Interconnection of Waste Chicken Feather Biodegradation and Keratinase and mcl-PHA Production Employing Pseudomonas putida KT2440

Background and objective: Waste chicken feather is an important waste product of the poultry processing industry and is annually produced in substantial amounts. Hence, wise management of this waste is desirable. In this work we aimed at feathers biodegradation by a selected bacterial strain capable of utilizing chicken feathers as sole carbon source Pseudomonas putida KT2440. To utilize feather, the bacterial culture excrete keratinase, which can be easily isolated after biodegradation process and which, therefore, represents an interesting side product of the intended technology. Moreover, bacterial culture of employed for feather degradation is also capable of mcl-PHA accumulation.Materials and methods: Bacterial culture of Pseudomonas putida KT2440 was cultivated in presence of waste chicken feathers as the only carbon source; during the cultivation keratinase activity and biomass growth were monitored. Metabolically active biomass after feather degradation was used for mcl-PHA production.Results and conclusion: During cultivation on waste feathers, bacteria did not accumulate detectable amounts of medium-chain length polyhydroxyalkanoate (mcl-PHA); nevertheless, when metabolically active bacterial cells after feather biodegradation were transferred into nitrogen limited mineral media, a high medium-chain length polyhydroxyalkanoate content of 61% of cell dry weight in microbial cells was reached. The polymer consisted of 3- hydroxyhexanoate (27.2% mol) and 3-hydroxyoctanoate (72.8% mol) monomer units. Therefore, this work demonstrates a possible interconnection of feather biodegradation with keratinase and medium-chain length polyhydroxyalkanoate production.Conflict of interest: The authors declare no conflict of interest

Proizvodnja biomase obogaćene karotenoidima ili ergostorolom s pomoću crvenoga kvasca Rhodotorula glutinis izloženog stresnim uvjetima

The aim of this study is to compare the production of biomass enriched with carotenoids and ergosterol by yeast strain Rhodotorula glutinis CCY 20-2-26 grown under optimal growth conditions and in the presence of exogenous stress factors. R. glutinis cells were exposed to UV irradiation, oxidative stress (2–10 mmol/L H2O2) and osmotic stress (2–10 % NaCl). During the experiment, growth characteristics and the production of biomass, carotenoids and ergosterol were evaluated. Experiments were carried out in Erlenmeyer flasks and in laboratory fermentor. First, R. glutinis cells were exposed to higher concentration of stress factors added into the production medium. Further, low concentrations of NaCl and H2O2 were added to the inoculum medium or to both inoculum and production media. Exposure of red yeast cells to all tested stress factors resulted in higher production of carotenoids as well as ergosterol, while biomass production was changed only slightly. Under high stress, 2–3 times increase of β-carotene was observed. The addition of low salt or peroxide concentration into the inoculation media led to about 2-fold increase of carotenoid production. In Erlenmeyer flasks the best effect on the carotenoid and ergosterol production (3- to 4-fold increase) was exhibited by the combined stress: the addition of low amount of NaCl (2 mmol/L) into the inoculum medium, followed by the addition of H2O2 (5 mmol/L) into the production medium. The production of ergosterol in most cases increased simultaneously with the production of carotenoids. Cultivation of R. glutinis carried out in a 2-litre laboratory fermentor was as follows: under optimal conditions about 37 g/L of yeast biomass were obtained containing approx. 26.30 mg/L of total carotenoids and 7.8 mg/L of ergosterol. After preincubation with a mild stress factor, the yield of biomass as well as the production of carotenoids and ergosterol substantially increased. The best production of enriched biomass was obtained in the presence of peroxide in the inoculation medium (52.7 g/L of biomass enriched with 34 mg/L of carotenoids) and also under combined salt/peroxide or salt/salt stress (about 30–50 g/L of biomass enriched with 15–54 mg/L of total carotenoids and about 13–70 mg/L of ergosterol). R. glutinis CCY 20-2-26 could be used as a potential biotechnological producer of carotenoid-rich biomass.Svrha je ovoga rada usporediti proizvodnju biomase, obogaćene karotenoidima ili ergosterolom s pomoću crvenoga kvasca Rhodotorula glutinis CCY 20-2-26 u optimalnim uvjetima rasta, s proizvodnjom u uvjetima vanjskoga stresa. Stanice R. glutinis bile su izložene UV-zračenju, oksidativnom (2-10 mmol/L H2O2) i osmotskom stresu (2-10 % NaCl). Tijekom pokusa, provedenog u Erlenmeyerovim tikvicama i laboratorijskom fermentoru, procijenjene su karakteristike rasta te proizvodnja biomase, karotenoida i ergosterola. Stanice R. glutinis najprije su izložene većim koncentracijama soli i vodikova peroksida dodanih podlozi za uzgoj, a zatim malim koncentracijama tih spojeva dodanih inokulumu ili inokulumu i podlozi za uzgoj. Izlaganjem stanica kvasca stresnim uvjetima dobivene su veće količine karotenoida ili ergosterola, dok se količina proizvedene biomase nije bitno povećala. Dodatkom veće koncentracije soli ili vodikova peroksida količina se β-karotena povećala 2-3 puta, a dodatkom malih koncentracija tih spojeva inokulumu količina je karotenoida dvostruko porasla. U pokusu provedenom u Erlenmeyerovim tikvicama dobiveno je 3-4 puta više karotenoida i ergosterola (u usporedbi s kontrolnim uzorkom) pri ovim uvjetima: 2 % NaCl je dodano inokulumu, a zatim je 5 mmol/L H2O2 dodano podlozi za uzgoj. Uzgojem R. glutinis u laboratorijskom fermentoru volumena 2 L pri optimalnim je uvjetima dobiveno 37 g/L biomase, obogaćene s 26,3 mg/L karotenoida i 7,8 mg/L ergosterola. Nakon prethodne inkubacije stanica kvasca pod blagim se stresom bitno povećala proizvodnja karotenoida i ergosterola. Najbolji su rezultati postignuti u ovim uvjetima: dodatkom vodikova peroksida dobiveno je 52,7 g/L biomase obogaćene s 34 mg/L karotenoida, a dodatkom soli i vodikova peroksida ili dviju različitih koncentracija soli dobiveno je 30-50 g/L biomase obogaćene s 15-54 mg/L karotenoida i 13-70 mg/L ergosterola. Na kraju je pokusa zaključeno da se R. glutinis CCY 20-2-26 može uspješno upotrijebiti za proizvodnju biomase obogaćene karotenoidima

Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganisms

Background Monitoring and control of both growth media and microbial biomass is extremely important for the development of economical bioprocesses. Unfortunately, process monitoring is still dependent on a limited number of standard parameters (pH, temperature, gasses etc.), while the critical process parameters, such as biomass, product and substrate concentrations, are rarely assessable in-line. Bioprocess optimization and monitoring will greatly benefit from advanced spectroscopy-based sensors that enable real-time monitoring and control. Here, Fourier transform (FT) Raman spectroscopy measurement via flow cell in a recirculatory loop, in combination with predictive data modeling, was assessed as a fast, low-cost, and highly sensitive process analytical technology (PAT) system for online monitoring of critical process parameters. To show the general applicability of the method, submerged fermentation was monitored using two different oleaginous and carotenogenic microorganisms grown on two different carbon substrates: glucose fermentation by yeast Rhodotorula toruloides and glycerol fermentation by marine thraustochytrid Schizochytrium sp. Additionally, the online FT-Raman spectroscopy approach was compared with two at-line spectroscopic methods, namely FT-Raman and FT-infrared spectroscopies in high throughput screening (HTS) setups. Results The system can provide real-time concentration data on carbon substrate (glucose and glycerol) utilization, and production of biomass, carotenoid pigments, and lipids (triglycerides and free fatty acids). Robust multivariate regression models were developed and showed high level of correlation between the online FT-Raman spectral data and reference measurements, with coefficients of determination (R2) in the 0.94–0.99 and 0.89–0.99 range for all concentration parameters of Rhodotorula and Schizochytrium fermentation, respectively. The online FT-Raman spectroscopy approach was superior to the at-line methods since the obtained information was more comprehensive, timely and provided more precise concentration profiles. Conclusions The FT-Raman spectroscopy system with a flow measurement cell in a recirculatory loop, in combination with prediction models, can simultaneously provide real-time concentration data on carbon substrate utilization, and production of biomass, carotenoid pigments, and lipids. This data enables monitoring of dynamic behaviour of oleaginous and carotenogenic microorganisms, and thus can provide critical process parameters for process optimization and control. Overall, this study demonstrated the feasibility of using FT-Raman spectroscopy for online monitoring of fermentation processes.Raman spectroscopy online monitoring of biomass production, intracellular metabolites and carbon substrates during submerged fermentation of oleaginous and carotenogenic microorganismspublishedVersio

The Role of Volunteers in a Swimming Organization for Persons with Disabilities

Participation in leisure time physical activity (LTPA) has considerable health-related, psychological, and social benefits. However, the involvement of individuals with disabilities is considerably less than that of their peers without disabilities. A higher rate of participation of individuals with disabilities in LTPA may be achieved by the active involvement of volunteers. This study aimed to describe the importance of volunteer involvement in a swimming organization focused on individuals with disabilities, as perceived by all participants, including swimmers with disabilities, their parents, volunteers, and coaches. The organization uses volunteers as swimming instructors who work individually with swimmers with disabilities. The data were obtained through 11 semi-structured interviews with swimmers with disabilities and their parents, volunteers, and coaches. The interviews were transcribed verbatim and analyzed using a five-step inductive thematic analysis. As a result of the cooperation with the volunteer swimming instructors, swimmers with disabilities felt an improved range of movement, greater independence, and higher self-esteem than before they started using the services of the swimming organization. Consequently, even individuals with severe disabilities can participate in LTPA. Membership to the organization also provided space for the establishment of new social relations, and the instructors described them accepting persons with disabilities as their equals. More importantly, the involvement of volunteers enables organizations to provide respite care for parents

Effect of Encapsulation on Antimicrobial Activity of Herbal Extracts with Lysozyme

Resistance of microorganisms to antibiotics has increased. The use of natural components with antimicrobial properties can be of great significance to reduce this problem. The presented work is focused on the study of the effect of encapsulation of selected plant and animal antimicrobial substances (herbs, spices, lysozyme and nisin) on their activity and stability. Antimicrobial components were packaged into liposomes and polysaccharide particles (alginate, chitosan and starch). Antimicrobial activity was tested against two Gram-positive (Bacillus subtilis and Micrococcus luteus) and two Gram-negative (Escherichia coli and Serratia marcescens) bacteria. Encapsulation was successful in all types of polysaccharide particles and liposomes. The prepared particles exhibited very good long-term stability, especially in aqueous conditions. Antimicrobial activity was retained in all types of particles. Liposomes with encapsulated herb and spice extracts exhibited very good inhibitory effect against all tested bacterial strains. Most of herbal extracts had very good antimicrobial effect against the tested Gram-negative bacterial strains, while Gram-positive bacteria were more sensitive to lysozyme particles. Thus, particles with co-encapsulated herbs and lysozyme are more active against different types of bacteria, and more stable and more effective during long-term storage. Particles with encapsulated mixture of selected plant extracts and lysozyme could be used as complex antimicrobial preparation with controlled release in the production of food and food supplements, pharmaceutical and cosmetic industries

Production of Carotenoid-/Ergosterol-Supplemented Biomass by Red Yeast Rhodotorula glutinis Grown Under External Stress

The aim of this study is to compare the production of biomass enriched with carotenoids and ergosterol by yeast strain Rhodotorula glutinis CCY 20-2-26 grown under optimal growth conditions and in the presence of exogenous stress factors. R. glutinis cells were exposed to UV irradiation, oxidative stress (2–10 mmol/L H2O2) and osmotic stress (2–10 % NaCl). During the experiment, growth characteristics and the production of biomass, carotenoids and ergosterol were evaluated. Experiments were carried out in Erlenmeyer flasks and in laboratory fermentor. First, R. glutinis cells were exposed to higher concentration of stress factors added into the production medium. Further, low concentrations of NaCl and H2O2 were added to the inoculum medium or to both inoculum and production media. Exposure of red yeast cells to all tested stress factors resulted in higher production of carotenoids as well as ergosterol, while biomass production was changed only slightly. Under high stress, 2–3 times increase of β-carotene was observed. The addition of low salt or peroxide concentration into the inoculation media led to about 2-fold increase of carotenoid production. In Erlenmeyer flasks the best effect on the carotenoid and ergosterol production (3- to 4-fold increase) was exhibited by the combined stress: the addition of low amount of NaCl (2 mmol/L) into the inoculum medium, followed by the addition of H2O2 (5 mmol/L) into the production medium. The production of ergosterol in most cases increased simultaneously with the production of carotenoids. Cultivation of R. glutinis carried out in a 2-litre laboratory fermentor was as follows: under optimal conditions about 37 g/L of yeast biomass were obtained containing approx. 26.30 mg/L of total carotenoids and 7.8 mg/L of ergosterol. After preincubation with a mild stress factor, the yield of biomass as well as the production of carotenoids and ergosterol substantially increased. The best production of enriched biomass was obtained in the presence of peroxide in the inoculation medium (52.7 g/L of biomass enriched with 34 mg/L of carotenoids) and also under combined salt/peroxide or salt/salt stress (about 30–50 g/L of biomass enriched with 15–54 mg/L of total carotenoids and about 13–70 mg/L of ergosterol). R. glutinis CCY 20-2-26 could be used as a potential biotechnological producer of carotenoid-rich biomass

Evaluation of Recombinant Kpkt Cytotoxicity on HaCaT Cells: Further Steps towards the Biotechnological Exploitation Yeast Killer Toxins

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows β-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25–2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries