Polyhydroxyalkanoates (PHA) production in bacterial co‐culture using glucose and volatile fatty acids as carbon source

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

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.

Waste paper as a resource for polyhydroxyalkanoate (PHA) production through anaerobic digestion is a low-cost strategy to produce bioplastic. In this study, volatile fatty acids (VFAs) produced from waste paper, one of the significant constituents of municipal solid waste, was utilized as a feedstock for polyhydroxyalkanoate (PHA) production. PHA production from synthetic VFAs by Cupriavidus necator was initially optimized under different VFAs concentrations, VFAs ratios, and nitrogen sources. VFAs concentration of 10 g/L, 5:1:4 ratio of acetic, propionic, and butyric acids (HAc:HPr:HBu) and NaNO3 as nitrogen source were considered the optimum conditions with 56.98% PHA and 0.31 g/g yield. Anaerobic digestion of shredded office paper (OP/S) produced the maximum VFAs (521.50 mg/L) after 15 days of incubation and were utilized for PHA synthesis. Almost 2.24-fold increase in the yield of PHA was achieved with limited nutrient medium compared to nutrient contained medium with a PHA content of 53.50 and 23.88%, respectively. PHA production using anaerobic effluent of waste paper is a promising approach where a series of pretreatment processes, the expensive enzymatic hydrolysis, and detoxification were no longer required, suggesting an environmentally friendly way of biopolymer production.

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters with comparable properties to some petroleum-based polyolefins. PHA production can be achieved in open, mixed microbial cultures and thereby coupled to wastewater and solid residual treatment. In this context, waste organic matter is utilised as a carbon source in activated sludge biological treatment for biopolymer synthesis. Within the EU project Routes, the feasibility of PHA production has been evaluated in processes for sludge treatment and volatile fatty acid (VFA) production and municipal wastewater treatment. This PHA production process is being investigated in four units: (i) wastewater treatment with enrichment and production of a functional biomass sustaining PHA storage capacity, (ii) acidogenic fermentation of sludge for VFA production, (iii) PHA accumulation from VFA-rich streams, and (iv) PHA recovery and characterisation. Laboratory- and pilot-scale studies demonstrated the feasibility of municipal wastewater and solid waste treatment alongside production of PHA-rich biomass. The PHA storage capacity of biomass selected under feast-famine with municipal wastewater has been increased up to 34% (g PHA g VSS(-1)) in batch accumulations with acetate during 20 h. VFAs obtained from waste activated sludge fermentation were found to be a suitable feedstock for PHA production.

Polyhydroxyalkanoates (PHAs) production from lignocellulosic biomass using mixed microbial cultures (MMC) is a potential cheap alternative for reducing the use of petroleum-based plastics. In this study, an MMC adapted to acidogenic effluent from dark fermentation (DF) of exhausted sugar beet cossettes (ESBC) has been tested in order to determine its capability to produce PHAs from nine different synthetic mixtures of volatile fatty acids (VFAs). The tests consisted of mixtures of acetic, propionic, butyric, and valeric acids in the range of 1.5–9.0 g/L of total acidity and with three different valeric:butyric ratios (10:1, 1:1, and 1:10). Experimental results have shown a consistent preference of the MMC for the butyric and valeric acids as carbon source instead other shorter acids (propionic or acetic) in terms of PHA production yield (estimated in dry cell weight basis), with a maximum value of 23% w/w. Additionally, valeric-rich mixtures have demonstrated to carry out a fast degradation process but with poor final PHA production compared with high butyric mixtures. Finally, high initial butyric and valeric concentrations (1.1 g/L and 4.1 g/L) have demonstrated to be counterproductive to PHA production.

Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated from alkaline excess sludge fermentation

Plastic production and accumulation have devastating environmental effects, and consequently, the world is in need of environmentally friendly plastic substitutes. In this context, polyhydroxyalkanoates (PHAs) appear to be true alternatives to common plastics because they are biodegradable and biocompatible and can be biologically produced. Despite having comparable characteristics to common plastics, extensive PHA use is still hampered by its high production cost. PHAs are bacterial produced, and one of the major costs associated with their production derives from the carbon source used for bacterial fermentation. Thus, several industrial waste streams have been studied as candidate carbon sources for bacterial PHA production, including whey, an environmental contaminant by-product from the dairy industry. The use of whey for PHA production could transform PHA production into a less costly and more environmentally friendly process. However, the efficient use of whey as a carbon source for PHA production is still hindered by numerous issues, including whey pre-treatments and PHA producing strain choice. In this review, current knowledge on using whey for PHA production were summarized and new ways to overcome the challenges associated with this production process were proposed.

Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible thermoplastics that can be synthesized in various microorganisms. Volatile fatty acids (VFAs) are produced by anaerobic treatment of organic wastes that can be utilized as inexpensive substrates for PHA synthesis. In this study, several Ralstonia eutropha strains were grown on the mixture of VFAs (acetic, propionic, and butyric acid) as its carbon and energy source for growth and PHA synthesis. R. eutropha KCTC 2658 accumulated PHAs up to 50% of dry cell weight from total 5 g/L of mixed VFAs (acetic acid: propionic acid: butyric acid=1: 2: 2). In batch culture of R. eutropha KCTC2658 in a 5 L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from 20 g/L glucose as a sole carbon source with dry cell weight of 8.4 g/L and PHA content of 30%. In fed-batch culture, two feeding strategies, pulse or pH-stat, were applied to add VFAs to the fermentor. When VFAs were fed using pH-stat feeding strategy after 40 h, a copolymer of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] was produced with dry cell weight of 8.1 g/L, PHA content of 50%, and 3HV fraction of 20 mol%.

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 .

Waste of industrial origin produced from synthetic materials are a serious threat to the natural environment. The ending resources of fossil raw materials and increasingly restrictive legal standards for the management of plastic waste have led to research on the use of biopolymers, which, due to their properties, may be an ecological alternative to currently used petrochemical polymers. Polyhydroxyalkanoates (PHAs) have gained much attention in recent years as the next generation of environmentally friendly materials. Currently, a lot of research is being done to reduce the costs of the biological process of PHA synthesis, which is the main factor limiting the production of PHAs on the industrial scale. The volatile fatty acids (VFAs) produced by anaerobic digestion from organic industrial and food waste, and various types of wastewater could be suitable carbon sources for PHA production. Thus, reusing the organic waste, while reducing the future fossil fuel, originated from plastic waste. PHA production from VFAs seem to be a good approach since VFAs composition determines the constituents of PHAs polymer and is of great influence on its properties. In order to reduce the overall costs of PHA production to a more reasonable level, it will be necessary to design a bioprocess that maximizes VFAs production, which will be beneficial for the PHA synthesis. Additionally, a very important factor that affects the profitable production of PHAs from VFAs is the selection of a microbial producer that will effectively synthesize the desired bioproduct. PHA production from VFAs has gained significant interest since VFAs composition determines the constituents of PHA polymer. Thus far, the conversion of VFAs into PHAs using pure bacterial cultures has received little attention, and the majority of studies have used mixed microbial communities for this purpose. This review discusses the current state of knowledge on PHAs synthesized by microorganisms cultured on VFAs.

Valorization of food waste derived anaerobic digestate into polyhydroxyalkanoate (PHA) using Thauera mechernichensis TL1

Purpose: To isolate polyhydroxyalkanoates (PHA)-producing bacterial strains from contaminated soil using industrial wastewater and glucose as carbon soured by Macrogen sequencing. Two different sources, namely, glucose and wastewater were used to ces.Methods: The strains were isolated and identified as Pseudomonas, Bacillus, Enterobacter, Exiguobacterium and Stenotrophomonas using biochemical tests and further confirmevaluate and compare the use of wastewater as a carbon source for PHA production. The biomass obtained was analyzed by Fourier transform infra-red (FTIR) to identify the presence of PHA in it. Afterwards, PHA extraction was carried out and then gas chromatography (GC) performed to identify PHA monomers.Results: Utilization of glucose resulted in the production of PHB, while wastewater yielded copolymers poly-3 hydroxybutyrate-co-3hydroxyvalerate P(3HB-co-3HV) due to its content of volatile fatty acids such as acetic acid, propionic acid and butyric acid, which led to the production of different types of polymers. The maximum PHA production was 41 ± 0.22 % obtained for Stenotrophomonas (SM03) using 2 % glucose as carbon source while for wastewater, maximum production was achieved by the Pseudomonas strain (SM01).Conclusion: Wastewater is produced in large quantities daily during various activities and therefore can be used as a cheap carbon source for the production of valuable products such as PHA.Keywords: Polyhydroxyalkanoates, Wastewater, Glucose, Pseudomonas strain, Stenotrophomonas

Reducing the effect of non-volatile fatty acids (non-VFAs) on polyhydroxyalkanoates (PHA) production from fermented thermal-hydrolyzed sludge

Production of biopolymers from renewable carbon sources provides a path towards a circular economy. This review compares several existing and emerging approaches for polyhydroxyalkanoate (PHA) production from soluble organic and gaseous carbon sources and considers technologies based on pure and mixed microbial cultures. While bioplastics are most often produced from soluble sources of organic carbon, the use of carbon dioxide (CO2) as the carbon source for PHA production is emerging as a sustainable approach that combines CO2 sequestration with the production of a value-added product. Techno-economic analysis suggests that the emerging approach of CO2 conversion to carboxylic acids by microbial electrosynthesis followed by microbial PHA production could lead to a novel cost-efficient technology for production of green biopolymers.

Resource recovery from organic waste streams by microbial enrichment cultures