Environmental impacts of a novel biorefinery platform integrated with power-to-protein technology to decrease dependencies on soybean imports.

The consistent population growth is directly tied to the annual rise in livestock production, placing a substantial burden on the crop sector that supplies animal feed. The Danish government has been relying on importing soybeans and soybean meal to be used as animal feed. However, this sparked environmental concerns that require more environmentally friendly solutions, such as self-sufficiency in animal feed production. The rise of green biorefineries allows new avenues of animal proteinaceous feed production using green biomass to produce leaf protein concentrate (LPC) and utilize side-stream products, such as brown juice and press cake, for feed-quality products. This study evaluated the combination of grass-clover biorefinery and the power-to-X concept, including power-to-protein technology, for its environmental sustainability through a consequential life cycle assessment (CLCA). The production of protein concentrate from organic grass clover exhibits optimal environmental performance when press cake and brown juice are used for bioenergy recovery. The findings indicate that combining a green biorefinery with power-to-protein to fully valorize the carbon and nitrogen content of brown juice and press cake into feed-grade protein can increase the environmental benefits. Such an integration resulted in an avoided impact of -995.9 kg CO2-eq/tonne of protein concentrate. The avoided impacts of climate change could be higher within the first 20 years due to a higher carbon sequestration rate. However, even after 20 years when a new carbon balance in the soil is reached, the environmental gain could be big enough to encourage the production and use of organic grass-clover protein concentrate.

The composition and nutritional quality of biorefined lucerne protein depend on precipitation method.

BACKGROUND: Lucerne protein extract is a novel high-quality protein source with excellent amino acid (AA) composition of interest for human consumption. In this study, protein from screw-pressed lucerne juice was extracted by different precipitation methods to evaluate the effect on the chemical composition and nutritional quality of the extracted protein. Methods based on heat, acidification or fermentation were used for protein precipitation, and the nutritional value of protein was evaluated in a rat digestibility trial. RESULTS: Heat precipitation at 85 °C produced a protein product with a crude protein (CP) content of 589 g kg-1 dry matter (DM), a balanced AA composition and a high standardized nitrogen (N) digestibility (82.8%). Precipitation by acidification, at a lower temperature (60 °C) or by fermentation, resulted in lower CP content (425-488 g kg-1 DM). Nitrogen digestibility for the pH-adjusted precipitate was equal to the 85 °C heat-precipitated protein, while the fermented and 60 °C precipitated proteins showed lower N digestibility (76.5% and 78.6%, respectively). By applying a two-step heat precipitation method (60 °C followed by 80 °C), a protein content of 712 g kg-1 DM and an N digestibility of 93.6% was reached, which are comparable to high-quality animal-based protein sources such as milk, whey, casein, and eggs, covering the AA requirements for children >6 months. CONCLUSION: High-quality protein can be extracted from lucerne, but the future focus should be on increased yield as the current low yields of the refined product will challenge the environmental and economic sustainability of production. © 2023 Society of Chemical Industry.

Green Biorefinery systems for the production of climate-smart sustainable products from grasses, legumes and green crop residues.

Grasses, legumes and green plant wastes represent a ubiquitous feedstock for developing a bioeconomy in regions across Europe. These feedstocks are often an important source of ruminant feed, although much remains unused or underutilised. In addition to proteins, these materials are rich in fibres, sugars, minerals and other components that could also be used as inputs for bio-based product development. Green Biorefinery processes and initiatives are being developed to better capitalise on the potential of these feedstocks to produce sustainable food, feed, materials and energy in an integrated way. Such systems may support a more sustainable primary production sector, enable the valorisation of green waste streams, and provide new business models for farmers. This review presents the current developments in Green Biorefining, focusing on a broad feedstock and product base to include different models of Green Biorefinery. It demonstrates the potential and wide applicability of Green Biorefinery systems, the range of bio-based product opportunities and highlights the way forward for their broader implementation. While the potential for new products is extensive, quality control approval will be required prior to market entry.

Nutritional values of forage-legume-based silages and protein concentrates for growing pigs.

In organic pig production systems, one of the main challenges is to meet the demand for resources rich in protein. Among the resources available, temperate green plants, such as forage legumes, are potential sources of energy and protein. The aim of the study was to determine the nutritional value of silages (S) from the whole plant of lucerne (L) and red clover (R) and protein pastes (PPs) obtained from L and R leaves. In a first trial, 30 pigs were used in a factorial design to determine the total tract digestibility (TTD) of dietary nutrients and energy in five dietary treatments. The control group was fed a control diet (C1). The lucerne silage (LS) and red clover silage (RS) groups were fed a 78%:22% mixture (on a DM basis) of the C1 diet and LS or RS. The lucerne protein paste (LPP) and the red clover protein paste (RPP) groups were fed an 81%:19% mixture (on a DM basis) of the C1 diet and LPP or RPP. In the second trial, five pigs were used in a 5 × 5 Latin square design to evaluate the standardised ileal digestibility (SID) of amino acids (AAs) in the four legume products. The control diet (C2) was formulated with casein as the sole protein source. The LS and RS groups were fed an 85%:15% mixture (on a DM basis) of the C2 diet and LS or RS. The LPP and RPP groups were fed an 80%:20% mixture (on a DM basis) of the C2 diet and LPP or RPP. Regardless of the plant species, silages obtained from L and R leaves contained less AA and more fibre than protein pastes. While the fresh forages contained the same percentage of protein N in total N (63.6%), lucerne lost more protein N during ensiling than red clover (-75.5 vs -33.8%). The calculated TTD coefficient of energy was higher in silages than in protein pastes and lower in R than in L products (72.8, 71.5, 67.7, and 61.3 for LS, RS, LPP and RPP, respectively). The SID of total essential AA was higher in LPP than in RPP (87.2 vs 79.2%) whereas it was lower in LS than in RS (33.2 vs 56.8%). The lower SID values in silages were explained by the protein degradation during the ensiling process and a high proportion of AA linked to the NDF fraction. The results of the present study show that protein pastes obtained from lucerne and red clover are valuable protein sources for pig. In contrast, legume silages have to be considered as an energy source rather than a protein source.

Biorefinery of Green Biomass─How to Extract and Evaluate High Quality Leaf Protein for Food?

There is a growing need for protein for both feed and food in order to meet future demands. It is imperative to explore and utilize novel protein sources such as protein from leafy plant material, which contains high amounts of the enzyme ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCo). Leafy crops such as grasses and legumes can in humid climate produce high protein yields in a sustainable way when compared with many traditional seed protein crops. Despite this, very little RuBisCo is utilized for foods because proteins in the leaf material has a low accessibility to monogastrics. In order to utilize the leaf protein for food purposes, the protein needs to be extracted from the fiber rich leaf matrix. This conversion of green biomass to valuable products has been labeled green biorefinery. The green biorefinery may be tailored to produce different products, but in this Review, the focus is on production of food-grade protein. The existing knowledge on the extraction, purification, and concentration of protein from green biomass is reviewed. Additionally, the quality and potential application of the leaf protein in food products and side streams from the green biorefinery will be discussed along with possible uses of side streams from the protein production.

Biorefined grass-clover protein composition and effect on organic broiler performance and meat fatty acid profile.

Protein extracted from green biomass can be a sustainable and valuable feed component for organic poultry production. Earlier studies in rats have shown high digestibility of laboratory-scale extracted protein. The aim of this study was to test the effect of upscaling the biorefining process on composition of protein extracted from organic grass-clover and on performance of organic broilers when including grass-clover in the feed. Crude protein content of the extracted grass-clover protein was 36.2% of dry matter (DM) with a higher methionine content, but lower lysine and total sulphur-containing amino acids than that in soybean. Acid-insoluble residue constituted a major fraction of the dietary fibre content, and a large proportion of total CP was bound in this fraction. Alpha-linolenic acid was the dominating fatty acid in the extracted grass-clover protein. One-day-old organic Colour Yield broiler chicks were included in a dose-response trial with grass-clover protein constituting 0%, 8%, 16% or 24% of the feed from day 12 and until slaughter at day 57. Increasing levels of grass-clover protein extract reduced feed intake, growth and slaughter weight; however, at 8% inclusion feed intake and performance were not affected. The fatty acid composition in broiler breast meat reflected the composition of grass-clover protein extract; thus, the increasing dietary addition increased meat alpha-linolenic acid content. A lowered tocopherol content in meat from broilers fed increasing grass-clover protein demonstrated the need for increased amounts of antioxidants due to the high content of unsaturated fat. In conclusion, the study shows that broilers can grow on grass-clover protein from an upscaled biorefining process, but highlights the importance of further optimisation with focus on increased protein content and on avoiding formation of insoluble protein complexes, as these most likely reduce protein digestibility.

Ensiled pulp from biorefining increased milk production in dairy cows compared with grass-clover silage.

The objective of the current study was to examine the effect of fibrous pulp and partial substitution of soybean meal with green protein concentrate from biorefining of grass-clover on dry matter intake, milk production, digestibility, and eating behavior in dairy cows compared with untreated grass-clover silage and soybean meal. Biorefining of grass-clover occurred right after harvest in a production-scale twin-screw press. The twin-screw pressing separated the grass-clover into a pulp and a green juice. The green juice was fermented using lactic acid bacteria for protein precipitation and then decanted, and the precipitate was heat dried to constitute the green protein concentrate. From the same field, grass-clover was harvested 6 d later due to rainy weather and was prewilted before ensiling. The pulp and the grass-clover were ensiled in bales without additives. The production trial consisted of an incomplete 6 × 4 Latin square trial (3-wk periods; 12 wk total) including 36 lactating Holstein cows. The trial had 6 treatments in a 2 × 3 factorial design with 2 forage types (grass-clover silage and pulp silage) and 3 protein treatments (low protein, high protein with soybean meal, and high protein with a mixture of soybean meal and green protein). The trial was designed to test silage type, protein type, protein level, and the interaction between protein level and silage type. The forage:concentrate ratio was 55:45 in low protein total mixed rations (TMR) and 51:49 in high protein TMR. Low protein and high protein TMR were composed of 372 and 342 g/kg of DM of experimental silages, respectively, and green protein supplemented TMR was composed of 28.5 g/kg of DM of green protein. Silage type did not affect dry matter intake of cows. The average energy-corrected milk yield was 37.0 and 33.4 kg/d for cows fed pulp silage and grass-clover silage, respectively, resulting in an improved feed efficiency in the cows receiving pulp silage. Milk fat concentration was greater in milk from cows fed pulp silage, and milk protein concentration was lower compared with milk from cows fed grass-clover silage. The in vivo digestibility of crude protein and neutral detergent fiber was greater for pulp silage diets compared with grass-clover silage diets. Eating rate was greater, whereas daily eating duration was lower, for pulp silage diets compared with grass-clover silage diets. The partial substitution of soybean meal with green protein did not affect dry matter intake, milk yield, or eating behavior. The in vivo digestibility of crude protein in green protein supplemented diets was lower compared with soybean meal diets. The results imply that extraction of protein from grassland plants can increase the value of the fiber part of grassland plants.

Effects of Heat Treatment of Green Protein on in Situ Protein Disappearance and in Vitro Fatty Acid Biohydrogenation.

Soluble protein extracted from leaves and stems of grasses and forage legumes is defined as green protein. The present study was conducted to evaluate in situ green protein degradability, intestinal protein disappearance, and in vitro fatty acids biohydrogenation (BH) in dairy cows. Three green protein concentrates (red clover, ryegrass, and grass clover) were heat treated as follows: oven-drying at 70 °C, subsequent autoclaving at 121 °C for 45 min, and for grass clover also spin flash-drying. Freeze-dried green protein was considered as a control (untreated). Autoclaving and oven-drying of green protein reduced the crude protein and dry matter degradability. The linolenic acid BH rate was lowest in heat-treated grass clover concentrate ( P < 0.01). In conclusion, green proteins are heat sensitive, and oven-drying can be an appropriate method to increase the amount of protein and unsaturated fatty acids escaping from the rumen.

Techno-environmental assessment of the green biorefinery concept: Combining process simulation and life cycle assessment at an early design stage.

The Green biorefinery (GBR) is a biorefinery concept that converts fresh biomass into value-added products. The present study combines a Process Flowsheet Simulation (PFS) and Life Cycle Assessment (LCA) to evaluate the technical and environmental performance of different GBR configurations and the cascading utilization of the GBR output. The GBR configurations considered in this study, test alternatives in the three main steps of green-biorefining: fractionation, precipitation, and protein separation. The different cascade utilization alternatives analyse different options for press-pulp utilization, and the LCA results show that the environmental profile of the GBR is highly affected by the utilization of the press-pulp and thus by the choice of conventional product replaced by the press-pulp. Furthermore, scenario analysis of different GBR configurations shows that higher benefits can be achieved by increasing product yields rather than lowering energy consumption. Green biorefining is shown to be an interesting biorefining concept, especially in a Danish context. Biorefining of green biomass is technically feasible and can bring environmental savings, when compared to conventional production methods. However, the savings will be determined by the processing involved in each conversion stage and on the cascade utilization of the different platform products.

Combined ensiling and hydrothermal processing as efficient pretreatment of sugarcane bagasse for 2G bioethanol production.

BACKGROUND: Ensiling cannot be utilized as a stand-alone pretreatment for sugar-based biorefinery processes but, in combination with hydrothermal processing, it can enhance pretreatment while ensuring a stable long-term storage option for abundant but moist biomass. The effectiveness of combining ensiling with hydrothermal pretreatment depends on biomass nature, pretreatment, and silage conditions. RESULTS: In the present study, the efficiency of the combined pretreatment was assessed by enzymatic hydrolysis and ethanol fermentation, and it was demonstrated that ensiling of sugarcane bagasse produces organic acids that can partly degrade biomass structure when in combination with hydrothermal treatment, with the consequent improvement of the enzymatic hydrolysis of cellulose and of the overall 2G bioethanol process efficiency. The optimal pretreatment conditions found in this study were those using ensiling and/or hydrothermal pretreatment at 190 °C for 10 min as this yielded the highest overall glucose recovery yield and ethanol yield from the raw material (0.28-0.30 g/g and 0.14 g/g, respectively). CONCLUSION: Ensiling prior to hydrothermal pretreatment offers a controlled solution for wet storage and long-term preservation for sugarcane bagasse, thus avoiding the need for drying. This preservation method combined with long-term storage practice can be an attractive option for integrated 1G/2G bioethanol plants, as it does not require large capital investments or energy inputs and leads to comparable or higher overall sugar recovery and ethanol yields.

Combination of ensiling and fungal delignification as effective wheat straw pretreatment.

BACKGROUND: Utilization of lignocellulosic feedstocks for bioenergy production in developing countries demands competitive but low-tech conversion routes. White-rot fungi (WRF) inoculation and ensiling are two methods previously investigated for low-tech pretreatment of biomasses such as wheat straw (WS). This study was undertaken to assess whether a combination of forced ensiling with Lactobacillus buchneri and WRF treatment using a low cellulase fungus, Ceriporiopsis subvermispora, could produce a relevant pretreatment effect on WS for bioethanol and biogas production. RESULTS: A combination of the ensiling and WRF treatment induced efficient pretreatment of WS by reducing lignin content and increasing enzymatic sugar release, thereby enabling an ethanol yield of 66 % of the theoretical max on the WS glucan, i.e. a yield comparable to yields obtained with high-tech, large-scale pretreatment methods. The pretreatment effect was reached with only a minor total solids loss of 5 % by weight mainly caused by the fungal metabolism. The combination of the biopretreatments did not improve the methane potential of the WS, but improved the initial biogas production rate significantly. CONCLUSION: The combination of the L. buchneri ensiling and C. subvermispora WRF treatment provided a significant improvement in the pretreatment effect on WS. This combined biopretreatment produced particularly promising results for ethanol production.

Ensiling and hydrothermal pretreatment of grass: consequences for enzymatic biomass conversion and total monosaccharide yields.

BACKGROUND: Ensiling may act as a pretreatment of fresh grass biomass and increase the enzymatic conversion of structural carbohydrates to fermentable sugars. However, ensiling does not provide sufficient severity to be a standalone pretreatment method. Here, ensiling of grass is combined with hydrothermal treatment (HTT) with the aim of improving the enzymatic biomass convertibility and decrease the required temperature of the HTT. RESULTS: Grass silage (Festulolium Hykor) was hydrothermally treated at temperatures of 170, 180, and 190°C for 10 minutes. Relative to HTT treated dry grass, ensiling increased the solubilization of dry matter (DM) during HTT and gave increased glucan content, but lower lignin in the insoluble fiber fraction. Ensiling improved glucose yields in the enzymatic hydrolysis of the washed solid fiber fraction at the lower HTT temperatures. At 170°C glucose yield improved from 17 to 24 (w/w)% (45 to 57% cellulose convertibility), and at 180°C glucose yield improved from 22 to 29 (w/w)% (54 to 69% cellulose convertibility). Direct HTT of grass at 190°C gave the same high glucose yield as for grass silage (35 (w/w)% (77% cellulose convertibility)) and improved xylan yields (27% xylan convertibility). The effect of ensiling of grass prior to HTT improved the enzymatic conversion of cellulose for HTT at 170 and 180°C, but the increased glucose release did not make up for the loss of water soluble carbohydrates (WSC) during ensiling. Overall, sugar yields (C6 + C5) were similar for HTT of grass and grass silage at both 170 and 180°C, but at 190°C the overall sugar yield was better for HTT of dry grass. CONCLUSIONS: This study unequivocally establishes that ensiling of grass as a biomass pretreatment method comes with a loss of WSC. The loss of WSC by ensiling is not necessarily compensated for by providing a lower temperature requirement for HTT for high enzymatic monosaccharide release. However, ensiling can be an advantageous storage method prior to grass processing.

Ensiling of wheat straw decreases the required temperature in hydrothermal pretreatment.

BACKGROUND: Ensiling is a well-known method for preserving green biomasses through anaerobic production of organic acids by lactic acid bacteria. In this study, wheat straw is subjected to ensiling in combination with hydrothermal treatment as a combined pretreatment method, taking advantage of the produced organic acids. RESULTS: Ensiling for 4 weeks was accomplished in a vacuum bag system after addition of an inoculum of Lactobacillus buchneri and 7% w/w xylose to wheat straw biomass at 35% final dry matter. Both glucan and xylan were preserved, and the DM loss after ensiling was less than 0.5%. When comparing hydrothermally treated wheat straw (170, 180 and 190°C) with hydrothermally treated ensiled wheat straw (same temperatures), several positive effects of ensiling were revealed. Glucan was up-concentrated in the solid fraction and the solubilisation of hemicellulose was significantly increased. Subsequent enzymatic hydrolysis of the solid fractions showed that ensiling significantly improved the effect of pretreatment, especially at the lower temperatures of 170 and 180°C. The overall glucose yields after pretreatments of ensiled wheat straw were higher than for non-ensiled wheat straw hydrothermally treated at 190°C, namely 74-81% of the theoretical maximum glucose in the raw material, which was ~1.8 times better than the corresponding yields for the non-ensiled straw pretreated at 170 or 180°C. The highest overall conversion of combined glucose and xylose was achieved for ensiled wheat straw hydrothermally treated at 180°C, with overall glucose yield of 78% and overall conversion yield of xylose of 87%. CONCLUSIONS: Ensiling of wheat straw is shown to be an effective pre-step to hydrothermal treatment, and can give rise to a welcomed decrease of process temperature in hydrothermal treatments, thereby potentially having a positive effect on large scale pretreatment costs.