Green Biorefinery Research Articles

Effects of Harvest and Fertilization Frequency on Protein Yield and Extractability From Flood-Tolerant Perennial Grasses Cultivated on a fen Peatland

Paludiculture, and in particular the cultivation of perennial grasses as biomass feedstock for green biorefineries, may be an economic and environmentally sustainable option for agricultural peatlands in temperate regions. However, the optimal biomass quality for protein extraction from flood-tolerant grasses is largely unknown. The aim of this study was to define the combined effect of harvest and fertilization frequency, with one to five annual cuts, on protein yield and extractability for the grasses tall fescue (TF) and reed canary grass (RCG), cultivated on an agricultural fen peatland in Denmark.The content of protein fractions was determined according to the Cornell Net Carbohydrate and Protein System (CNCPS). We assessed protein extractability by lab-scale biorefinery techniques using a screw-press followed by acid precipitation of true protein. The two methods were compared to correlate potential extractable protein yields with actual biorefinery outputs. We found the highest annual biomass and crude protein (CP) yields in the two cut treatments, with 13.4 and 15.6 t dry matter (DM) ha−1 year−1, containing 2.9–3.4 t CP ha−1 year−1for TF and RCG, respectively. The highest neutral-extractable (fractions B1and B2) true protein yields of 1.1 and 1.5 t ha−1 year−1were found in the two cut treatments, representing 39% (TF) - 45% (RCG) of total CP. Using biorefining techniques, we were able to precipitate up to 2.2 t DM ha−1 year−1of protein concentrate, containing up to 39% CP. Significant correlations between methods were found, with a distinct relationship between CNCPS fractions B1+ B2and CP yield of the protein concentrate, indicating the suitability of the CNCPS as an indicator for extractable protein yields. Biomass and CP yields were not significantly improved beyond two annual cuts. However, timing and harvest frequencies significantly affected plant maturity and consequently extractable CP contents and protein concentrate yields. We conclude that TF and RCG are promising feedstocks for green biorefineries due to high biomass, extractable CP, and protein concentrate yields, and highlight the potential of flood-tolerant grasses, cultivated on wet agricultural peatlands, for an enhanced valorisation beyond the common utilisation for bioenergy.

Enhancement of lignin removal and enzymolysis of sugarcane bagasse by ultrasound-assisted ethanol synergized deep eutectic solvent pretreatment

A low-cost and green biorefinery will increase the economy and revenue from lignocellulosic biomass. This study demonstrated the effect of two-pot sequential pretreatment, comprising of ultrasound ethanol (USEL) synergized ternary deep eutectic solvent (TDES, choline chloride: glycerol: FeCl3·6H2O), with the aim to investigate the effects of USEL, TDES, and USEL + TDES pretreatments on enhancing delignification of sugarcane bagasse (SCB). The results showed that under the optimum TDES pretreatment conditions (FeCl3·6H2O, 120 °C, 3 h), the cellulose content and lignin removal rate (LRR) of SCB reached 47.67 and 82.71%, respectively. Under the optimum synergy conditions (USEL conditions: 100% ethanol, 20 + 28 + 40 kHz, 240 W, 60 min; TDES conditions: FeCl3·6H2O, 120 °C, 3 h), the cellulose content and LRR of SCB reached 66.17 and 86.39%, respectively. After enzymatic hydrolysis, the SCB pretreated with USEL + TDES shows a saccharification rate of 90.31%, which was substantially higher than that of the SCB pretreated with TDES alone (85.68%). X-ray diffraction, particle size, brunauer-emmet-teller, scanning electron microscopy, and optical microscopy analyses were conducted to confirm the efficiency of USEL + TDES on pretreating SCB. Meanwhile, 2D-HSQC NMR analysis revealed that regenerated lignin exhibited well-preserved structures (β-O-4, β-β linkages), making it suitable for depolymerization into monoaromatic compounds. Overall, this work demonstrated that biomass pretreatment with the USEL + TDES was promising for a low-cost biorefinery to efficiently fractionate lignocellulosic biomass into glucose and high-quality lignin with tailored chemical structures.

Pilot-scale anaerobic digestion of by-product liquid (brown juice) from grass protein extraction using an un-heated anaerobic filter

The present study investigated the feasibility of using pilot-scale anaerobic filter to treat by-product liquid (brown juice, BJ) from grass protein extraction in a green biorefinery, and produce biogas via anaerobic digestion without co-digestion substrate. Prior to feed into the un-heated anaerobic filter reactor, the BJ was warmed up to 55−60 °C in order to maintain the digestion temperature. The influence of retention time and feeding frequencies were tested in order to optimize the overall performance. The study demonstrated that anaerobic filter is feasible for converting BJ to biogas. The specific CH production reached 230 mL gCOD−1 d −1 while up to 80 % of influent COD was removed within a hydraulic retention time of 5.5 days. In addition, higher feeding frequencies enhanced the process stability as it narrowed the temperature variation and decreased the instant impact on buffer capacity.

Dynamic Sustainability Assessment Tool: Case Study of Green Biorefineries in Danish Agriculture

In this work, a novel, dynamic sustainability assessment tool is presented and validated in a case study. This tool combines two methods—system dynamics (SD) and temporal soil carbon modelling. The case study for sustainability analysis of Danish agriculture and green biorefineries supply chains in Denmark is used. The development of the Danish agriculture sector is simulated and assessed in relation to the ecosystem’s carrying capacity until 2050, defined as 1.4 livestock units per hectare. The results show that under the current development, the agriculture sector would exceed this carrying capacity shortly after 2030. The results obtained from the dynamic sustainability assessment tool show a more precise and less optimistic projection of future development than the assessment using constant soil carbon modelling values only. The study, therefore, suggests that the use of the temporal aspects in the sustainability assessment should be included and further developed.

Combining the production of L-lactic acid with the production of feed protein concentrates from alfalfa

The production of L-lactic acid was investigated in combination with the production of protein concentrates in the frame of a green biorefinery for efficient utilization of grasses and legume crops. Alfalfa green juice was the sole substrate utilized for initial lactic acid fermentation with Lactobacillus salivarius, Lactobacillus paracasei or Bacillus coagulans in order to drop the pH and precipitate the plant proteins present in the juice. Afterwards, proteins were separated by microfiltration with 40-42% of protein recovery into protein concentrates, suited for feeding monogastric animals. The (residual) brown juice was investigated as source of nutrients for producing L-lactic acid from glucose or xylose with B. coagulans A107 or B. coagulans A166, respectively. Fermentation of glucose (30, 60, 100 g L-1) resulted in productivities of 2.8-4.0 g L-1 h-1 and yields of 0.85-0.91 g LA per g consumed glucose. Fermentation of xylose (30, 60 g L-1) resulted productivities of 1.1-2.3 g L-1 h-1 and yields of 0.83-0.88 g LA per g consumed xylose. Comparing different brown juices, initial green juice fermentation with B. coagulans is recommended if the brown juice is to be used for producing L-lactic acid. Based on our results, it is possible to combine protein recovery with lactic acid production, and the brown juice proved to be a good nutrient source for L-lactic acid production with high optical purities.

Green proteins: An energy‐efficient solution for increased self‐sufficiency in protein in Europe

AbstractThe heavy reliance of the livestock industry of the European Union (EU) on feed protein imports has initiated a transition to alternative protein sources such as grass proteins. Green biorefineries (which process grass into protein and other related bio‐products) are gaining interest in the EU as the EU searches for ways to cut its import of feed proteins, to reduce its reliance on fossil fuels, and to combat climate change. However, the vulnerability of green biorefineries to fossil energy constraints has not been studied. We estimated the energy conversion efficiencies (EE) and the energy return on investment (EROI) of bio‐products from standalone (SGBR) and integrated grass refinery (IGBR) systems using scenario and energy analysis. The base scenario assumes an SGBR that processes grass into protein, fiber, and brown juice. The three IGBR scenarios assume that grass is processed into protein, fiber, and biomethane (Scenario 1); into protein, fiber, heat, and electricity (Scenario 2); or into protein, fiber, heat, and biomethane (Scenario 3). We found that the EE of the IGBR (83%–85%) largely exceeded that of the SGBR (77%) in all cases. Energy returns on investment were lower for grass than for clover‐grass because of the high fertilizer needs of the former. The standard EROIs (EROIstd) for grass protein ranged from 1.6 to 5.4 over the various feedstocks and scenarios evaluated. The EROIstd decreased when the system boundary was expanded to the point of use (EROIpou), or when they were adjusted for quality (EROIqly). Other bioproducts from both SGBR and IGBR also had high EROIstd, and showed similar patterns to that of grass protein (i.e., EROIstd > EROIpou > EROIqly). Although Scenario 1 had a high EE relative to the base scenario, its heavy reliance on auxiliary energy inputs reduced the EROIs of its products. Our analysis showed the strong impacts of brown‐juice recycling in the energy performance of green biorefinery. It thus deserves close attention when designing and implementing a green biorefinery in a given region. With favorable economic conditions, green biorefineries could contribute to the reduction of food and energy insecurity in Europe in a sustainable way. © 2020 Society of Chemical Industry and John Wiley & Sons, Ltd

Efficient removal of lignin from vegetable wastes by ultrasonic and microwave-assisted treatment with ternary deep eutectic solvent

A low-cost and green biorefinery will increase the economy and revenue from cellulose food waste biomass. Ultrasound and microwave-assisted Ternary deep eutectic solvents (TDES) pretreatment were developed to deconstruct the recalcitrant structure of garlic skin (GS) and green onion root (GOR) for further lignocellulose fractionation and cellulose enzymatic hydrolysis. In this study, the TDES consists of choline chloride (ChCl) as hydrogen bond acceptor, glycerin, oxalic acid, urea and metal chloride as hydrogen bond donor. The results showed that ultrasound (room temperature, 30 min) and microwave (80 °C, 20 min)-assisted TDES pretreatment resulted in notable removal of lignin. Under the optimum conditions (ultrasound frequency: 20+28+40 kHz; TDES: ChCl-Gly-AlCl3·6H2O (1:2:0.2); heating method: ultrasound microwave), the lignin removal rate of GS and GOR reached 90.14 and 92.34 %, respectively. Simultaneously, the cellulose content of GS and GOR was increased by 48.52 and 50.28 %, respectively, which was significantly enhanced by 1.94 and 1.45 times, respectively, compared to that of the original biomass without pretreatment. Besides, the content of the reducing sugar in the enzymatic hydrolysate of GS and GOR was 2.63 and 3.11 %, respectively. All these structural characteristics (FT-IR, XRD and SEM) confirmed the efficient fractionation of GS and GOR by ultrasonic and microwave pretreatment. Overall, this work demonstrated a novel and high-performance pretreatment method for eff ;ective lignocellulose deconstruction and cellulose digestibility.

A Novel 2,5-Furandicarboxylic Acid Biosynthesis Route from Biomass-Derived 5-Hydroxymethylfurfural Based on the Consecutive Enzyme Reactions

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block as a green alternative to petroleum-based terephthalate in polymer production. Most of FDCA is produced by the oxidation of 5-hydroxymethylfurfural (HMF), which is derived from hexose. Although the chemical conversion is widely applied, the biocatalytic conversion is expected due to the relatively mild condition and fewer toxic chemicals consumption. However, it's difficult to catalyze the conversion of HMF to FDCA by a single enzyme. Here, a newly enzymatic cascade reaction process was introduced with a yield of 94.0% by the combination of 5-hydroxymethylfurfural oxidase (HMFO) and lipase. Briefly, a flavine adenosine dinucleotide independent (FAD-independent) HMFO of Methylovorus sp. MP688 was used to convert HMF to 2,5-diformylfuran (DFF) and 5-formylfuroic acid (FFA), which consecutively transformed to FDCA by a lipase Novozym 435. To facilitate the purification, a coupled alkali precipitation was developed to recover FDCA from organic solvent with an improved purity from 84.4 to 99.0% and recovery of 78.1%. This work will help to construct the green biorefinery route for the bulk FDCA from biomass by enzymes.

Kinetic Studies and Optimization of Heterogeneous Catalytic Oxidation Processes for the Green Biorefinery of Wood

In the present work, a kinetic study and optimization of the process of spruce wood peroxide oxidation in “acetic acid–water” medium in the presence of suspended TiO2 catalyst at temperatures 70–100 °C were accomplished for the first time. The effect of wood species and organic solvent nature on the features of the processes of catalytic peroxide fractionation of wood biomass on microcrystalline cellulose and soluble organic products from lignin and hemicelluloses is described. Solid products of wood peroxide oxidation were characterized by FTIR, XRD, SEM, solid state 13C CP-MAS NMR and soluble products were identified by GC–MS. The experimental optimization of the process of birch wood oxidation by oxygen in “water–alkaline” medium in the presence of suspended Cu(OH)2 catalyst was carried out at temperature range 160–180 °C. The scheme of biorefinery of birch wood, based on catalytic oxidative fractionation of wood biomass with the production of pentosans, vanillin, syringaldehyde and levulinic acid was developed. The resulting products are in demand in many areas, including food, pharmaceutical, chemical, cosmetic industries, synthesis of new functional and biodegradable polymers.

Grass silage pulp as a dietary component for high-yielding dairy cows

Green biorefineries provide novel opportunities to use the green biomass efficiently and utilize the ecosystem services provided by grasslands more widely. The effects of the inclusion of fractionated grass silage solid fraction (pulp) on feed intake, rumen fermentation, diet digestion and milk production in dairy cows were investigated. Pulp was separated from grass silage using a screw press simulating a green biorefinery. Partial removal of liquid from forage increased DM concentration from 220 to 432 g/kg and NDF from 589 to 709 g/kg DM while CP decreased from 144 to 107 g/kg DM. A feeding trial using an incomplete changeover design with 24 Nordic Red cows and two 3-week periods was conducted. The pulp replaced grass silage in the diet at 0 (P0), 25 (P25) and 50 (P50) percentage of total forage, which was fed ad libitum with 13 kg of concentrate for all treatments. The forage DM intake was highest on P25 (14.1 kg/day) while P0 and P50 did not differ from each other (13.2 and 13.0 kg/day, respectively). There were no differences between the treatments in rumen pH or ammonia N, but the proportion of acetate increased with increasing pulp inclusion. The digestibility was measured using acid insoluble ash and indigestible NDF (iNDF) as internal markers. Neither of the markers detected differences in NDF digestibility, but according to iNDF, apparent total tract organic matter digestibility decreased with increasing pulp inclusion. The cows maintained milk production at P25, but it showed some decline at P50 (energy-corrected milk at P0 and P25 was 39.8 kg/day while for P50, it was 38.5 kg/day, P = 0.056) and the milk protein yield significantly declined with higher pulp inclusion. Simultaneously, the nitrogen use efficiency in milk production increased. It seems that the fibrous grass-based fraction from a biorefinery process has potential to be used as a feed for ruminants.

Enhancing Protein Recovery in Green Biorefineries by Lignosulfonate-Assisted Precipitation

Biorefining of green biomass to produce proteins for feed and food provides an important challenge in relation to development of a future sustainable and climate-neutral agriculture. In order to make it a viable value chain, all parts of the process need to be optimized. This includes careful selection of the source of biomass, the procedure used for protein extraction as well as the separation and recovery of proteins from the press juice. We here focus on recovery of proteins from juice made from screw press fractionation of four green biomasses, viz. ryegrass, red clover, ryegrass-white clover mixture and spinach. Separating out protein from the juice is a key step and several different methods have routinely been applied. We demonstrate that lignosulfonates aid in the precipitation of proteins in the juice. The optimal loading of lignosulfonate was 0.6-0.7 g per g crude protein in the juice. For ryegrass and red clover juice, this loading resulted in 10-25 % increase in precipitated protein compared to the classical precipitation by heat treatment or acidification. For spinach, up to 60% increase in protein recovery was obtained by lignosulfonate addition. We conclude that lignosulfonate-assisted precipitation holds potential for improving the protein yield in biorefining of green biomass.

Fibrolytic enzyme treatment prior to ensiling increased press-juice and crude protein yield from grass silage

Grass is a versatile raw material for green biorefineries and preserving it as silage provides a year-round feedstock. The objective of the current study was to evaluate the effect of fibrolytic enzyme application on silage as a feedstock for a biorefinery. Two batches of grass (mixture of timothy and meadow fescue) silages were ensiled in pilot scale after fibrolytic enzyme was applied to them at four levels. Enzyme application increased fibre degradation linearly during ensiling and increased lactic and acetic acid concentrations in the silage. Simultaneously, silage fermentation quality improved as indicated by decreasing pH and ammonia values. Press-juice and crude protein yields increased in response to the fibrolytic enzyme application, which is beneficial in a biorefinery concept for retrieving valuable nutrients from grass matrix. Optimized ensiling methodology can be considered as a pretreatment for a biorefinery process.

Beyond ruminants: discussing opportunities for alternative pasture uses in New Zealand

The New Zealand government has set ambitious goals for primary sector growth and of zero net carbon emissions by 2050. This presents an opportunity and obligation to develop new ideas for grassland production systems to increase export value and generate new job opportunities, while reducing environmental impacts. The aim of this paper is to draw on recent research in Europe to investigate some of the alternative and complementary uses for pasture as a feedstock for a green biorefinery. A biorefinery is a facility, or a series of processes, that convert biomass into a spectrum of value-added products. For example, protein can be extracted mechanically from green biomass once harvested. The residual fibre fraction could be used as a low-nitrogen feed for ruminants to reduce urinary nitrogen, while the liquid protein fraction could be processed to make it suitable for mono-gastric or human consumption. Enzymes can promote protein extraction and controlled conversion of insoluble plant fibres and oligosaccharides to foster gut-health promoting prebiotic food ingredients. Anaerobic digestion of residues can then be used to create energy and soil-improving products. Research and demonstration of these approaches in practice, along with the results of feasibility studies, will be required to see which of these opportunities is a good fit for New Zealand pasture systems.

Potential Nutrient Recovery in a Green Biorefinery for Production of Feed, Fuel and Fertilizer for Organic Farming

Nutrient recovery from organic green biomass after processing in a green biorefinery concept for the production of protein feed, biogas and fertilizer for organic farming was investigated. Mass balances in terms of wet weight, TS, VS, C, N, P, K and S were evaluated for processing red clover and clover grass into protein concentrate, press cake and brown juice according to the green biorefinery concept. Depending on the biomass, between 60 and 79% of C and between 52 and 63% of N, P and S in the fresh biomass were found in the press cake, while only 6–10% of C, 8–15% of N, but up to 27% of P and up to 26% of S were found in the brown juice. In contrast, less than 45% of K was transferred to the press cake and 19–31% of K ended up in the brown juice. Moreover, nutrient recovery in the digestate after anaerobic co-digestion of press cake and brown juice produced from clover grass in a pilot-scale trial was assessed in a bench-scale biogas reactor. The analysis of the digestate from the AD process revealed that 56% of the influent total-C was converted into biogas and that the share of ammonia-N of total-N was increased from 9.4 to 43% during the biogas process. Therefore, the proportion of plant available N was improved in the digestate. The digestate to be applied as organic fertilizer presented a C:N ratio of 7 and a nutrients N:P:K ratio of 4:1:12.

Organic solvent-free extraction of carotenoids from carrot bio-waste and its physico-chemical properties

The bio-wastes (like peels, seeds, etc.) from food industry are rich source of bio-active components, but are poorly managed. In present study, carotenoids were extracted from carrot pomace using ultrasonication and high shear dispersion techniques and flaxseed oil as green solvent (green biorefinery approach). Various combinations of time and temperature were used and final selection was made on the basis of maximum recovery of carotenoids. High shear disperser yielded maximum carotenoids (recovery 94.8 ± 0.08%). The total carotenoid content, antioxidant activity as ABTS, DDPH and FRAP and β-carotene of carotenoid rich extract from carrot pomace (CREP) were 82.66 ± 0.06μg/g, 1596.04 ± 69.45μg Trolox eq./ml, 380.21 ± 39.62μg Trolox eq./ml, 941.20 ± 19.91μM Trolox eq./ml, 78.37μg/g, respectively were significantly higher (p < 0.05) when compared with the extracting medium. The L*, a* and b* values of CRE were 18.65 ± 0.037, 19.42 ± 0.21, 27.947 ± 0.65 and were significantly higher than extracting medium. The CRE could be used as a natural source of β-carotene and natural colorant for food applications.

Life‐cycle assessment of green biorefinery process options

AbstractIn this study, the biorefinery process for generating amino acids (AA) from grass silage is analyzed ecologically. Life‐cycle assessment is conducted in accordance with ISO 14040 and ISO 14044. A kilogram of amino acid is selected as a functional unit. The system boundary is drawn around silage production and includes both agricultural and conversion processes. The technology setup is modeled based on data from a demonstration amino acid production plant that uses the silages of permanent grassland in Austria (GBR 1), and data from the experimental work conducted within the European‐funded GRASSFinery project (GBR 2) located in the Czech Republic and Germany, using life‐cycle assessment software (GaBi®).Different scenarios are developed with different system boundaries. The baseline scenario for GBR 1 and the primary and central processing units of GBR 2 includes the green biorefinery processes without the disposal of waste flows to the biogas plant. By enlarging the system boundaries, the waste flows to the biogas plant and the energy yield from anaerobic digestion are considered as well as the transportation of intermediate products.The global warming potential (GWP) of the whole AA production process ranges from 11.4 to 16.8 kg CO2eq/kg AA, depending on the scenario. The GWP excludes biogenic carbon so the bound carbon in the grass silage is not included. High quality grass silage is crucial to achieve high yields of amino acids. Closed loops are a sensible step to reduce the environmental impact. © 2019 Society of Chemical Industry and John Wiley &amp; Sons, Ltd

Testing different ensiling parameters to increase butyric acid concentration for maize silage, followed by silage separation and methane yield potential of separated solids residues

In a Green Biorefinery system, the production of organic acids, fibres and biogas is in focus. Silage is separated into a press juice and press cake. During the ensiling process, organic acids like lactic acid, acetic acid and butyric acid can be formed, depending on the used substrate and ensiling conditions. Different treatments were applied to maize to increase the butyric acid content. The silage was separated and the specific methane yield of the resulting press cake was measured. The combination of an increased buffering capacity through carbonated lime and a low dry matter content leads to high butyric acid concentrations of up to 75.06 g kg−1 DM in the silage. Silage separation with this treatment resulted in an 18% loss in the methane yield per hectare.

Grass silage for biorefinery—A meta‐analysis of silage factors affecting liquid–solid separation

AbstractThis meta‐analysis based on 19 studies from Finland comprising 43 grass silages was undertaken to evaluate the effect of silage quality on liquid yield, liquid composition and retained compounds in liquid using four different liquid–solid separation methods. Silages were classified according to species (grass, clover or a mixture of them), additive treatment (no, biological or formic acid‐based additive) and harvest (primary growth or regrowth). A mixed model regression analysis with random study effect was used to evaluate the impact of silage characteristics on biorefinery efficiency. There was a large variation in silage quality in the data set. Silage dry‐matter concentration was the characteristic most highly correlated with liquid yield for all separation methods, and when used as an independent variable in the model, it resulted in the best predictions. The liquid–solid separation methods presented a great variation in the liquid yield, ranging from 0.26 to 0.56 when silage dry‐matter concentration was standardized to 250 g/kg. There was no effect of additive treatment and harvest in the estimation of the biorefinery potential, but species was a significant variable in predicting liquid yield for the laboratory‐scale presses with higher liquid yield for mixed grass and legume. The high correlation between silage quality and liquid yield and liquid composition provides potential to predict the biorefinery potential based on equations developed for each separation method. This information can be used to modify the silage production systems so that they best meet the requirements of a green biorefinery process.

Targeted grassland production – A Danish case study on multiple benefits from converting cereal to grasslands for green biorefinery

Current research on, e.g. agricultural supply-chains, engages in implementing new technologies enabling extraction of protein from other sources than soya such as grass, clover, or alfalfa. Still, it is not obvious where to initiate production of the biorefinery crop without damaging existing ecological systems or maybe even to strengthen local environments.This paper uses a targeted approach to identify the most suitable agricultural areas to convert from cereal to grassland production while gaining multiple environmental benefits. For the Danish region, three individual biomass goals were set: 1) reach 3 × 106 t dry matter from organically produced non-fertilized grass-clover mixtures, and 2) reach 5 × 106 t dry matter from grass fertilized with 300 kg N/ha, or 3) 450 kg N/ha. Areas for conversion were selected based on coastal nitrogen (N) loads and a surrounding nature index in a stepwise manor until the three biomass goals were reached separately.Our results reveal a spatial distribution of some of the areas selected for conversion close to existing nature areas and vulnerable aquatic environments for all goals. Producing 5 × 106 t dry matter fertilizing grass with 450 kg N/ha requires less agricultural area (23% of total agricultural area) compared to fertilizing with 300 kg N/ha (28%) and even less than producing only 3 × 106 t dry matter without fertilizing (24% of total agricultural land). Despite less land area, goal 3 still has a higher coastal N load compared to goal 2, and consequently goal 2 reaches 50–60% of the national N reduction goal to reduce 13,000 t N compared to 21–28% for goal 3. Furthermore, surrounding nature shows a higher index value for goal 2 compared to goal 3. Hence, in our case goal 3 would be preferable if land scarcity was the heaviest weighing factor. Still, the environmental gain resulting from using goal 1 and 2 would increase potential multifunctionality of converting from cereal to grass production.

Application of lactic acid bacteria in green biorefineries

Lactic acid bacteria (LAB) have extensive industrial applications as producers of lactic acid, as probiotics, as biocontrol agents and as biopreservatives. LAB play a large role in food fermentation and in silage processes, where crops such as grass, legumes, cereals or corn are fermented into high-moisture feed that is storable and can be used to feed cattle, sheep or other ruminants. LAB also have great applications within green biorefineries, with simultaneous production of protein-rich feed for monogastric animals, silage or feed pellets for ruminants and production of lactic acid or specific amino acids. In green biorefineries, fresh or ensiled wet biomass is mechanically fractionated into green juice and solid residues (press cake), where the plant juice, for example, can be used for production of lactic acid using LAB. In a process named 'ENLAC', recovery of protein and chlorophyll from silage by simultaneous lactic acid fermentation and enzyme hydrolysis has been developed. Furthermore, a process for protein recovery was recently developed by applying a specific LAB starter culture to green juice from freshly harvested crops. This paper focuses on reviewing LAB for their applications within biorefining of 'green' crops such as clover, alfalfa, grasses and other green plant materials.