can be calculated from .

Polyhydroxyalkanoate (PHA) production via resource recovery from industrial waste streams: A review of techniques and perspectives

The utilization of fermentation media derived from waste and by-product streams from biodiesel and confectionery industries could lead to highly efficient production of bacterial cellulose. Batch fermentations with the bacterial strain Komagataeibacter sucrofermentans DSM (Deutsche Sammlung von Mikroorganismen) 15973 were initially carried out in synthetic media using commercial sugars and crude glycerol. The highest bacterial cellulose concentration was achieved when crude glycerol (3.2 g/L) and commercial sucrose (4.9 g/L) were used. The combination of crude glycerol and sunflower meal hydrolysates as the sole fermentation media resulted in bacterial cellulose production of 13.3 g/L. Similar results (13 g/L) were obtained when flour-rich hydrolysates produced from confectionery industry waste streams were used. The properties of bacterial celluloses developed when different fermentation media were used showed water holding capacities of 102–138 g·water/g·dry bacterial cellulose, viscosities of 4.7–9.3 dL/g, degree of polymerization of 1889.1–2672.8, stress at break of 72.3–139.5 MPa and Young’s modulus of 0.97–1.64 GPa. This study demonstrated that by-product streams from the biodiesel industry and waste streams from confectionery industries could be used as the sole sources of nutrients for the production of bacterial cellulose with similar properties as those produced with commercial sources of nutrients.

Process optimization for polyhydroxyalkanoate (PHA) production from waste via microbial enrichment cultures

Polyhydroxyalkanoate (PHA) production by Bacillus thuringiensis EGU45 and defined mixed culture of Bacillus spp. were studied by using crude glycerol (CG) and hydrolyzed biowastes as feed material. Hydrolysates from onion peels (OP), potato peels, pea-shells (PS), apple pomace 2% total solids obtained with defined mixed hydrolytic cultures (MHC2) were inoculated with B. thuringiensis EGU45 and defined mixed bacterial cultures (5MC1), which produced PHA at the rate of 40-350 and 65-450mg/L, respectively. Addition of CG (1%, v/v) to these hydrolysates resulted in 1.8-fold and 4.5-fold enhancement in PHA production from OP by B. thuringiensis EGU45 and 5MC1, respectively. Co-utilization of OP and PS (in 2:1 ratio) supplemented with CG (1%, v/v) by B. thuringiensis EGU45 resulted in 2-fold increase in PHA production in comparison to OP+CG. This co-metabolism of OP and PS also enabled PHA co-polymer production (1300mg/L), having an enhanced HV content of 21.2% (w/w).

Polyhydroxyalkanoate production from crude glycerol by newly isolated Pandoraea sp.

Crude glycerol has emerged as a suitable feedstock for the biotechnological production of various industrial chemicals given its high surplus catalyzed by the biodiesel industry. Pseudomonas bacteria metabolize the polyol into several biopolymers, including alginate and medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs). Although P. putida is a suited platform to derive these polyoxoesters from crude glycerol, the attained concentrations in batch and fed-batch cultures are still low. In this study, we employed P. putida KT2440 and the hyper-PHA producer ΔphaZ mutant in two different fed-batch modes to synthesize mcl-PHAs from raw glycerol. Initially, the cells grew in a batch phase (μmax 0.21 h–1) for 22 h followed by a carbon-limiting exponential feeding, where the specific growth rate was set at 0.1 (h–1), resulting in a cell dry weight (CDW) of nearly 50 (g L–1) at 40 h cultivation. During the PHA production stage, we supplied the substrate at a constant rate of 50 (g h–1), where the KT2440 and the ΔphaZ produced 9.7 and 12.7 gPHA L–1, respectively, after 60 h cultivation. We next evaluated the PHA production ability of the P. putida strains using a DO-stat approach under nitrogen depletion. Citric acid was the main by-product secreted by the cells, accumulating in the culture broth up to 48 (g L–1) under nitrogen limitation. The mutant ΔphaZ amassed 38.9% of the CDW as mcl-PHA and exhibited a specific PHA volumetric productivity of 0.34 (g L–1 h–1), 48% higher than the parental KT2440 under the same growth conditions. The biosynthesized mcl-PHAs had average molecular weights ranging from 460 to 505 KDa and a polydispersity index (PDI) of 2.4–2.6. Here, we demonstrated that the DO-stat feeding approach in high cell density cultures enables the high yield production of mcl-PHA in P. putida strains using the industrial crude glycerol, where the fed-batch process selection is essential to exploit the superior biopolymer production hallmarks of engineered bacterial strains.

Polyhydroxyalkanoates (PHA) production from waste streams using mixed microbial cultures (MMC) can unlock the potential of PHA to substitute oil-based plastics. However, these processes are still at low technology readiness level (4–6). Demonstrating a better environmental performance would boost their deployment at industrial scale. Hence, including environmental guidance during their development, when there are still opportunities for major alterations, is essential. To the best of our knowledge, this work elucidates for the first time how waste-to-PHA biorefineries could develop in the future by combining prospective LCA with scenario methodology and where the attention of stakeholders should be focused. Four future scenarios were derived considering both surrounding (e.g., scale, environmental or bioeconomy policies) and technological parameters (e.g., acidification yield, PHA content in biomass or recovery yield). Those scenarios derived under ambitious environmental and bioeconomy policies shop up to 50% lower environmental impacts than those under business-as-usual policies. These differences are caused by the different background processes’ environmental burdens (e.g., electricity mix with low renewable energies share) and the higher consumption of chemicals and utilities. However, the environmental impacts caused by lower yields can be partially mitigated by valorizing the intermediate waste streams into biogas. Sensitivity analysis results pointed out recovery yield and PHA content as the parameters that influence most the environmental performance, being responsible for up to 60% of variance in environmental performance. These parameters determine the chemicals and utilities consumption in PHA downstream processing, which is confirmed as the main environmental hotspot. This work goes beyond previous LCA studies on PHA production and quantifies the influence of different parameters on the environmental performance.

Volatile fatty acids (VFA) may serve as building blocks for Polyhydroxyalkanoates (PHA) production and can be derived from waste streams. Ideal streams for PHA production contain a high Chemical Oxygen Demand (COD) to nutrient ratio, such as (waste)water from a paper-mill factory or candy-bar factory. The (waste)water generated by these companies are usually treated anaerobically with the final product being methane containing biogas. Usually, the methane is burned to produce either heat or electricity. Potentially, more value can be added to these streams by producing VFA and/or PHA. PHA can be produced using microbial enrichment cultures that can be established by cultivation in a selective environment that favours the growth of PHA producing microorganisms. Some advantages of using open cultures are that no sterilization and expensive equipment is required compared to pure culture biotechnology. Open culture biotechnology can be effectively applied when the right selection pressure for a specific microbial trait is identified. The microorganism that is most effective in the given conditions will win the competition, i.e. the strongest will survive. A selection criteria for PHA productis is consuming substrate very fast by first making a storage polymer (in this case PHA) from the supplied substrate. The PHA producers prefer VFA as substrate, hence it is important to maximize the VFA content in the substrate stream. For the production of VFA in the product chain towards PHA it is important to minimize the solid content in the feedstock for PHA production. The objective of the research described in this thesis was to gain more insight in the two-step upstream process for PHA production from agricultural waste streams. The first step concerns the maximization of the VFA concentration in the feedstock. Optimization of VFA production was investigated using the granular sludge process in order to maximize the volumetric VFA production capacity and to minimize the solids concentration in the effluent. Two process variables were investigated regarding the PHA production process. Firstly, the influence of the presence of nutrients on PHA production was investigated using PHA producing enrichment cultures. Secondly, the production of PHA was investigated using the leachate of the organic fraction of municipal solid waste (OFMSW) at pilot scale.

Tuna condensate, an organic-rich by-product from the tuna canning industry, was assessed as a substrate for polyhydroxyalkanoate (PHA) production using Cupriavidus necator TISTR 1095. The effect of cultivation parameters on PHA accumulation was studied, including substrate concentration, carbon to nitrogen (C/N) ratio, initial pH-value control and fermentation strategies. For the bacterium, a biomass of 3.8 ± 0.1 g/L, PHA of 1.64 ± 0.1 g/L with PHA productivity of 0.027 g/L.h were obtained under batch cultivation using 100% tuna condensate with a C/N ratio of 88:1 and no control of pH. However, the PHA production was increased 1.3-fold when repeated-batch cultivation was applied. The highest biomass (7.5 ± 0.1 g/L) and PHA (3.8 ± 0.1 g/L) with 0.063 g/L.h of PHA productivity were achieved after the third cycle of repeated-batch cultivation. High chemical oxygen demand (COD) removal efficiency of 70% under the optimal condition was also demonstrated. The polymers generated by C. necator TISTR 1095 were characterised. The size of polymer granules was in the range of 0.7-0.8 μm. The polymer produced in the optimal medium under batch and repeated-batch cultivation was identified as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with a 20 mol% of 3-hydroxyvalerate. The molecular mass (Mn) and polydispersity of the polymer were 2 × 106 Da and 2.5, respectively. The results demonstrated that tuna condensate could be used as a cheap substrate for PHA production on an industrial scale.

Production and recovery of polyhydroxyalkanoates (PHA) from waste streams – A review

Crude glycerol (CG), a by‐product from biodiesel production, is a carbon source with potential as feedstock for polyhydroxyalkanoate (PHA) production. PHAs are biological macromolecules synthesized by microorganisms as intracellular carbon and energy storage granules. PHA production and its properties were investigated using Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429 cultivated with CGs from different origins. The highest PHA extraction percentage (71.56% [w/v]) occurred when C. necator IPT 029 metabolized CG 3 (from the processing of biodiesel from castor bean oil). The gas chromatography–mass spectrometry analyses revealed novel PHA constituents as building blocks of medium (3‐hydroxytetradecanoate) and long (11‐hydroxyoctadecanoate) chains. Molar mass distribution revealed range of 121–6900 kDa. The initial degradation temperature ranged from 181.83 to 287.50°C and the crystallinity ranged from 35.30 to 66.70%. The results obtained indicate that C. necator IPT 029 from CG 3 could produce copolymers with industrially applicable thermophysical properties. Copyright © 2015 John Wiley & Sons, Ltd.

BackgroundOur study aimed to search for novel bacteria capable of producing polyhydroxyalkanoates (PHAs) using crude glycerol residue obtained from biodiesel production in which used cooking oils were the substrates.ResultsNewly isolated bacteria from soils in Thailand were screened for the efficient production of PHAs from crude glycerol. The bacterial strains were cultivated on glucose, refined glycerol, crude glycerol, or various cooking oils (canola oil, palm oil, soybean oil, sunflower oil, corn oil, grape seed oil, olive oil, rice bran oil, camellia seed oil) for growth and PHA production. The effects of the total organic carbon (TOC) concentration and the mole ratio of carbon to nitrogen were investigated in batch cultivation. 1H NMR, two dimensional-1H-correlation spectroscopy (2D-1H-COSY) and 13C NMR analyses confirmed four bacterial strains were capable of producing medium-chain-length PHAs (mcl-PHAs), consisting of 3-hydroxyoctanoate (3HO) and 3-hydroxy-5-cis-dodecanoate (3H5DD), from crude glycerol. On the basis of phenotypic features and genotypic investigations, the bacterial strains were assigned as: ASC1, Acinetobacter genus (94.9 % similarity); ASC2, Pseudomonas genus (99.2 % similarity); ASC3, Enterobacter genus (99.2 % similarity); ASC4, Bacillus genus (98.4 % similarity). The highest amount of mcl-PHAs, 17.5 ± 0.8 g/L (content 61.8 ± 3.3 % wt), with 3HO (14.7 ± 2.2 mol %), 3H5DD (85.3 ± 2.2 mol %), and a total biomass of 32.3 ± 0.3 g/L, was obtained from Pseudomonas sp. ASC2 in batch cultivation after 36 h. The mcl-PHAs recovered had a number-average molecular weight (MN) of 3.6 × 104 Da. Homopolymeric 3H5DD was obtained when the cultivation time was prolonged to 96 h.ConclusionsNovel PHA-producing strains were isolated and identified. These bacterial strains are able to produce mcl-PHAs from crude glycerol. The mcl-PHAs produced contained a high percentage of 3H5DD, which suggests their future application as softeners mixed with other biomaterials. The unsaturated side chain of 3H5DD monomers containing double bounds offers additional potential for improving the properties of the mcl-PHAs or extending their applications to the food industry.

Microbial community-based polyhydroxyalkanoate (PHA) production has been demonstrated repeatedly at pilot scale. Ammonium, normally present in waste streams, might be oxidized by nitrifying bacteria resulting in additional aeration energy demand. The use of low dissolved oxygen (DO) concentrations allowed to reduce nitrifying rates by up to 70% compared to non-oxygen limiting conditions. At lower DO concentrations nitrate was used as alternative electron acceptor for PHA production and therefore, a constant PHA production rate could only be maintained if nitrate was sufficiently available. An optimum DO concentration (0.9 mgO2/L) was found for which nitrification was mitigated but also exploited to supply requisite heterotrophic nitrate requirements that maintained maximum PHA production rates. PHA accumulations with such DO control was estimated to reduce oxygen demand by about 18%. This work contributes to establish fundamental insight towards viable industrial practice with the control and exploitation of nitrifying bacteria in microbial community-based PHA production.