Technological feasibility and environmental assessment of polylactic acid-nisin-based active packaging

Sustainable Materials and Technologies 33 (2022) e00460 Available online 23 June 2022 2214-9937/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Technological feasibility and environmental assessment of polylactic acid-nisin-based active packaging Ana Arias * , Gumersindo Feijoo, Maria Teresa Moreira CRETUS. Department of Chemical Engineering, School of Engineering, Universidade de Santiago de Compostela, Spain ARTICLE INFO Keywords: Active packaging Functional film PLA-nisin Life cycle assessment ABSTRACT The new consumption patterns based on the “use and throw away” trend has led to an increase in the production of petroleum-based plastics for their application as food packaging films, leading to significant environmental impacts. In the search for alternatives to conventional plastics, polylactic acid (PLA) stands out due to its renewable origin, its mechanical properties and its biodegradability potential. In this regard, this manuscript addresses the large-scale simulation of a bio-technological process for the valorization of wheat straw residues for the co-production of nisin and lactic acid through a batch-type fermentation process. Subsequently, lactic acid will be used for the production of PLA films, to which nisin will be applied to convert them into functional antimicrobial films. To assess the degree of environmental impacts and sustainability of this biotechnological process, Life Cycle Assessment, considering a cradle-to-gate approach, and Greenness Grid methodologies have been applied. The environmental profiles obtained showed that the pre-treatment and downstream stages are the ones leading to the highest impact, being the energy requirements the main hot spots. Moreover, seeking an environmental communication perspective, useful for researchers and stakeholders, the Green Index indicator value obtained leads to the consideration of a potentially sustainable process. 1. Introduction In response to the consumption habits of today’s society, the agrifood industry has progressively implemented production and preservation technologies that guarantee the hygienic quality of food [1,2] towards the use of biodegradable packaging systems that displace the massive use of plastic [3–5]. According to data from the European Bioplastics Association, the global production capacity of bioplastics will increase from around 2.11 million tonnes in 2020 to approximately 2.87 million tonnes in 2025. Among the different types of biopolymers, the group of biodegradable plastics, including PLA (Poly lactic acid), PHA (polyhydroxy alkanoate), starch blends and others, represent the largest share with almost 60% (more than 1.2 million tonnes) of the global bioplastics production, which will facilitate the supply of raw material for various industrial sectors and the possibility of lowering their price. The ability to be biodegradable, compostable, low-cost manufacturing and environmentally friendly have made PLA biofilms good candidates for the development of food packaging films. However, certain reinforcement strategies need to be considered when it comes to using such biofilms as food packaging materials. Barrier properties, i.e., water and heat resistance, need to be improved to ensure proper food preservation. However, there are studies that address the development of techniques that, in a simple way, can significantly improve these weaknesses, without forgetting the “bio/natural” concept. For example, the addition of bio-nanocomposite materials in biofilm shows that water resistance can be increased by 60% [6–8]. Beyond the traditional “packaging” approach, new food preservation technologies are emerging under the umbrella of “active packaging” [9–11]. These are systems designed to intentionally incorporate components that either release beneficial substances such as antimicrobials, antioxidants, flavorings, or absorb substances from the packaged food or its environment such as unwanted or harmful substances such as oxygen, moisture or unpleasant odours. Through the technological development of active packaging systems, it is possible to correct the shortcomings of traditional preservation technologies for packaged foods, improving their quality, extending their shelf life and avoiding food waste [4,9]. In addition to the potential of bioplastics as a food packaging material, there is a renewed interest in fresh and “natural” products, i.e., products without artificial additives and preservatives that retain their * Corresponding author. E-mail address: [email protected] (A. Arias). Contents lists available at ScienceDirect Sustainable Materials and Technologies journal homepage: www.elsevier.com/locate/susmat https://doi.org/10.1016/j.susmat.2022.e00460 Received 4 October 2021; Received in revised form 18 November 2021; Accepted 18 June 2022 Sustainable Materials and Technologies 33 (2022) e00460 2 nutritional and organoleptic properties after processing. Beyond thermal processes and chemical additives, bacteriocins emerge as potent antimicrobial agents and offer the possibility of preserving foods and especially their organoleptic quality. In fact, several research articles have evaluated the use of nisin for the production of functional food packaging materials, reaching its efficacy against foodborne pathogens [13–16]. In particular, nisin stands out not only as a natural biopreservative [10,11] but also in the biomedical and pharmaceutical field as it reduces the undesirable effects of certain antibiotics [19] and is effective in the treatment of infections related to drug-resistant, gastrointestinal and respiratory pathogens, among others [20]. As an example of cutting-edge research, the inclusion of bacteriocins in nanomaterials has shown positive preliminary results as a potential anticancer treatment, reducing the growth of tumor cells [21]. This manuscript addresses the environmental evaluation of the production process of functional biofilms based on PLA and nisin from the valorization of wheat straw as a source of fermentable sugars after enzymatic hydrolysis of the cellulose present in the biomass. According to a recent study addressing the development of a linear regression model to estimate the generation of post-harvest wheat residues [22], it is concluded that a total of 58.3 million tonnes of wheat crop residues will be produced in the European Union in 2030, a value that shows the wide availability of this resource for use beyond its use on farms as fodder, animal bedding or as amendment for compost. Thus, the biotechnological valorization process proposed in this scientific article is developed from modelling criteria and tools based on the conceptual design of the production process on a demonstrative or real scale. This stage is key in the collection of inventory data necessary to carry out the environmental assessment using the Life Cycle Assessment methodology [23]. It is considered a standardized methodology that allows a quantitative analysis of the environmental impacts of a product/process throughout its life cycle [23]. Its applicability and use are widespread for the evaluation of production processes, both those that are under development and those that already exist, as it allows researchers and policy makers to develop an exhaustive knowledge of their potential for environmental damage [24–27]. On the other hand, it is a tool recommended by renowned institutions, such as the European Commission and the United Nations Environment Programme, as it is the basis for the development of Environmental Product Declarations (EDPs) (ISO 14025), for the calculation of the Carbon Footprint and for the realization of Eco-efficiency Analysis, among others. This methodology has proven to be appropriate, effective and accurate in the development of environmental impact analysis of bioplastics applied as packaging for food products. Just to give some examples, LCA has been applied for assessing a modified starch-PLA biofilm [28,29], a chitosan-based film [30], a biodegradable carbohydrate polymer film [31], soy-protein and chitosan-agar films prepared form waste streams [26] and also a wheat gluten-based packaging film [32]. In addition, this methodology has been used to analyze how wastes and food losses could affect the environmental sustainability of food packaging [33]. Although at first glance bio-based processes seem to be more environmentally friendly, this is not always the case. The main “disadvantage” or weak point in the development and implementation of new production processes based on the valorization of agro-industrial waste streams is that they lack the specialization, optimization and development of conventional processes, many of which are based on the use of non-renewable fossil resources for the production of raw materials or energy. Even so, the feasibility of an approach based on circular economy principles needs to be assessed, so that a process is being proposed in which a certain fraction of waste is valorized into a high added value product such as a functional packaging. Moreover, from the perspective of end-of-life management, the biodegradable character of PLA biofilms has to be taken into account, which implies a great ecological and environmental advantage. On the other hand, looking for define the overall sustainability associated with the active packaging formulated with PLA, as reference bioplastic, and nisin, as antimicrobial, which plays the role of active ingredient that ensures the quality of the stored food, authors have considered the combination of LCA with the Greenness Grid (G2) methodology [34]. This methodology is based on the 12 principles of Green Chemistry [34] and it allows a direct comparison between those principles by a calculation criterion based on standardized models of a qualitative and quantitative nature. In addition, it gives a single dimensionless value that allows a global assessment of the sustainability of the process under study through the development of a ranking and a color code [34]. On the other hand, it is important to mention that, despite the effectiveness of this methodology, the authors themselves consider that certain aspects related to raw materials or products cannot be analyzed in detail using this metric and, therefore, consider that the combination of the LCA methodology together with the G2 is not only desirable, but also recommended. 2. Materials and methods In order to develop an environmental analysis of the biotechnological production of a functional biofilm based on PLA and nisin, a large-scale simulation of the process from the valorization of wheat straw was proposed. In this way, it would also be possible to compare it with the production of conventional plastic films and identify the main advantages in the development of the biofilm. In order to determine the environmental profile associated with the production of active packaging, it is necessary to identify the different stages of the process as well as the main input and output streams together with the assumptions and methodologies of calculation and evaluation, which are put forward for the conceptual design of the process using the SuperPro Designer software. Once the basic information for the estimation of the environmental impacts has been compiled using the life cycle methodology and the SimaPro software, a study will be carried out on the adequacy of the PLA-nisin biofilm production process to the principles of green chemistry, based on the calculation of the Green Index, as proposed by Pinto et al. (2020) [34]. The Greenness Grid (G2) methodology has been recently described with the aim of assessing the sustainability of new processes developed within the framework of green chemistry, such as this case study, from a quantitative and specific perspective. This methodology compiles the numerical output of 15 metrics associated with the principles of green chemistry, allowing a score to be reached for the different chemical processes/products when they are evaluated. 2.1. Process description and simulation procedure In order to evaluate the environmental profile associated with PLAbased active packaging, the production process has been divided into four sub-systems: SS1. Wheat straw pre-treatment, SS2. Fermentation of wheat straw, SS3. PLA film production comprising 4 main stages: Oligomerization (SS3.1), Pre-polymerization (SS3.2), Polymerization (SS3.3) and Purification (SS3.4) and finally the subsystem SS4 Active production of packaging film. 2.1.1. SS1. Pre-treatment of wheat straw The first stage of this biotechnological process for the valorization of wheat straw residues starts with a pre-treatment to promote the release of sugars present in the molecular structure of the lignocellulosic biomass (Fig. 1). Wheat straw is characterized by a high content of cellulose (28–39%) and hemicellulose (23–24%) [35], which makes it an excellent source of fermentable sugars. The SS1 subsystem starts with an autohydrolysis stage at high temperature, 210 ◦C, and high pressure, leading to the release of fermentable sugars from cellulose and hemicellulose with conversion percentages of around 22 and 31%, respectively [36]. Next, filtration A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 3 equipment allows the separation of a solid stream, rich in cellulose, and a liquid stream, rich in hemicellulose. While the solid stream will undergo enzymatic hydrolysis treatment, the liquid stream will be treated with a chemical process after hydrolysis. The enzymatic process is characterized using a cocktail of hydrolase enzymes, thus achieving a cellulose to glucose conversion yield of 93.7%, and the conversion of hemicellulose to xylose with a yield of 38.5%. As for the required operating conditions, 50 ◦C and continuous agitation during a residence time of 48 h are required [36]. Regarding the post-hydrolysis chemical process, sulfuric acid will be used, requiring a temperature of 121 ◦C to be maintained for a period of 1 h [36]. With these operating conditions, yields of around 97% are achieved for the conversion of cellulose into glucose, and 22% for hemicellulose to xylose. After these two pretreatment stages, a flow rich in sugars is obtained, which will be used in the fermentation stage for the co-production of nisin and lactic acid. 2.1.2. SS2. Fermentation of wheat straw The fermentation process is based on the combined production of nisin and lactic acid (as a base element for the production of PLA) by the strain Lactococcus lactis [37]. One of the most important aspects when considering a fermentation process is the formulation of the culture medium as it not only affects the regulation of the microbial metabolism and thus the productivity and yield of the process, but also the economic viability of the biotechnological process as the production costs are usually strongly related to the formulation of the medium, with percentages between 25 and 40% of the total [38]. While the carbon source will be provided in the form of glucose and xylose, obtained after the wheat straw pre-treatment process, it is necessary to add a source of nitrogen (NH 4 OH) and phosphorus (K 2 HPO 4 ). As for the ideal operating conditions to promote a higher yield of the fermentation process, it is necessary to operate at 30 ◦C under an aeration flow of 0.5 L⋅h −1 for a period of 24 h [39] (Fig. 2). 2.1.3. SS3. Downstream process After the previous fermentation process, two fermentation products, i.e. nisin and lactic acid, are obtained as well as biomass and the residual fraction of the culture medium that has not been depleted. Therefore, a further step for the separation and purification of the two co-products is required (Fig. 2). First, to separate the biomass, a vacuum filtration system is used, followed by a chemical precipitation in an acid medium, using ammonium sulphate [40]. This purification system was chosen on the basis of the high nisin recovery achieved. The precipitated nisin will be separated from the stream by a centrifugation system and then the final product will be obtained after spray drying with a purity of more than 95% by weight. After the centrifugation, a lactic acid rich stream is obtained, which will be processed for separation and purification. Three stages can be distinguished in the purification of lactic acid: a first concentration stage to remove excess water, a second esterification stage with methanol, in which methyl lactate is obtained, which will be reconverted into purified lactic acid in the third stage of hydrolysis [41]. 2.1.4. SS4. PLA film production The production process of high molecular weight PLA from lactic acid is carried out in a sequence of 4 steps (Fig. 3), which are described below. 2.1.4.1. SS4.1. Oligomerization. The formation of high molecular weight polymers requires the use of highly concentrated monomer solutions, i.e., with low water content [42–44]. Consequently, the lactic Fig. 1. SS1.Pre-treatment flow diagram of PLA-nisin-based functional film process. A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 4 acid monomer will be subjected, in a first stage (SS4.1), to a process under high temperature and vacuum operating conditions, where it will undergo self-esterification, leading to the formation of oligomers (low molecular weight PLA) [45]. The model reactions that have been considered for the large-scale simulation of this first subsystem (SS4.1) are those developed in Table 1, where the yields in the production of lactic acid and oligomers after this first stage are also included. Oligomerization of lactic acid at 180 ◦C and 533 mbar for a residence time of 20 min yields a mixture of lactic acid monomer (43 wt%), the oligomer LA1, corresponding to (SS)-2-[(2-hydroxypropanoyl(oxy)] propanoic acid (24.6 wt%) and LA2, which is (SSS)-3-hydroxybut-3-en2-[(2-hydroxypropanoyl(oxy)] propanoate (9.96 wt%). It must be ensured that the stream has a low water content, as the intermediate cyclic dimer, lactide, required for subsequent PLA production, is an unstable compound in the presence of water, thus affecting the overall yield of the production process. 2.1.4.2. SS4.2. Pre-polymerization. Once the lactic acid oligomers have been obtained, the pre-polymerization stage is carried out. The product obtained by this second subsystem is lactide, a cyclic lactic acid diester with the molecular formula C 6 H 8 O 4 , obtained from the dehydration of two lactic acid molecules in the presence of a SnO (Tin(II) Oxide) catalyst [46]. One of the characteristics of lactide is that there are two stereocenters in its molecular structure, which leads to the formation of three optically active stereoisomers: L-lactide, meso-lactide and D-lactide [47]. From the model reactions, it is possible to progress on the largescale simulation of the process (Table 2). Lactic acid and its oligomers lead to the formation of three stereoisomers of lactide, together with some side products with different molecular structures [46]. All three lactide stereoisomers can be converted to PLA by catalysis. Although semi-crystalline PLA structures would be obtained using any of the three stereoisomers presented, all of which are suitable for application as bioplastics, the yield strength, heat resistance and other mechanical properties of the biofilm obtained depend on the proportions of each stereoisomer. The ratio of lactide stereoisomers in the final PLA film will significantly affect its mechanical and morphological properties. As a lot of L-lactide is present in the PLA film, high crystallinity is achieved, which favors obtaining a less amorphous biofilm, with improved properties, highlighting the strength and stability of the biofilm [48]. PLA obtained from the L-lactide stereoisomer, called isotactic Fig. 2. SS2. Fermentation of Wheat Straw and SS3. Downstream Process flow diagram of PLA-nisin-based functional film process. Fig. 3. SS4.PLA film production flow diagram of PLA-nisin-based functional film process. Table 1 Reaction considered for the large-scale simulation of the process subsystem SS4.1. SS1 Oligomerization Weight % Lactic acid C 3 H 6 O 3 65.43 Oligomer LA1 2C 3 H 6 O 3 → C 6 H 10 O 5 +H 2 O 24.60 Oligomer LA2 3C 3 H 6 O 3 → C 9 H 14 O 7 +2H 2 O 9.96 Table 2 Reactions considered for large-scale simulation of process subsystem SS4.2. SS2 Pre-polymerization Lactic acid >Lactide 2C 3 H 6 O 3 → C 6 H 8 O 4 +2H 2 O LA1 >Lactide C 6 H 10 O 5 → C 6 H 8 O 4 +H 2 O LA2 >Lactide C 9 H 14 O 7 → C 6 H 8 O 4 +C 3 H 6 O 3 Side Products (C 3 H 6 O 3 ) n A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 5 PLLA, is the most abundant commercially [49], i.e. the one that is produced industrially in larger quantities, given the excellent mechanical properties of the bioplastic, which is widely spread in its applications. Therefore, it is necessary to select those operating conditions that favor a higher production of the L-lactide stereoisomer. Thus, a temperature of 240 ◦C and a pressure of 32.5 mbar have been considered, which are maintained for a time of 49 min, resulting in a yield of 91.7% [46], and thus obtaining the following proportion of lactide stereoisomers: 73.7% of L-lactide, 16.5% of meso-lactide and 4.1% of D-lactide. 2.1.4.3. SS4.3. Polymerization. After obtaining lactide, the stereoisomers are converted into high molecular weight PLA from a ring-opening polymerization process in the presence of a precursor and a catalyst. As for the precursor for the lactide ring-opening process, most of them are alcohol-based compounds, such as methanol or 1-dodecanol. However, the high potential of poly(butylene succinate) (PBS) as an initiating agent leads to process yields of up to 84% with 0.001 mol% PBS, as opposed to a yield of 42% using 0.1 mol% 1-dodecanol [50]. On the other hand, as for the most efficient catalyst for this stage of the process, the most used is 0.1 mol% tin (II) 2-ethylhexanoate (C 16 H 30 O 4 Sn). Operating conditions of 0.3 bar, 160 ◦C and a reaction time of 4 h have been considered as requirements to achieve a yield of 84% [50]. 2.1.4.4. SS4.4. Purification. The separation of the catalyst required in the pre-polymerization and polymerization stages is based on the acidbase reaction between SnO and sodium hydroxide (NaOH), resulting in the formation of sodium stannite (Na 2 [Sn(OH) 6 ]), with reduced environmental impacts, thus being less harmful compared to the SnO catalyst [51]. On the other hand, with regard to the purification of the polymer produced, it is based on a simple evaporation procedure in which all oligomers that have not been bound to the molecular structure of the PLA polymer are removed, selecting 220 ◦C and 0.35 bar as the appropriate operating conditions [52]. 2.1.4.5. SS5. Active packaging film production. The SS5 subsystem has not been simulated on a large scale, nor has it been considered within boundaries of the LCA, since it is considered, a subsequent stage developed by the food packaging company itself, which would be supplied with PLA and nisin films. The process of introducing the bacteriocin into the PLA film layers is based on a simple diffusion coating method [53], which consists of immersing the PLA films in a solution at pH 2 containing 1% nisin for 18 h, with constant agitation and at room temperature. After this time, the functional films would be washed with a solution of 1 N NaCl and deionized water before drying for subsequent use as food packaging [54]. 2.2. Environmental analysis using LCA methodology In the framework of the evaluation and comparison between the different alternatives of bio-based production processes and their petrochemical analogues, it is important to use a tool that allows the identification of the environmental impacts associated with them [55]. In this way, it will be possible to analyze the potential and weaknesses of biotechnological processes for replacing those based on the use of fossil resources. Therefore, LCA has been considered for the environmental assessment of the production of PLA-nisin biofilm. For this purpose, according to ISO 14040 and ISO 14044, the development of four interrelated phases is required: Stage I. Goal & Scope definition, Stage II. Inventory Analysis, Stage III. Impact assessment and Stage IV. Results Interpretation 2.2.1. Goal & Scope definition The objective of this report is the evaluation of the environmental profile of the functional biofilm based on PLA and nisin. As there is no data available regarding this production process, a simulation based on experimental data at laboratory scale is required. SuperPro Designer V11.2 has been the selected software, as it allows to perform material and energy balances, which values will be used as inputs for the development of the LCA methodology. The production of 1 kg of PLAnisin biofilm was the functional unit selected to report the environmental results. In terms of the scope of the study, an attributional cradleto-gate approach has been considered, which includes the extraction of raw materials, the biofilm manufacture and management of waste and emissions within the system boundaries. 2.2.2. Impact assessment Inventory data is provided through large-scale simulation of the production process, including raw materials, chemicals, energy needs, both electricity and heat, waste streams and emissions. The Ecoinvent v3.5 database has been chosen as the secondary data source to consider the background activities associated with raw materials, chemicals and waste treatments. Regarding the methodologies selected for assessing the environmental impacts associated with the manufacture of the biofilm, ReCiPe 2016 hierarchist Midpoint method V1.03, to obtain the characterization impact values, and ReCiPe 2016 hierarchist Endpoint method V1.03H/ H, for comparing the single score values obtained for each process subsystem, have been selected. 2.3. Green Index calculation using G2: greenness grid methodology The G2 methodology is based on the calculation of the Green Index, obtained as a result of the sum of a total of 15 metrics encompassing the 12 principles of Green Chemistry. In this way, a value between 0 and 15 is obtained, with 0 being the worst result, therefore representing an unsustainable process, and 15 the best, that is, a sustainable process. For the qualification of the final value of the Green Index obtained, once the metric has been developed, the color code developed by Pinto et al., (2020) [34] will be used (Fig. 4). 3. Results and discussion Process simulation using SuperPro Designer provides input and output flows, as well as electricity and heat requirements, associated with the biotechnological production of functional PLA-nisin biofilms. With this high quality data, it is possible to perform life cycle inventories, essential information in the development of the LCA. 3.1. Inventory analysis The global inventory data for SS1, SS2 and SS3 are shown in Table 3, considering as functional unit the production of 1 kg of PLA. On the other hand, Table 4 shows the main source of data considered for life Fig. 4. Color code and Green Index categories considered in the application of G2: greenness grid methodology. A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 6 cycle inventories. 3.2. Environmental assessment The ReCiPe Midpoint methodology has been used to provide environmental profiles for each of the subsystems. In this way, it is possible to assess the relative contributions of the inputs and outputs involved in each process sub-system. A total of 12 impact categories have been selected for analysis, being the following: global warming (GW), stratospheric ozone depletion (SOD), ionization radiation (IR), ozone formation, human health (OF, HH), fine particulate matter formation (FPMF), ozone formation, terrestrial ecosystems (OF, TE), terrestrial acidification (TA), freshwater eutrophication (FE), marine eutrophication (ME), terrestrial ecotoxicity (TET), freshwater ecotoxicity (FET), marine ecotoxicity (MET), human carcinogenic (HC), human non-carcinogenic (HNCT), land use (LU), mineral resource scarcity (MRS), fossil resource scarcity (FRS) and water consumption (WC). Table 3 Life Cycle Inventory for the bio-technological production of PLA nisin functional biofilm. Inputs from technosphere Outputs to technosphere SS1. Pre-treatment of wheat straw Materials/Fuels Products Wheat Straw 10.60 kg SS1 44.16 kg Sulfuric Acid 0.01 kg Ammonium Sulfate 2.14 g Enzymes 1.31 kg Water 30.44 kg Emissions to air Sodium Salts 0.34 kg Carbon dioxide 0.79 kg Electricity/Heat Heat, steam 71.98 kg Final Waste Flows Electricity 94.94 kWh Non-hazardous waste 2.95 kg SS2. Fermentation of wheat straw Materials/Fuels Products SS1 44.16 kg SS2 116.16 kg Dipotassium Phosphate 7.89 kg Ammonium Hydroxide 8.48 kg Electricity/Heat Emissions to air Heat, steam 51.03 kg Carbon dioxide 0.95 kg Electricity 60.54 kWh Methanol 0.02 kg SS3. Downstream Process Materials/Fuels Products SS2 116.16 kg SS3 5.64 kg Ammonium Sulfate 2.52 kg Sulfuric Acid 0.03 kg Methanol 0.25 kg Hydrochloric Acid 21.96 kg Final Waste Flows Water 21.38 kg Non-hazardous waste 0.88 kg Electricity/Heat Heat, steam 310.67 kg Waste to treatment Electricity 156.17 kWh Wastewater 124.87 l SS4. PLA film production Materials/Fuels Products SS3 5.64 kg PLA 1.00 kg Sodium Hydroxide 0.23 kg Water 3.95 kg Emissions to air Catalyst 22.35 g Lactic acid 0.14 kg L-lactic acid 0.25 kg Electricity/Heat Water 4.43 kg Heat, steam 6.35 kg Emissions to water Electricity 4.65 kWh Tin Oxide 7.48 g Cooling energy 1226 kJ Sodium Stannite 21.72 g Table 4 Main EcoInvent data source considering for Life Cycle Inventories. Data required Data souce Materials/fuels Wheat Straw Straw {RoW}| wheat production | Cut-off, U Sulfuric Acid Sulfuric acid {RER}| market for sulfuric acid | Cut-off, U Ammonium Sulfate Ammonium sulfate, as N {GLO}| market for | Cut-off, U Enzymes Enzyme, Cellulase, Novozyme Celluclast/kg/RER Water Tap water {Europe without Switzerland}| market for | Cutoff, U Sodium Salts Sodium bicarbonate {GLO}| market for sodium bicarbonate | Cut-off, U Dipotassium Phosphate Potassium hydroxide {GLO}| market for | Cut-off, U Ammonium Hydroxide Ammonia, liquid {RER}| market for | Cut-off, U Methanol Methanol {GLO}| market for | Cut-off, U Hydrochloric acid Hydrochloric acid, without water, in 30% solution state {RER}| market for | Cut-off, U Sodium Hydroxide Sodium hydroxide, without water, in 50% solution state {GLO}| market for | Cut-off, U Catalyst Tin dioxide {GLO}| market for | Cut-off, U Electricity/Heat Electricity Electricity, medium voltage {Europe without Switzerland}| market group for | Cut-off, U Heat, steam Steam, in chemical industry {RER}| market for steam, in chemical industry | Cut-off, U Cooling energy Cooling energy {GLO}| market for | Cut-off, U Waste to treatment Wastewater Wastewater, average {Europe without Switzerland}| market for wastewater, average | Cut-off, U Table 5 Characterization results per impact category for each process subsystem. Impact category Unit Total SS1 SS2 SS3 SS4 GW kg CO 2 eq 257.50 66.75 74.66 111.71 1 4.38 SOD mg CFC 11 eq 110.37 35.24 31.07 41.93 1 2.13 IR kBg Co-60 eq 46.59 20.64 1 14.85 10.03 1.08 OF, HH mg NO x eq 369.44 95.24 117.97 148.61 1 7.61 FPMF mg PM 2.5 eq 292.31 80.65 95.29 109.30 1 7.07 OF, TE mg NO x eq 376.83 96.96 120.26 151.88 1 7.72 TA mg SO 2 eq 774.84 220.29 224.60 316.05 1 13.90 FE g P eq 102.46 41.54 1 33.16 25.07 2.68 ME g N eq 9.01 3.74 1 2.35 2.75 0.17 TET kg 1.4DCB 368.80 53.27 128.13 166.67 1 20.72 FET kg 1.4DCB 3.35 1.09 1 0.96 0.99 0.31 MET kg 1.4DCB 4.74 1.53 1 1.39 1.44 0.39 HCT kg 1.4DCB 5.95 2.22 1 1.94 1.62 0.17 HNCT kg 1.4DCB 151.52 51.01 1 44.52 44.12 11.87 LU m 2 a crop eq 6.06 3.25 1 1.27 1.44 0.09 MRS g Cu eq 636.33 29.91 32.51 417.98 1 155.94 FRS kg oil eq 80.20 18.92 23.19 36.79 1 1.30 WC m 3 2.64 0.90 1.05 1 0.63 0.05 1 Highest contribution values on impact categories highlighted in bold. A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 7 3.2.1. Characterization and relative contributions per subsystem Table 5 includes the characterization results and Fig. 5 shows the relative contribution of each process sub-system. In addition, the subsystem with the highest contribution in each of the impact categories is highlighted in bold in Table 5. The results show that the main contributors to the environmental profile obtained for the process are subsystems SS1 and SS3, with subsystem SS4 being the smallest contributor. Regarding the SS1 and SS2 subsystems, the environmental profiles obtained by applying the ReCipe methodology are shown in Figs. 6 and 7, respectively. As can be seen, the main hotspot of the environmental profiles obtained for both SS1 and SS2 is the electricity requirements. As for SS1, the equipment with the highest energy demand is the P-14b seed fermenter (Fig. 1), which represents 53.6% of the total electricity required for this process subsystem, followed by P-9, the reactor in which the enzymatic hydrolysis of the solid fraction takes place. On the other hand, the contribution of steam in the TET and FRS impact categories is also noteworthy due to its non-renewable nature and the background activities required for its production. With regard to the SS2 subsystem, the one referring to microbial fermentation, the main reason for the high-power demand is based on the need to maintain a homogeneous fermentation medium, through a constant stirring system, during the whole fermentation time, i.e., 24 h. On the other hand, potassium phosphate also exhibits some impact on the environmental profile obtained, as well as ammonium hydroxide in the TET category. The environmental contributions of these chemical agents are the result of their non-renewable character, which implies the consumption of non-renewable fossil and mineral resources, as well as due to their synthesis processes, which lead not only to the consumption of other chemical agents, but also to energy needs, emissions and waste streams, which entail environmental impacts in the categories studied. A detailed analysis of the SS3 subsystem has also been carried out (Fig. 8), where it has been observed that, unlike the previously analyzed subsystems, the calorific requirements are the main hotspot of the profile for most of the impact categories studied, with the exception of the MRS. In the case of this category, ammonium sulfate is the main contributor, given the significant energy demand required by its production process, obtained by the use of non-renewable resources, causing It depletion. Regarding SS4, the only category in which the SS4 subsystem presents a certain contribution is that of MRS and, in order to find out which component of the subsystem is responsible for this contribution to the overall environmental profile, a detailed analysis of the SS4 subsystem has been carried out, obtaining the profile shown in Fig. 9. As can be seen, the use of the catalyst is the main hotspot in the MRS impact category, deriving from the background activities associated with its non-renewable origin. On the other hand, its impact on the ecotoxicity categories, both FET and MET, and its impact on human health, especially in the HNC category, is also remarkable. Continuing with the analysis of this subsystem, the electrical requirements give rise to an important contribution in the profile, being the main contributor in the impact categories of GW, SOD, TA, FE, ME and FRS, together with the steam requirements. Its environmental implication is derived from its non-renewable fossil nature, in addition to the production process necessary to obtain them. On the other hand, it highlights the contribution of on-site emissions in the TET impact category, with liquid emissions containing traces of catalyst being the reason for this high environmental contribution. 3.2.2. EndPoint environmental results The EndPoint methodology has been used to compare the environmental contribution of each of the subsystems, grouping the results into three damage categories: human health, ecosystems and resourcesThe sum of these categories gives rise to obtaining the single score, a dimensionless value indicator in which the overall environmental characterization scores are weighted. As can be seen in Table 5, the SS3 subsystem is the most damaging in all the categories, being that of human health where the greatest impact is observed. In contrast, the SS4 subsystem is the one that, globally, can be considered the most environmentally friendly since its damage values are relatively low. 3.2.3. G2: greenness grid results Obtaining the value of the Green Index requires the development of a series of steps and the calculation of each of the metrics separately. In this way, Table 6 includes the main data that have been used for the development of the GS methodology: greenness grid. Once the data has been processed and each of the metrics has been calculated, they are summed, thus obtaining a value between 0 and 15. Table 7 shows the values obtained, as well as the final resulting value for the Green Index, reaching a score of 10.93, which is indicative of a potentially sustainable process. This methodology also makes it possible to identify the aspects that need to be addressed in order to obtain a higher value in the index, in other words, a process with a higher degree of sustainability. Two metrics have been identified on which special attention should be paid, Fig. 5. Relative environmental contribution of each process subsystem. A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 8 given their low degree of sustainability: energy efficiency and the degradability of the components that form part of the production process. Regarding energy efficiency, the LCA results obtained in the environmental profiles already indicated an environmental problem, given the large impact of the energy consumption required in each of the subsystems. It should be noted that it is possible to develop a large degree of improvement in this aspect with the optimization of processes and energy efficiency, through the selection of less energy-demanding equipment and by carrying out Pinch Analysis, a methodology used to identify the minimum energy consumption necessary for the development of large-scale processes. 4. Conclusions The PLA-nisin functional biofilm obtained from the biotechnological valorization of wheat straw has proven to be a sustainable alternative for the replacement of fossil-based plastic films. The large-scale production process has been divided into four subsystems, to which the LCA methodology has been applied, SS3: Downstream stage being the main environmental contributor, with energy requirements identified as the main critical points. In order to reduce the energy requirements of the process, different strategies could be carried out. The first of these, and perhaps the simplest, would be to valorize the biomass obtained after the fermentation process, given that its calorific value is sufficiently high for its valorization in a cogeneration system. In this way, the energy produced could be used in the production process itself, thus reducing the consumption of external fossil energy resources. Besides, a second appropriate strategy would be to carry out a pinch analysis, with the aim of using the process streams, which are at different temperatures, as heat transfer agents, thus reducing the consumption of steam and cooling agents. In addition, the G2 methodology has been used to analyze the sustainability score of the proposed biotechnological process, obtaining a Green Index value that is indicative of its potential to be considered as a sustainable agricultural waste recovery process. This report provides valuable information for the development of future biotechnological procedures aiming at the valorization of lignocellulosic wastes through the production of active food packaging films. The environmental profiles obtained, together with the main critical points analyzed, could help to identify which actions should be implemented to reduce the Fig. 6. Relative contribution per unit of the process subsystem SS1. Fig. 7. Relative contribution per unit of the process subsystem SS2. A. Arias et al. Sustainable Materials and Technologies 33 (2022) e00460 9 impacts associated with the process. Moreover, the use of the Green Index value could be very useful for stakeholders, as it is a unique value, easily understandable by citizens, for the dissemination of new sustainability criteria in food packaging. Declarations Availability of data and materials Not applicable. Competing interests Not applicable. Funding This research has been financially supported by the European project iFermenter (Grant Agreement 79057). iFermenter is a project funded under the “Bio-Based Industries Joint Undertaking under the European Union’s Horizon 2020 research and innovation programme”. The authors belong to the Galician Competitive Research Group (GRC ED431C 2017/29) and to the Cross-disciplinary Research in Environmental Technologies (CRETUS Research Center, ED431E 2018/01). Author’s contribution A. Arias has conducted the methodology, formal analysis, investigation and writing-original the draft manuscript. G. Feijoo provided writing review and editing. MT Moreira has conducted the conceptualization, supervision, writing-review and manuscript edit. All authors read and approved the final manuscript. Authors’ information The authors belong to the Galician Competitive Research Group (GRC ED431C 2017/29) and to the Cross-disciplinary Research in Environmental Technologies (CRETUS Research Center, ED431E 2018/ 01). Fig. 8. Relative contribution per unit of the process subsystem SS3. Fig. 9. Relative contribution per unit of the process subsystem SS4. A. Arias et al.