Antonija Trontel

Lignocellulolytic enzyme cocktails for biomass saccharification are commonly produced using solid-state fermentation (SSF) or submerged fermentation (SmF), whereas sequential solid state and submerged (SSF→SmF) co-cultivation remain poorly explored for synergistic fungal enzyme production. In this study, enzyme production strategies (SSF, SmF, and SSF→SmF) were evaluated using Fusarium oxysporum f. sp. lagenariae JCM 9293, Laetiporussulphureus DSM 1014, and Irpexlacteus DSM 9595 cultivated on dilute acid-pretreated common reed and ryegrass in shaking flasks. Sequential co-cultivation, where I. lacteus was grown during SSF followed by F. oxysporum during the SmF phase, yielded the highest specific enzyme activities in shake flasks after 9 days, reaching 5.24 U mg⁻¹ cellulase, 26.1 U mg⁻¹ CMC-ase, and 43.1 U mg⁻¹ xylanase. Enzyme production on common reed exceeded ryegrass by 1.2–2.5 fold, consistent with its higher glucan content (52 %). Partial diafiltration further enriched xylanase activity to 61.8 U mg⁻¹. Scale-up experiments in a stirred-tank bioreactor and an air-lift bioreactor confirmed a successful SSF→SmF transfer. The highest productivity was achieved in a horizontal rotating tubular bioreactor, which achieved up to 75 % of shake-flask enzyme productivity. Lower aeration and shear conditions in the stirred-tank and air-lift bioreactors reduced performance. These results demonstrate that time separated and optimized fungal co-cultivation enables the scalable production of lignocellulolytic enzyme cocktails from abundant wetland biomass, thus supporting sustainable biorefinery applications.

Lignocellulosic biomass represents an abundant and renewable carbon source, and its valorization through microalgal cultivation offers a sustainable route to resource-efficient bioprocessing. This study examined the effects of various carbon and nitrogen sources on the growth and lipid metabolism of Desmodesmus subspicatus, with a focus on ryegrass enzymatic hydrolysates as an alternative carbon source. Cultures were supplied with glucose, xylose, or arabinose at different concentrations, along with sodium nitrate or yeast extract, under different carbon-to-nitrogen ratios. Additionally, the impacts of alkaline- and acid-pretreated enzymatic ryegrass hydrolysates were evaluated. Growth was assessed by optical density and gravimetric analysis, and fatty acid profiles by gas chromatography. Glucose supplementation enhanced lipid accumulation, yielding fatty acid profiles dominated by C16 and C18 fatty acids, which are favorable for the quality of the produced biodiesel. Nitrogen limitation further promoted lipid accumulation; cultures supplied with sodium nitrate achieved higher total lipid content, while yeast extract favored greater proportions of PUFAs. Alkaline-pretreated ryegrass hydrolysate supported dose-dependent biomass formation reaching approximately 12 g L⁻¹ at 50%, whereas the acid-pretreated hydrolysate exhibited inhibitory effects at the same concentration. Scale-up in a 1 L photobioreactor yielded lower biomass but higher lipid content with a fatty acid profile shifted to SFA. These results support ryegrass as a viable alternative carbon source and highlight cultivation parameters that influence growth and lipid quality relevant for biofuel applications.

This study evaluates the enzymatic hydrolysis of pretreated corn cobs (PCCs) using a blend of commercial enzymes (Cellulase enzyme blend and Viscozyme L), followed by simultaneous saccharification and fermentation (SSF) with Mucor indicus DSM 2185 for ethanol production. A combination of 2% (vol vol⁻¹) Cellulase enzyme blend and 5.18% (vol vol⁻¹) Viscozyme L, corresponding to an enzyme loading of 48.9 FPU/g_PCCs, enabled near-complete hydrolysis of 40 g L⁻¹ PCCs within 6–48 h, achieving 92.66% total carbohydrate conversion into fermentable sugars. In SSF experiments conducted in Erlenmeyer flasks, optimal ethanol production in matrix nutrient medium (MNM) reached 14.95 g L⁻¹, with a conversion coefficient of 0.373 g g⁻¹ at 30 °C over a 48 h period. Scale-up of the bioprocess in a 1.5 L stirred-tank bioreactor at 30 °C resulted in an ethanol concentration of 16.46 g L⁻¹, a total carbohydrate conversion of 86.27%, and a substrate-to-ethanol conversion coefficient of 0.44 g g⁻¹ within 22 h. Minor secondary metabolites, including 0.88 g L⁻¹ xylitol and 0.26 g L⁻¹ glycerol, were also detected. Overall, the results demonstrate the potential of M. indicus in combination with commercial enzyme blends as a scalable strategy for industrial ethanol production.

Utilization of various biomasses as raw materials in biorefineries represents a promising alternative for the production of valuable chemicals and biofuels. This study investigates the potential of the fungus Mucor indicus DSM 2158, cultivated on media containing the liquid phase of grass hydrolysates (LGH) and various nitrogen sources (yeast extract and corn steep liquor), for the production of valuable metabolites, such as ethanol, chitin, chitosan, and fatty acids. The ethanol yield varied depending on the cultivation media and conditions. The highest substrate-into-ethanol conversion coefficients (0.14–0.2 g g⁻¹) were achieved during M. indicus cultivation on the LGH medium containing 5 g L⁻¹ CSL in Erlenmeyer flasks and a bubble column bioreactor. In these cultivations, the highest fungal biomass concentrations (5.61–5.91 g L⁻¹) were also observed. In flask cultivations, the highest content of total lipids in fungal dry biomass (15.76%) was observed. The obtained fungal biomass contained up to 22 fatty acids, with oleic acid (≈50%) being the most predominant. Chitin and chitosan yields were from 0.1 g g⁻¹ to 0.3 g g⁻¹ of dry biomass depending on the cultivation media and conditions. The residual media from the cultivation of M. indicus were used for the growth of the non-sulfur purple bacterium Rhodovulum adriaticum DSM 2781. Cultivations of R. adriaticum DSM 2781 on the residual media, in Erlenmeyer flasks and a stirred-tank bioreactor, resulted in a biomass yield of 0.50 to 2.26 g L⁻¹. After extraction of bacterial biomass, total pigments (expressed as bacteriochlorophyll-a) were obtained in the range from 1.8 to 48.1 mg g⁻¹ dry biomass depending on the media and cultivation conditions. The highest titer of bacteriochlorophyll-a was achieved during cultivation on the exhausted LGH medium with 5 g L⁻¹ yeast extract. The established biorefinery system has to be optimized in order to reach capacity for transfer to a larger scale.

The biomass of the unicellular green microalga Parachlorella kessleri is considered as one of the valuable sources of easily digestible protein, polysaccharides, pigments, and high-molecular fatty acids in the composition of synthesized lipids. The quality and quantity of these compounds vary based on intraspecific differences in strains, as well as growing conditions. Nitrogen is one of the most critical nutrients affecting cell growth and biochemical composition of microalgae, ultimately determining the lipid productivity for biofuels. In order to investigate the effect of nitrogen sources on newly isolated microalga Parachlorella kessleri MDC 6524 cell growth, lipid synthesis, and fatty acid composition, the strain was simultaneously grown on wastewater-simulating nutrient medium with and without a nitrogen source in bubble column photobioreactors. After 14 days of cultivation in the medium devoid of nitrogen sources, the total fatty acid content was 31.2% of the dry weight of the biomass, while in the medium rich in nitrogen sources, the fatty acid content was only 12.5%. Two methods for producing biodiesel were investigated: (i) lipid extraction followed by transesterification and (ii) direct transesterification from algal biomass. As a result, lipid extraction followed by transesterification increased biodiesel yield (per unit of biomass) by 1.2 times compared to the direct transesterification method. However, the direct transesterification method is significantly less time-consuming. Despite this advantage, the method has some limitations due to the interaction of lipids with the acid catalyst used in the process.

Water kefir is a product obtained through the fermentation of sucrose solution, usually with some kind of dried fruit addition, by a combined culture of micro-organisms which are contained within kefir grains. Its popularity is rising because of the simplicity of its preparation and its anti-inflammatory, antioxidant, probiotic, and antibacterial effects. In this research, the water kefir production was studied in 250 mL jars, as well as in a horizontal rotating tubular bioreactor (HRTB). The first part of the research was conducted in smaller-scale (jars), wherein the optimal fruit and fruit portions were determined. These experiments included the addition of dried plums, apricots, raisins, dates, cranberries, papaya, and figs into 150 mL of initial sugar solution. Also, the optimal ratio between dried fruit and sucrose solution (0.2) at the beginning of the bioprocess was determined. The second part of this research was conducted using HRTB. The experiments in the HRTB were carried out by using different operational modes (constant or interval bioreactor rotation). A total of six different bioreactor setups were used, and in all experiments, figs were added at the beginning of the bioprocess (0.2 ratio between dried figs and sucrose solution). On the basis of the obtained results, the interval bioreactor rotation mode proved to be the better HRTB mode for the production of the water kefir, as the yield of the main fermentation products was higher, and their ratios were the most adequate for the quality of water kefir drink. The optimal results were obtained via HRTB setup 3/57 (3 min rotation, 57 min pause within 1 h) and rotation speed of 3 rpm. Furthermore, it is clear that HRTB has great potential for water kefir production due to the fact that HRTB experiments showed shorter fermentation times (at least five times) than water kefir production in jars.

Research background. An innovative integrated bioprocess system for bioethanol production from raw sugar beet cossettes (SBC) and arabitol from remaining exhausted sugar beet cossettes (ESBC) was studied. This integrated three-stage bioprocess system is an example of the biorefinery concept to maximise the use of raw SBC for the production of high value-added products such as sugar alcohols and bioethanol. Experimental approach. The first stage of the integrated bioprocess system was simultaneous sugar extraction from SBC and its alcoholic fermentation to produce bioethanol in an integrated bioreactor system (vertical column bioreactor and stirred tank bioreactor) containing a high-density suspension of yeast Saccharomyces cerevisiae (30 g/L). The second stage was the pretreatment of ESBC with dilute sulfuric acid to release fermentable sugars. The resulting liquid hydrolysate of ESBC was used in the third stage as a nutrient medium for arabitol production by non-Saccharomyces yeasts (Spathaspora passalidarum CBS 10155 and Spathaspora arborariae CBS 11463). Results and conclusions. The obtained results show that the efficiency of bioethanol production increased with increasing temperature and prolonged residence time in the integrated bioreactor system. The maximum bioethanol production efficiency (87.22 %) was observed at a time of 60 min and a temperature of 36 °C. Further increase in residence time (above 60 min) did not result in the significant increase of bioethanol production efficiency. Weak acid hydrolysis was used for ESBC pretreatment and the highest sugar yield was reached at 200 °C and residence time of 1 min. The inhibitors of the weak acid pretreatment were produced below bioprocess inhibition threshold. The use of the obtained liqiud phase of ESBC hydrolysate for the production of arabitol in the stirred tank bioreactor under constant aeration clearly showed that S. passalidarum CBS 10155 with 8.48 g/L of arabitol (YP/S=0.603 g/g and bioprocess productivity of 0.176 g/(L·h)) is a better arabitol producer than Spathaspora arborariae CBS 10155. Novelty and scientific contribution. An innovative integrated bioprocess system for the production of bioethanol and arabitol was developed based on the biorefinery concept. This three-stage bioprocess system shows great potential for maximum use of SBC as a feedstock for bioethanol and arabitol production and it could be an example of a sustainable ‘zero waste’ production system.

Značajne količine raznovrsnih ostataka (odnosno lignocelulozne biomase) nastaju u poljoprivredi, prehrambenoj industriji i šumarstvu. Stoga je važno podići svijest o mogućnostima primjene takvih materijala koji se u današnje vrijeme ne bi trebali tre...

Alternative to the use of fossil fuels are biofuels (e.g., bioethanol, biodiesel and biogas), which are more environmentally friendly and which can be produced from different renewable resources. In this investigation, bioethanol production from raw sugar beet cossettes (semi-solid substrate) by yeast Saccharomyces cerevisiae in a horizontal rotating tubular bioreactor (HRTB) was studied. Obtained results show that HRTB rotation mode (constant or interval) and rotation speed have considerable impact on the efficiency of bioethanol production in the HRTB. The main goal of this research was to develop a non-structural mathematical model of bioethanol production from raw sugar beet cossettes in the HRTB. The established mathematical model of bioethanol production in the HRTB describes substrate utilization and product formation (glycerol, ethanol and acetate) and presumes negative impact of high substrate concentration on the working microorganism (substrate inhibition) by using Andrews inhibition kinetics. All simulations of bioethanol production in the HRTB were performed by using Berkeley Madonna software, version 8.3.14 (Berkeley Madonna, Berkeley, CA, USA). The established non-structural bioprocess model describes relatively well the bioethanol production from raw sugar beet cossettes in the HRTB.