Research Article
ISSN: 2475 3432

Polyhydroxyalkanoates (PHAs) from Household Food Waste: Research Over the Last Decade

Klempetsani Stavroula*1, Malamis Simos2, Haralambous Katherine-Joanne1
1School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou St., 15780 Athens, Greece
2Department of Water Resources and Environmental Engineering, School of Civil Engineering, National Technical University of Athens, 5 Iroon Polytechniou St., 15780 Athens, Greece
Corresponding author: Klempetsani Stavroula
School of Chemical Engineering, National Technical University of Athens, 9 Iroon Polytechniou St., 15780 Athens, Greece. E-mail: klepetsani@windowslive.com
Received Date: December 05, 2019 Accepted Date: January 02, 2020 Published Date: February 11, 2020
Citation:Klempetsani et al. (2020), Polyhydroxyalkanoates (PHAs) from Household Food Waste: Research Over the Last Decade. Int J Biotech & Bioeng. 6:2, 26-36
Copyright:©2020 Klempetsani Stavroula et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited


Polyhydroxyalkanoates (PHAs) are the ideal candidates for the substitution of the conventional plastics, as they present similar properties with them, but are also environmentally friendly. Regardless of their benefits, these biopolymers still face challenges today, due to their high market price, which is related to the raw material selected for their production. The utilization of low cost and readily available substrates - like organic wastes - for PHA production is a promising solution, which can decrease the total biopolymer production cost. However, PHA products from waste-derived materials need to have a consistent quality. This currently remains a challenge. The last years, PHA production from organic wastes, like food waste, has acquired growing attention. This work reviews the literature of the last decade on the use of household food waste as feedstock for PHA production. Household food waste has been divided in three categories: composite food waste, spent oils and spent coffee grounds. Both pure and mixed microbial cultures have been employed. The review focuses on the feedstock’s and the culture’s pre-treatment methods, the biopolymer’s production and the purification of the final product. It also refers to the PHA content obtained in each scientific work. Household food waste has proven to be a good substrate, especially when combined with pure microbial cultures, like Cupriavidus necator that resulted in PHA accumulation from around 37% to 90%. This scientific work also provides informations concerning PHA applications, industrial production and market prices.

Abbreviations

®, registered trademark symbol; ATCC, American Type Culture Collection; FFW, fermented food waste; FWC, composite food waste; GC, gas chromatography; GC-MS, gas chromatography-mass spectrometry; HA, hydroxyalkanoates; HDPE, high density polyethylene; HPLC, high performance liquid chromatography; HRT, hydraulic retention time; LB, Luria-Bertani; LDPE, low-density polyethylene; MM, mineral salt medium; MMC, mixed microbial cultures; MSB, minimal salt basal medium; OLR, organic loading rate; PHAs, polyhydroxyalkanoates; PHB, poly(3-hydroxybutyrate); P(HB-HV), poly(3-hydroxybutyrate-co-3-hydroxyvalerate); PHO, poly(3-hydroxy-octanoate); PLA, poly (lactic-acid); PP, polypropylene; SBRs, sequencing batch reactors; SCG, spent coffee grounds; SEC, size-exclusion chromatography; SRT, sludge retention time; TM, unregistered trademark symbol; TSA, tryptone soya agar; TSB, tryptone soya broth; UASB, upflow anaerobic sludge blanket; UCO, used cooking oil; UFW, unfermented food waste; VFAs, volatile fatty acids; WCO, waste cooking oil

Keywords:  Household food waste, Biopolymers, Polyhydroxyalkanoates (PHAs), Microbial Cultures, Commercial PHAs, Market prices




Introduction:


PHAs are unique polyesters synthesized naturally by many species of bacteria within their cellular structure under growth-limiting conditions imposed by the scarcity of a nutrient, electron donor or acceptor[1]. These biopolymers are suitable for the replacement of synthetic plastics in many sectors, as they present similar properties with them. Nowadays, they have penetrated mostly in packaging, agriculture and in the medical sector. Besides their good characteristics, PHAs are also environmentally friendly. Unlike conventional plastics that are fossil fuel-based, PHAs come from renewable carbon sources and they decompose naturally in the environment after their disposal. Suitable environments for PHA degradation are those with high microbial activity, like soil and sewage sludge.

HAs industrial production today is mainly based on the use of sugar-based compounds and the employment of pure microbial cultures. Both the raw material and the microorganisms raise the biopolymers production cost, making them less competitive in the market due to their high price. The utilization of low-cost organic wastes as feedstock for PHA production is a promising solution for the reduction of the biopolymers production cost, even though organic wastes - due to their variable composition - may not allow the production of a uniform product. Agricultural wastes, agro-industrial wastes, food wastes etc.[2] have been used a lot in the recent literature for PHA production achieving high PHA accumulation, especially when combined with pure microbial cultures. Mixed microbial cultures have also been employed in many cases. As opposed to pure microbial cultures, they do not need aseptic conditions for their use, though they result in lower PHA accumulation than the pure cultures.

Food waste is a rich organic material capable of providing needful compounds to the microorganisms for their maintenance, growth and the accumulation of PHAs. Its high moisture content and high biodegradability[3], turn it into a troublesome waste material, as it is responsible for the creation of odors, water-polluting leachate and greenhouse gas emissions. In the European Union approximately 88 million metric tons of food waste are generated annually[4], so food waste management is of great importance today. Its valorization for PHA production contributes in the reduction of the food waste amounts that end up in landfills. Though, it should be mentioned that in the literature, the estimation of the PHA content is based on the biomass weight unit, i.e. grams PHAs per grams of biomass and not on the food waste weight unit, which would be a helpful information for the correlation of the food waste amount used for the production of PHAs and the amount of the produced biopolymer.

This review focuses on recent literature advances of PHA production from household food waste with the employment of both pure and mixed microbial cultures. Household food waste comprises of inhomogeneous food wastes (composite food waste) and homogeneous waste streams, like cooked oils and spent coffee grounds. Food waste in general includes also the side streams of the food processing industries, like whey and molasses[5], but this review focuses only on the first two mentioned streams arising from a household. The overall procedure comprises of the substrate’s and the culture’s pre-treatment, the biopolymer’s production and the downstream processes, such as the extraction and the purification of the final product.

Applications, industrial production and market prices

The composition of the PHA, which is directly related to the biopolymer’s structural and mechanical properties, depends on the carbon substrate, the metabolic pathway utilized and the specificity of the PHA synthase[6], the key enzyme that polymerizes the monomeric hydroxyalkanoates[7]. PHAs are categorized in small, medium and long-chain-length PHAs according to the number of the carbon atoms in the monomeric block. They are also divided in homopolymers, that consist of one type of PHA throughout their structure, copolymers that are made up of two different types of monomers and heteropolymers that contain 3-hydroxy fatty acids of many different chain lengths[8]. PHB, which is a typical short-chain-length PHA, is brittle and stiff, while the medium-chain-length PHO is more flexible. So, each one of them can be applicable in different areas in accordance with its characteristics. Different monomers can be combined to form a copolymer with the desirable properties[9]. For example, the combination of HB and HV monomers forms the copolymer P(HB-HV), which is more flexible than the PHB[10]. In 2014, Bugnicourt et al. referring to the PHA general characteristics described them as biocompatible, nontoxic, soluble in chloroform, resistant to ultraviolet radiation and insoluble to water[10]. Figure 1 demonstrates the economic fields in which PHAs are commercialized today.

Fig 1: PHAs applications [11,12].

PHAs are suitable for a large number of applications and it is estimated that the PHA market will be worth 290 million USD by the end of 2025[13]. The industrial production of PHAs is generally comprised of several steps including fermentation, the separation of the biomass from the broth, the biomass drying, the extraction of the PHA, its drying and its packaging[14]. The most common industrially produced PHAs are limited to the combination of PHB and another PHA as the co-monomer[15]. Table 1 shows a list of industrially produced PHAs that are available in the market today and provides a short description of their applications.

Table 1: Industrially produced PHAs, available in the commerce today.

PHAs drawback is their high market price. Table 2 demonstrates a comparison of the market prices of PHAs, PLA and petroleum-based polymers, as it was reported by the Organization for Economic Co-operation and Development (OECD) in 2014. OECD also provides the information that PE (polyethylene) cost is €1.58/kg and PET (polyethylene terephthalate) cost is € 1.73/kg. As it is seen, PHAs are approximately four times more expensive than the petroleum-based polymers. So, the development of cost-effective technologies for the generation of PHAs in order to make them more competitive in the market is of high priority.

Table 2: Prices of biopolymers [18].

PHA production from household food waste the last decade

Household food waste categories

The utilization of food waste has been reported in the literature for the production of high added value products, such as lactic acid and PHAs. Prior to its use for PHA production, food waste must undergo a suitable pre-treatment process in order to be available to the microorganisms. The most frequent pre-treatment process is fermentation. The last decade, scientific papers referring to PHA production from household food waste have focused on composite food waste, spent coffee grounds and spent oils like waste frying oil, used cooking oil and spent palm oil. As it is derived from tables 3 and 4, composite food waste was the most employed waste for PHA production (47%), while spent oils and spent coffee grounds were used at a percentage of 41% and 11%, respectively. The methods employed for the food waste pre-treatment depend on its characteristics (Fig. 2).

Fig 2: Pre-treatment methods employed for each category of household food waste.

Spent coffee grounds:
Spent coffee grounds are an important waste product of the coffee industry [19]. It is estimated that 6 million tonnes of spent coffee grounds are generated worldwide every year [20]. They contain large amounts of organic compounds like fatty acids, amino acids, polyphenols, minerals and polysaccharides [21]. In recent studies, it is seen that the use of spent coffee grounds for PHA production has resulted in very high PHA content, approximately from 70% to 90%. Their useful part for PHA production is their oil. So, it first needs to be extracted in order to be utilized in the procedure. In one study, the spent coffee grounds were dried and then their oil was extracted through supercritical extraction in a semi-continuous high pressure extraction pilot unit [22] and in another, it was extracted with the use of n-hexane in an extractor apparatus [23].
Spent oils:
Spent oils are considered to be very good candidates for PHA production, as it is cheaper to use a mixture of fatty acids found in plants, than oily purified acids [24]. The advantage of utilizing plant oils is their high carbon content as well as their high conversion rate to PHA [25–27]. Due to the fact that oils compose a much higher number of carbon atoms per weight, low flow rate streams can be applied reducing the dilution of the fermentation broth and optimizing the product concentration [28]. Besides the above, spent oils require no pre-treatment [28–32] or they may just require to be sterilized before the PHA production step [33].

Composite food waste:
Composite food waste does not provide easily biodegradable substances directly in the culture, so it needs to be fermented for the production of a liquid full of easily consumable volatile fatty acids. In the literature, the composite food waste was directly fermented or it was pre-treated prior to fermentation. Eshtaya et al. (2013) diluted the composite food waste in water before its fermentation, while in other scientific works, it went through mastication, filtration, oil removal and pH adjustment [34] or dilution in sewage sludge to the required OLR [35]. The fermentations took place at ambient temperature [35,36], mesophilic temperature [37,38] or the reactor was subjected in different temperature and pH regimes for the investigation of their effect on the fermentation process [39]. In some cases, the acidogenic fermentation was conducted with the use of anaerobic consortia [35,38]. The reactor operated in a fed-batch mode [35,37] or in a batch mode [38]. Nevertheless, in most cases anaerobic conditions prevailed in the reactors [34,35,38], but microaerobic conditions have been also reported [37]. The pH value was usually left uncontrolled throughout the procedure, but it is also reported that it was adjusted to 6 prior to fermentation [34]. Some fermentations were accompanied with agitation [39] and others took place with no agitation [36]. After fermentation, the produced liquid was either filtered [36] or the organic acids were recovered by the freezing thawing method [39]. Finally, the hydrolysate obtained from the acidogenic fermentation was used as influent substrate for the culture’s enrichment step and the production of PHA. Figure 2 illustrates schematically the pre-treatment methods that were employed for each category of household food waste before its use in PHA production.

The production of PHAs is a biological procedure consisting of three major phases. The first phase is the substrate’s pre-treatment, in this case the household food waste’s pre-treatment, for the obtainment of a liquid rich in volatile fatty acids, the precursors of PHAs. In the second phase, the culture is cultivated under specific environmental conditions for the production of a large homogeneous microbial population that is capable of producing PHAs. If the culture is pure, which means that it consists of only one species of bacteria that are already known in the literature as PHAs accumulators, homogeneity is achieved. This homogeneous population only needs to multiply before PHA production for the obtainment of a sufficient amount of active biomass capable of producing PHAs. The multiplication of the bacterial population is achieved through the acclimatization phase (Fig. 3). If the culture is mixed, consisting of many species of microbes, a selective pressure needs to be applied in the reactor for the enrichment of the culture in PHA-storing microorganisms before its utilization in PHA production. In this case, the second phase is referred to as the culture’s selection and enrichment step (Fig. 4). In the third phase, the cells harvested from the second phase are fed with the rich in organic acids liquid obtained from the first phase, usually without the addition of nutrients, for the optimization of the culture’s capacity in accumulating PHAs. After the accumulation, PHAs are extracted from the microorganisms and purified through chemical methods.

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

A wide variety of bacteria is capable of accumulating PHAs. Alcaligenes latus, Cupriavidus necator and Pseudomonas putida are the most commonly used species in the industrial production of PHAs today[40]. In the recent bibliography referring to PHA production from household food waste, pure microbial cultures were much more employed than the mixed ones for the production of these biopolymers. As it results from tables 3 and 4, C. necator of the genus Cupriavidus was the most widely employed culture used for PHA production (53%), followed by the activated sludge and aerobic consortia equally used (12% each). The rest scientific works employed E. Coli, Pseudomonas and isolates obtained from the environment (23% in total). E. Coli is uncapable of producing PHAs when it is in its natural form, but it is the most studied genetically modified strain in the research of PHA biosynthesis[41]. Eshtaya et al. (2013) that employed recombinant E. Coli for PHA production with the use of organic acids from fermented restaurant waste reported PHB accumulation equal to 44% (w/w)[39]. Isolates are not considered to be competitive with the commercial strains for the production of PHAs in the literature[42], though Vijay et al. (2019) observed a slightly higher PHA accumulation from a bacterial isolate with the utilization of onion peels as substrate in comparison with the PHA content obtained from a reference strain with the use of the same substrate[43].

Before the acclimatization phase, a pure culture is usually maintained in a synthetic medium, which is mainly composed of peptone, meat or yeast extract, sodium chloride and the solidity provider - agar [23,31,44]. These ingredients offer key compounds to the microorganisms to support their growth like carbohydrates, nutrients and salts[45]. The exact composition of the medium is related to the strain requirements[41]. Besides the culture preservation, a nutrient broth is also used for the culture development in the acclimatization phase[23]. The medium usually contains nutrients and an easily biodegradable carbon source[9,30,36,44]. In most of the cases, the carbon source is the VFAs-rich stream obtained from the first stage[22,30,31]. Many types of media have been used for the acclimatization phase in the recent literature, such as a mineral medium supplemented with the liquid obtained from the food waste fermentation[22,31], a nutrient rich medium[9], a Luria-Bertani medium[31,39], a tryptone soya broth[33] etc. The incubation of the cultures mostly took place at 30OC[9,31,33,44] and it lasted 16 h[9], 24 h[22,33] or 48 h[30,31].

In the PHA accumulation stage, the hydrolysate from the composite food waste fermentation, the extracted oil from the spent coffee grounds or a waste oil obtained from the first phase was added in the reactor containing the cells from the second phase. In some cases, an extra carbon source was supplemented along with the principle raw material for the achievement of feedstock availability[9]. Both batch and fed-batch cultivations were performed for the accumulation of PHAs. In one study, the fed-batch fermentation for PHA production resulted in much higher PHB content than the batch one[44], while in another one three feeding regimes were investigated – pulse, stepwise and continuous feeding – and the highest PHA accumulation was achieved from the continuous feeding regime[36]. Temperature was mostly maintained at 30℃[30,43,46,48]. In the scientific paper of Eshtaya et al. (2013), the temperature increased from 34 to 38OC to induce PHB production, while in Hafuka et al. (2011) it was maintained at 20℃. The pH value was maintained at 6.8[31], 7 [9,23,39,43,44] and 7.5[36]. Aerobic conditions prevailed throughout the process[30,31,36].

The downstream processes for PHA extraction and purification are not related with the type of the carbon source and the microbial culture used for PHA production, as they take place after the biological production of PHA. The cells from the third phase are harvested-mainly by centrifugation-and then subjected in a pre-treatment method for the softening of the cell structure that is around the PHA granules. Pre-treatment can be chemical, physical or biological[10]. The most common pre-treatment methods are the thermal drying and the lyophilization. Chemical pre-treatment requires the use of sodium chloride (NaCl) or sodium hypochlorite (NaOCl), while physical methods require high temperature and ultrasonication methods[10]. The extraction and the precipitation of PHAs in a lab scale are mostlly achieved with a use of a solvent – mainly chloroform – and the addition of an alcohol, mostly acidified methanol or propanol, respectively. After their extraction from the biomass, the biopolymers are analyzed with the use of GC, HPLC or SEC.

In the recent literature referring to the generation of PHAs from bacterial strains, genetic modified strains or isolates from the environment with the use of household food waste, high percentages of PHA content were reported. 66% of the scientific works demonstrated in table 3 reported more than 50% PHA accumulation, while the highest PHA contents were observed for the employment of C. necator. Table 3 presents the overall procedure of PHA production with the employment of pure microbial cultures from scientific works that dealt with PHA production from household food waste the last decade.

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

Fig. 3: Flow diagram of PHA production from household food waste and pure microbial cultures

The employment of mixed microbial cultures, such as the bacterial populations from the activated sludge process, has gained growing attention the last years mostly for the scaling up of the process, as it results in lower production cost and provides the advantage of the procedure’s integration in the wastewater treatment process. For the selection of a culture with high PHA storage capacity, the mixed microbial population must undergo through a particular pre-treatment method. In all scientific works that dealt with PHA production from household food waste by applying mixed microbial cultures, the cultures employed were subjected in the feast and famine regime for the selection of PHA-storing bacteria [34,37,38]. In this regime, when the reactor operates under periods of substrate excess (feast phase) and relatively long periods of substrate limitation (famine phase), the microorganisms undergo through an internal growth limitation. During the latter, they use external nitrogen and phosphate ions and organic carbon stored internally as PHA for growth and maintenance. Those organisms that do not store PHAs are seriously affected by the long famine period and are eliminated from the reactor, while those that have the capacity to store bigger amounts of PHAs become the prevailing species in the reactor.

The culture’s selection and enrichment step was often carried out in sequencing batch reactors (SBRs), compact systems where the full feast and famine cycle were performed in one single reactor and the length of each phase could be varied[49]. During the feast period, the reactor was filled with sludge, carbon substrate and nutrients. The carbon substrate in all cases was the hydrolysate obtained from the food waste fermentation, while the nutrients comprised of ammonium and phosphate ions. If the seed is obtained from the aeration tank of an activated sludge process it is usually aerated for some time, so that the original existing substrate can be exhausted[38]. The reactor operation is divided in cycles. Each cycle lasts for 12 h[38] or 24 h[34] and usually consists of four discrete phases: the influent filling, the aerated phase, the settling of the biomass and the withdrawal of the supernatant. Besides of the cycle length, the sludge retention time, the hydraulic retention time, the organic loading rate and the C:N ratio are also important parameters for the reactor operation.

PHA accumulation requires the hydrolysate from phase I as carbon substrate, which is full of volatile fatty acids and the enriched culture from phase II as microbial population. Batch mode[37] and fed-batch mode[35] were employed. In most cases of MMC PHA production, aerobic conditions were applied[37,38]. Venkateswar et al. (2012) tested both the aerobic and the anoxic environment, though. The temperature was usually ambient[38] or it was maintained around 30OC[37] and the pH value was adjusted to 7[35,37], 8.8[34] or it fluctuated from 5.5 to 7.5 [38]. Finally, it is reported that, in order to avoid nitrification into the reactor, thiourea was added along with the substrate and the enriched culture[37]. Analytical description of all stages of PHA production from household food waste and mixed microbial cultures is demonstrated in Table 4. The accumulation of PHAs by the mixed microbial populations was lower than that obtained from the pure ones.

Fig. 4: PHA production from household food waste and mixed microbial cultures.

Fig. 4: PHA production from household food waste and mixed microbial cultures.

Conclusion and suggestions for further research

Household food waste has shown a good potential as a substrate for PHA production. The review of existing works indicated PHA accumulation from around 19% to 90% (g-PHAs/g-biomass*100%). So, the performance of PHA production from household food waste can be very high, but also presents great fluctuations that may be connected with the different operating parameters employed by each scientific work and the variability of the substrate and should be taken into consideration for the scaling up of the process. Nevertheless, in the scientific works that used spent coffee grounds oil for PHA production, the PHA content was over 70%. Waste cooking oils have also been a promising substrate, as they produced PHAs up to 85%. Pure microbial cultures exhibit higher performance than the mixed ones, but their pre-treatment is more expensive, which is a drawback for the overall production cost in a large scale system. The use of Cupriavidous necator for PHA production from household food waste resulted in high PHA accumulation from around 37% to 90%, while the corresponding percentage when mixed microbial cultures were employed was 23-47%.

A key challenge in PHA production is that they have to be cost competitive with the fossil fuel-based polymers. PHA industrial production nowadays is mainly carried out with refined sugar substrates as raw materials and pure cultures or genetically modified strains as microbial populations for the creation of a final material with specific characteristics; this is an expensive choice. The use of low cost organic wastes - like household food waste - for PHA production, is a promising choice for the reduction of their market price, but also a challenge to the production of a uniform product having consistent properties.

Organic wastes are composite raw materials and they provide to the microbial population a variety of different carbon sources like proteins, carbohydrates and lipids making it difficult to foresee or control the composition of the produced biopolymer. Thus, PHAs produced from waste feedstocks are characterized by lack of consistency in properties. Furthermore, the extraction process is currently a bottleneck which increases the overall production cost of PHAs. The current situation results in a limited market uptake for PHAs, in comparison with the market uptake for conventional plastics that are both inexpensive and consistent in properties and therefore still have a primary role in commerce.

Ending, PHAs are of great significance today, as their production aims in decoupling economic growth from resource depletion and environmental degradation. PHA production from food waste offers the extra advantage of utilizing waste for the creation of another product. Subsequently, the food waste amounts that end up in landfills producing large quantities of methane, a powerful greenhouse gas, which is responsible – among others - for global warming and climate change, are reduced. The scientific community and the industrial sector should pay much more attention in PHA production from food waste the following years for the benefit of the environment and the improvement of the quality of life.


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