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Lignocellulosic biomass comprises the crop residues, which remain in large quantities from various stages of crop processing. Its main constituents, cellulose, hemicellulose, and lignin, are interlinked by hydrogen and covalent bonds and form a robust and intricate matrix making it resistant to enzymatic degradation. Pretreatment is crucial in lignocellulosic biomass processing, aiming to remove lignin and enhance enzyme access to polysaccharides. This study examines how microwave-assisted peroxide pretreatment affects the delignification of corncobs, aiming to enhance the efficiency of utilizing this agricultural residue in fermentation processes. Taguchi orthogonal array was used to optimize pretreatment conditions and assess the effects of individual parameters (temperature, H 2 O 2 dose, and pretreatment time) on delignification efficiency. The results showed the significant influence of pretreatment conditions on lignin removal from corncobs, with the highest delignification achieved at an H 2 O 2 dose of 500 mg/g, temperature of 100 °C, and pretreatment time of 2 minutes. H 2 O 2 dose had the most substantial impact on the delignification, followed by temperature and pretreatment time. The observed 81.6% delignification and 61.9% increase in cellulose content are pivotal for enzymatic hydrolysis efficiency. This improvement suggests enhanced enzyme availability during hydrolysis and reversible binding to polysaccharide active sites, potentially leading to higher sugar yields.

Traditional extraction methods involve the use of polar solvents and/or high temperatures. Both are often energy or timeconsuming and require additional purification steps. This compromises the quality of the extracts obtained. Non-thermal plasma pretreatment for extraction is a promising technique that could increase the sustainability of the process while following the principles of hurdle technology. In this work, we investigated the effects of plasma treatment on the aqueous extraction of antioxidant compounds from herbal tea by-products. Stinging nettle, hoary willowherb, and St. John's Wort extracts were treated with plasma at different time intervals. The extracts obtained were analyzed for their antioxidant capacity and total phenolic content. Their potential use as growth media for Ligilactobacillus sp. was also investigated. Hoary willowherb extracts showed the highest total phenolic content and antioxidant activity. While plasma treatment slightly decreased the antioxidant capacity of these extracts, it improved microbial growth. In contrast to hoary willowherb, the antioxidant capacity of stinging nettle extracts' was not affected by plasma treatment. These extracts were also better for bacterial growth. When added to 25% v/v Mrs broth, stinging nettle extracts achieved a growth rate similar to that of pure Mrs broth (~10⁹ CFU/ml), demonstrating their potential for microbial biomass production and food and feed supplementation.

Lignocellulosic biomass is a cheap raw material that, thanks to its high carbohydrate content, can be used in fermentation to produce biofuels, biogas and other compounds. Its complex structure, including cellulose, hemicellulose and lignin, requires prior treatment of the biomass to facilitate hydrolysis to simple sugars. Today, biomass is only partially utilized and generates about 14% of the world's energy. This is because the most commonly used physical, chemical and physicochemical treatments are not sustainable. They are energy-consuming but still low in productivity and toxic inhibitors formed during these treatments could hinder later steps of fermentation. Biomass treatment with advanced oxidation techniques has great potential as an environmentally friendly, so-called "green" treatment. These processes generate reactive species (radicals, electrons, ions and peroxides) that attack cellulose, hemicellulose, and lignin components. In this work, the effects of non-thermal plasma, the Fenton process, and the combined treatment of corn stalks with non-thermal plasma/Fenton were compared. Grounded biomass of corn stalks was mixed with Fenton reagent and hydrogen peroxide at different ratios and subjected to non-thermal plasma treatment. Carbohydrate content was decreased in non-thermal plasma treated samples both with and without Fe2+. However, a specific biomass: Fe2+:H2O2 ratio was required to achieve the highest rate of lignocellulose decomposition. The cellulose and hemicellulose fractions were affected and reduced by the treatments studied but resulted in almost no changes in the cellulose crystallinity index. The lower lignin content and cellulose crystallinity allow for more efficient enzyme hydrolysis of the treated lignocellulose and new options for valorization in fermentations.

This study provides a detailed analysis of the lignocellulolytic activity of a new isolate Stereum gausapatum F28, a Serbian autochthonous fungi, on beechwood sawdust supplemented with cheap waste, sugar beet molasses stillage. Advanced multiple response optimization techniques were applied to improve ligninolytic and reduce hydrolytic activity as a requirement for potential biorefinery use. The applied techniques were supposed to select cultivation conditions that would give manganese peroxidase and laccase activities above 0.84 and 0.12 U g-1 substrate, respectively, and cellulase and xylanase activities below 1.12 and 1.4 U g-1 substrate. The optimal cultivation conditions that met the set requirements included molasses stillage concentration of 10 %, substrate moisture content of 53 %, incubation temperature of 23.5 °C, and pH 5.2. The research showed that the addition of molasses stillage had a positive effect on enzyme production and that the optimal stillage concentration differed depending on the enzyme type (for laccase it was

This research examined the potential use of isolated Serbian autochthonous fungi in lignocellulosic biomass pretreatment. Among 12 isolated fungi, the isolates identified as Trametes hirsuta F13 and Stereum gausapatum F28 stood out as ligninolytic enzyme producers and were selected for potential use in the pretreatment of a waste lignocellulosic biomass. An isolate identified as Myrmaecium fulvopruinatum F14 showed high hydrolytic activity, but negligible ligninolytic activity, and it was selected as a potential producer of important industrial hydrolytic enzymes. Further, the breakdown of lignocellulosic waste, beechwood sawdust, by T. hirsuta F13 and S. gausapatum F28 was examined. Both isolates efficiently degraded biomass, but T. hirsuta F13 exhibited greater selectivity (selectivity coefficient of 1.7) than S. gausapatum F28 (1.1). The isolate F13 was considered a better candidate for the pretreatment, and it was selected for further analysis which involved the use of molasses stillage as a supplement to improve the pretreatment.

Brewer's spent grain (BSG) was used in this study as a support for the immobilization of Lactobacillus paracasei NRRL B-4564, thus enabling the recirculation of immobilized biomass in repeated-batch fermentation. The chemical composition and the energy parameters of the fermented and non-fermented BSG were analyzed and compared. Moreover, the probiotic features of L. paracasei were analyzed to examine the possibility of using fermented BSG as a functional ingredient in ruminant diets. The results obtained indicate that the fermented BSG had significantly higher protein and ash contents, as well as a significantly lower content of fiber fractions. Furthermore, the fermentation process increased the BSG energy content. The analysis of probiotic potential revealed a high tolerance of L. paracasei to pH 2.5 and bovine bile, autoaggregation ability and antimicrobial activity, suggesting that the fermented BSG with immobilized microbial biomass can be used as functional feed in ruminant diets.

Stillage is a main by-product of the bioethanol industry and, depending on the origin of substrates for bioethanol production, it can be a significant pollutant affecting the profitability of bioethanol production. Directing the stillage towards the production of bio-based chemicals or high-quality feed is a preferred strategy. In this paper, a complete utilization of stillages of different origins was assessed. Thin stillages from bioethanol production from molasses, wasted bread and corn were chemically characterized, evaluated and compared as substrates for lactic acid (LA) and probiotic biomass production by Lactobacillus rhamnosus ATCC 7469, while solid fractions of wasted bread and corn stillages were analyzed for feed. The impact of pH control using CaCO3 or NaOH was also examined, both in terms of LA production and valorization of the remains generated in each process.A maximal LA productivity of 1.14 g/(L h) was obtained on thin wasted bread stillage with pH control by NaOH while the number of viable probiotic bacterial cells was above 109 CFU/mL. The composition of the solid fraction of the wasted bread stillage was complementary with the needs of monogastric animals, while the solid fraction of corn stillage was more adequate for the nutritional requirements of ruminants. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR 31017]

Pathological formation of bone in non-skeletal soft tissues or heterotopic ossification (HO), for which there is currently no effective treatment, is considered to be mediated by activation of Hedgehog (Hh) signaling pathway. Moreover, the biochemical mechanism of this pathological process is not fully understood. Here, we tested the efficacy of three chemical inhibitors of the Hh signaling pathway, arsenic trioxide (ATO), lovastatin (Lov) and cholecalciferol (Vitamin D) to hamper differentiation of mesenchymal stem cells (MSC) into osteoblasts or osteogenesis. Each of the three Hh inhibitors potently decreased alkaline phosphatase activity, suggesting effective suppression of osteogenic activity in Hh-impaired MSC. Gene expression analysis revealed a significant reduction in mRNA levels of chief Hh signaling marker, Gli1, following administration of Hh small molecule inhibitors. A functional link between Hedgehog and osteogenesis in native MSC cells is further established in studies involving the mix of three Hh inhibitors acting at different checkpoints of the Hh signaling pathway. Thus, a combination of small molecule inhibitors of the Hh pathway at their lower concentrations could be a novel approach for HO prophylaxis with increased efficacy and potentially reduced side effects.

The aim of this study was to assess utilization of distillery stillage in a two-stage fermentation using Bacillus licheniformis TFUNS and Lactobacillus paracasei NRRL B-4564. In the first stage the stillage was pretreated with B. licheniformis which is an efficient producer of proteolytic enzymes, in order to increase the content of free α-amino nitrogen in the waste substrate. In the subsequent stage the lactic acid (LA) fermentation by L. paracasei was performed. The results of the fermentation of proteolytically pretreated stillage (i.e. of two-stage fermentation) were compared with the untreated stillage (one-stage fermentation). The results have shown that the amount of free α-amino nitrogen in pretreated media was 107 % higher compared to the initial value. The concentration of the LA obtained in the second stage by L. paracasei was 48 % higher than in untreated stillage. In addition, the growth of L. paracasei in pretreated stillage was also better supported compared to that in untreated media. The process enabled economical and sufficient supply of easily assimilative nitrogen sources needed for lactic acid bacteria, thus avoiding addition of costly sources in the media commonly performed in LA production.

The objective of this study was to evaluate the possibility of application of two triticale varieties, NS Paun and Odisej, as partial substitutes for barley malt in wort production, based on the triticale analyses, micromalting, and analyses of the produced triticale malts, with a highlight on the activity of amylases during the malting process. The analyses of two triticale varieties showed higher extract content and diastatic power in comparison to barley malt. In the varieties NS Paun and Odisej, the extract was by 11.4% and 6.3% higher than the standard values for barley malt, respectively. Diastatic power in varieties NS Paun and Odisej was by 100% and 111.9% higher than in barley malt, respectively. In both varieties, α- and β-amylases activities were also high. The α-amylase activity increased during the micromalting 34 and 20 times, respectively, in comparison to native triticale grain. The β-amylase activity also increased during micromalting, but to a lesser extent. The produced triticale malts had good technological parameters and could be used as a partial substitute for barley malt in wort production. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR-31066]

Lactic acid (LA) has gained considerable importance in the global market due to a wide range of applications. Because of the growing demand for food and feed, the use of lignocellulosic residues, by-products and waste streams is highly needed for commercial LA production. This study investigated the possibility of using potato stillage and sugar beet molasses for LA and biomass production by Lactobacillus paracasei NRRL B-4564 immobilized on sugar beet pulp. Adsorption of L. paracasei enabled easy separation of bacterial biomass from the fermentation media and its efficient reuse in three successive batch cycles. Total LA concentration of 146 g L-1 and average productivity of 1.03 g L-1 h-1 were achieved on waste substrate using bacterial cells immobilized on a natural carrier without mineral and nitrogen supplementation. The solid part of fermentation media remaining after LA fermentation is a valuable co-product, which could be used as animal feed rich in probiotic biomass.

In the last decays pulsed electric field techniques gained increasing importance in biotechnology and food production. Application of electric pulses to cells leads to increased permeability and conductivity of cell membranes and formation of pores in cell membrane - phenomenon called electroporation. Electroporation could be a very suitable technique for improvement of extraction or dying in both food processing and biorefinery. Non-thermal plasma treatment generates reactive species in the samples causing damage of the cell membrane and could be used for wastewater treatment, microbial inactivation and as a treatment prior to open fermentations on complex substrates. In this study, recent advances in pulsed electric field treatment of food, algal biomass and hospital wastewater as well as role of non-thermal plasma treatment in bio-decontamination of complex substrates will be addressed. Also, some environmental and economic aspects of these novel techniques in comparison to conventional treatments will be discussed.

In this study, the stillage from the bioethanol production on wasted bread has been used as a substrate for fermentation by lactic acid bacteria. Ultrasound has been studied as a physical treatment of wasted bread stillage before fermentation by Lactobacillus rhamnosus ATCC 7469. In the ultrasound treated sample a 15 % higher lactic acid concentration compared to control sample and lactic acid yield of 0.87 g g-1 were obtained. Ultrasound could significantly increase LA productivity without effect on cell growth. Moreover, the number of viable L. rhamnosus ATCC 7469 cells was found higher in ultrasound treated samples. The study indicated that the ultrasound pretreatment could be effectively applied and used as an alternative to sterilization in lactic acid fermentation of distillery stillage.

Distillery stillage is abundant industrial waste with a great potential for utilization as a substrate in production of valuable bio-based products. Processing of distillery stillage on cost effective way can significantly decrease the price of bioethanol as alternative fuel and provide a considerable benefit to the environment. In this paper, combined potato stillage and sugar beet molasses substrate is evaluated for integrated lactic acid and probiotic biomass production by Lactobacillus rhamnosus ATCC 7469. Waste substrate based on potato stillage and sugar beet molasses enabled lactic acid and biomass production with maximal volumetric lactic acid productivity of 1.11 g L-1 h-1 and maximal number of viable L. rhamnosus ATCC 7469 cells of 1.1×109 CFU mL-1. Residue after removal of lactic acid could be recommended as probiotics and betaine-enriched animal feed.

Brewers' spent grain (BSG) hydrolysates were used for lactic acid (LA) fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate possibilities of the BSG hydrolysate utilization as a substrate for LA fermentation as well as the effect of dry brewers' yeast addition in hydrolysate on lactic acid fermentation parameters (L-(+)-LA and reducing sugar concentration and number of viable cell-viability). Very high L. rhamnosus ATCC 7469 cell viability was achieved in all fermentations (9.26-9.52 log CFU/mL at the end of fermentation). L-(+)-LA concentration increased with an increase in brewers' yeast content in hydrolysate.

Vegetable oils are mainly esters of fatty acids and glycerol, which can be converted to fatty acid methyl esters (FAME), also known as biodiesel, by the transesterification reaction with methanol. In order to attain environmental benignity, a large attention has been focused in the last decades on utilizing heterogeneous catalysts for biodiesel production instead the homogenously catalyzed transesterification of vegetable oil. The pure CaO or CaO mixed with some other metal oxide due to its low solubility in methanol, FAME and glycerol, low cost and availability is one of the most promising among the proposed heterogeneous catalysts. Solid catalysts which contain CaO usually fulfill a number of important requirements, such as high activity at mild temperature, marginal leaching of Ca cations, long life activity, reusability in transesterification of vegetable oil and easy recovery from the final products of transesterification (FAME and glycerol). This review is focused to the recent application of pure CaO or CaO in complex catalyst structure and their use as heterogeneous base catalysts for biodiesel synthesis and suitability for industrial application. [Projekat Ministarstva nauke Republike Srbije, br. 45001]

In line with the goals of sustainable development and environmental protection today great attention is directed towards new technologies for waste and industrial by-products utilization. Waste products represent potentially good raw material for production other valuable products, such as bioethanol, biogas, biodiesel, organic acids, enzymes, microbial biomass, etc. Since the first industrial production to the present, lactic acid has found wide application in food, cosmetic, pharmaceutical and chemical industries. In recent years, the demand for lactic acid has been increasing considerably owing to its potential use as a monomer for the production of poly-lactic acid (PLA) polymers which are biodegradable and biocompatible with wide applications. Waste and industrial by-products such are whey, molasses, stillage, waste starch and lignocellulosic materials are a good source of fermentable sugars and many other substances of great importance for the growth of microorganisms, such as proteins, minerals and vitamins. Utilization of waste products for production of lactic acid could help to reduce the total cost of lactic acid production and except the economic viability of the process offers a solution of their disposal. Fermentation process depends on chemical and physical nature of feedstocks and the lactic acid producer. This review describes the characteristics, abilities and limits of microorganisms involved in lactic acid production, as well as the characteristics and types of waste products for lactic acid production. The fermentation methods that have been recently reported to improve lactic acid production are summarized and compared. In order to improve processes and productivity, fed-batch fermentation, fermentation with immobilized cell systems and mixed cultures and opportunities of open (non-sterilized) fermentation have been investigated.

Bioethanol, as a clean and renewable fuel with its major environmental benefits, represents a promising biofuel today which is mostly used in combination with gasoline. It can be produced from different kinds of renewable feedstocks. Whereas the first generation of processes (saccharide-based) have been well documented and are largely applied, the second and third generation of bioethanol processes (cellulose- or algae-based) need further research and development since bioethanol yields are still too low to be economically viable. In this study, the possibilities of bioethanol production from cotton fabrics as valuable cellulosic raw material were investigated and presented. Potential lignocellulosic biomass for bioethanol production and their characteristics, especially cotton-based materials, were analyzed. Available lignocellulosic biomass, the production of textile and clothing and potential for sustainable bioethanol production in Serbia is presented. The progress possibilities are discussed in the domain of different pretreatment methods, optimization of enzymatic hydrolysis and different ethanol fermentation process modes. [Projekat Ministarstva nauke Republike Srbije, br. 31017]

The cost of distillery wastewater treatment is an important issue in overall economy of bioethanol production. Besides traditional utilization of distillery wastewater or stillage in animal nutrition, its chemical complexity is offering other possibilities. Complete distillery wastewater and liquid stillage from bioethanol production were studied as substrates for batch, fed-batch and repeated batch lactic acid fermentation by Lactobacillus rhamnosus ATCC 7469 for parallel production of lactic acid, probiotics and feed. Both of the substrates, liquid stillage and complete stillage enabled effective production of lactic acid with the process productivity of approximately 1.80 g h-1L-1, however utilization of the remains was different. The first strategy enabled conversion of liquid distillery wastewater into lactic acid and high value feed additive with zeolite immobilized probiotic biomass. The second strategy enabled effective lactic acid production on the complete stillage with additional production of high quality fermented feed for monogastric animals.

In this study brewers' spent grain (BSG) hydrolysate was produced using optimal conditions. Hydrolysates were used for lactic acid fermentation by Lactobacillus rhamnosus ATCC 7469. The aim of this study was to evaluate possibilities of the BSG hydrolysate utilization as a substrate for lactic acid fermentation as well as the effect of dry brewers' yeast (1.0, 3.0, and 5.0 %) addition in hydrolysate on lactic acid fermentation parameters (L-(+)-lactic acid and reducing sugars concentration and number of viable cellsviability). Very high L. rhamnosus ATCC 7469cells viability was achieved in all fermentations (9.26-9.52 log CFU/mLat the end of fermentation). With the increase in brewers' yeast content in hydrolysate, L-(+)-lactic acid concentration increased. The highest L- (+)-lactic acid concentration and yield (89.01 %) were obtained in fermentation of hydrolysate with 5.0 % of brewers' yeast and 5.0 % of reducing sugars.

Maize (Zea mays L.) is one of the most important cereal crops. and one of the most significant renewable raw materials for the production of energy and many different products. Bioethanol is a biofuel that is mostly used as a replacement for fossil fuels worldwide. Major by­product that arises from the fermentation process of corn is dried distillers' grains (DDG). Due to its high feeding value it represents an excellent component for livestock feed mixtures. The main goal of this study was to determine the technological quality of maize grain and the influence of maize hybrid on bioethanol yield and quality of this process' by-product, dried distillers' grains. Five maize hybrids developed at the Maize Research Institute, Zemun Polje, were investigated in this study. The lowest concentration of bioethanol in the fermentation medium after 44h of alcoholic fermentation (8.64% w/w) was obtained by hybrid ZP 560, and the highest (9.10% w/w) by hybrid ZP 600, while compared to the maximum theoretical bioethanol yield, the highest scoring hybrid was ZP 606 (93.59%) and the lowest ZP 505 (87.33%). The highest in vitro dry matter digestibility of DDG was determined in a sample obtained from hybrid ZP 505 (82.41%) and the lowest (77.12%) in the DDG sample of ZP 606. The chemical composition and physical characteristics of the grain, as well as other parameters of the production process have influenced the overall bioethanol yield. Hybrids created in the Maize Research Institute represent unique starting material for research of the possibilities of bioethanol, starch, food and animal feed production.

Pretreatment is a necessary step in the process of conversion of lignocellulosic biomass to ethanol; by changing the structure of lignocellulose, enhances enzymatic hydrolysis, but, often, it consumes large amounts of energy and/or needs an application of expensive and toxic chemicals, which makes the process economically and ecologically unfavourable. Application of lignocellulolytic fungi (from the class Ascomycetes, Basidiomycetes and Deuteromycetes) is an attractive method for pre-treatment, environmentally friendly and does not require the investment of energy. Fungi produce a wide range of enzymes and chemicals, which, combined in a variety of ways, together successfully degrade lignocellulose, as well as aromatic polymers that share features with lignin. On the basis of material utilization and features of a rotten wood, they are divided in three types of wood-decay fungi: white rot, brown rot and soft rot fungi. White rot fungi are the most efficient lignin degraders in nature and, therefore, have a very important role in carbon recycling from lignified wood. This paper describes fungal mechanisms of lignocellulose degradation. They involve oxidative and hydrolytic mechanisms. Lignin peroxidase, manganese peroxidase, laccase, cellobiose dehydrogenase and enzymes able to catalyze formation of hydroxyl radicals (•OH) such as glyoxal oxidase, pyranose-2-oxidase and aryl-alcohol oxidase are responsible for oxidative processes, while cellulases and hemicellulases are involved in hydrolytic processes. Throughout the production stages, from pre-treatment to fermentation, the possibility of their application in the technology of bioethanol production is presented. Based on previous research, the advantages and disadvantages of biological pre-treatment are pointed out.