Crude glycerol from biodiesel as a carbon source for production of a recombinant highly thermostable β-mannanase by <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To explore an efficient use of crude glycerol for the production of a highly thermostable β-mannanase (ReTMan26) by Pichia pastoris X33.

Results

Cell growth was significantly inhibited by 4 and 6% (w/v) crude glycerol in 250 ml shake-flasks and in 5 l bioreactor batch cultures, respectively, but not affected by pure glycerol at the same concentrations. For further study, the impact of various impurities in crude glycerol on the cell growth of, and ReTMan26 production by, Pichia pastoris was investigated. Salts and methanol did not exert an inhibitory effect, but ≥ 0.2% and 0.3% (w/v) soap in shake-flask and bioreactor cultures, respectively, inhibited fermentation. Under identical conditions, the biomass and ReTMan26 activity produced by high-cell-density fermentation using 5% crude glycerol (glycerol at 80%, w/w) were slightly higher than those using 4% (w/v) pure glycerol.

Conclusions

Non-pretreated ≤ 5% (w/v) crude glycerol could be effectively utilized for industrial production of ReTMan26, and the total production costs using crude glycerol were ~ 4.2% lower than those using pure glycerol.

     

EFFECTS OF YEAST,FERMENTATION TIME,AND PRESERVATION METHODS ON TARHANA

The physicochemical properties of tarhana soup produced with different dough treatments, fermentation times, and preservation methods were examined. Tarhana doughs were prepared with yogurt (control) or baker's yeast (Saccharomyces cerevisiae) and fermented for 3 days. Samples were taken at 24, 48, and 72 hr. Samples were then preserved via one of four methods: sun dried, dried in the shade, vacumn dried, and frozen. Frozen samples produced lower organic acid levels after 72 hr of fermentation in both control (0.68 g/100 g) and yeast (0.61 g/100 g) applications than samples that were dried (0.94 g/100 g control samples; 0.81 g/100 g samples with yeast). Increasing fermentation time resulted in a significant effect on the formation of organic acid in the tarhana (p < .01). At 72 hr of fermentation, total acidity increased 11%, 17%, and 23% for tarhana samples vacumn-dried, sun-dried, and dried in the shade, respectively. Preservation methods also affected the moisture, ash, crude protein, total acidity, pH, salt, fat, reducing sugar levels, and the sensory assestment of tarhana soup (p < .01). Sensory characteristics were not significantly affected by baker's yeast in any of the preservation methods used (p > .01). However, sensory scores for tarhana prepared from the samples dried in a sheltered area showed a reduction in color desireablilty as the fermentation time increased. The soup prepared from frozen tarhana (72 hr fermentation, with yeast) had the highest scores with respect to color, mouth feel, flavor, and overall acceptability. Vacuum-dried samples' scores in these areas were also high in comparison to the two other drying methods.     

An integrative process of bioconversion of oil palm empty fruit bunch fiber to ethanol with on-site cellulase production

The aim of this study was to efficiently convert oil palm empty fruit bunch fiber (OPEFB), one of the most commonly generated lingo-wastes in Southeast Asia, into both cellulase and bioethanol. The unprocessed cellulase crude (37.29 %) produced under solid-state fermentation using OPEFB as substrate showed a better reducing sugar yield using filter paper than the commercial enzyme blend (34.61 %). Organosolv pretreatment method could efficiently reduce hemicellulose (24.3–18.6 %) and lignin (35.2–22.1 %) content and increase cellulose content (40.5–59.3 %) from OPEFB. Enzymatic hydrolysis of pretreated OPEFB using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g OPEFB at 50 °C, and pH 5.5 resulted in a OPEFB hydrolysate containing 36.01 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 17.64 g/L ethanol with 0.49 g/g yield from glucose and 0.088 g/g yield from OPEFB at 8 h using Saccharomyces cerevisiae.     

Co-utilization of Crude Glycerol and Biowastes for Producing Polyhydroxyalkanoates

Polyhydroxyalkanoate (PHA) production by Bacillus thuringiensis EGU45 and defined mixed culture of Bacillus spp. were studied by using crude glycerol (CG) and hydrolyzed biowastes as feed material. Hydrolysates from onion peels (OP), potato peels, pea-shells (PS), apple pomace 2% total solids obtained with defined mixed hydrolytic cultures (MHC2) were inoculated with B. thuringiensis EGU45 and defined mixed bacterial cultures (5MC1), which produced PHA at the rate of 40–350 and 65–450 mg/L, respectively. Addition of CG (1%, v/v) to these hydrolysates resulted in 1.8-fold and 4.5-fold enhancement in PHA production from OP by B. thuringiensis EGU45 and 5MC1, respectively. Co-utilization of OP and PS (in 2:1 ratio) supplemented with CG (1%, v/v) by B. thuringiensis EGU45 resulted in 2-fold increase in PHA production in comparison to OP + CG. This co-metabolism of OP and PS also enabled PHA co-polymer production (1300 mg/L), having an enhanced HV content of 21.2% (w/w).     

Influence of Inoculation with the Endomycorrhizal Fungi and Trichoderma viride on Morphological and Physiological Growth Parameters of Rauwolfia serpentina Benth. Ex. Kurtz

Two arbuscular mycorrhizal fungi, Glomus mosseae and Acaulospora laevis either alone or in combination with Trichoderma viride showed the dependence of Rauwolfia serpentina on endomycorrhizal fungi. After 60 days, G. mosseae singly or in combination with Trichoderma viride showed enhanced height increment compared to control plants. Maximum phosphorus content was shown by plants treated with G. mosseae plus T. viride (0.444 ± 2.62) in roots and (0.437 ± 4.71) in shoots. Phosphorus content in roots was more than that in shoots. Chlorophyll content and stomatal conductivity also showed similar trend.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

Solid-state fermentation of industrial solid wastes from the fruits of milk thistle Silybum marianum for feed quality improvement

The industrial solid wastes generated during the production of silymarin from the fruits of milk thistle Silybum marianum was used as the substrate. Preparation and evaluation of the feeds produced by solid-state fermentation (SSF) of the industrial solid wastes was carried out. The protein content of the fermented feed (FF) from a combination of Aspergillus niger and Candida tropicalis was the highest among the examined strains. The optimal process parameters for protein enrichment with SSF using A. niger and C. tropicalis included incubation temperature of 30.8 °C, fermentation time of 87.0 h, and initial moisture content of 59.7 %. Under these conditions, the value additions of FF occurred. The fiber of FF was decreased by 25.07 %, while the digestibility of protein, protein content, and the ratio of total essential amino acids to total amino acids were increased by 79.85, 16.22, and 8.21 %, respectively. The analysis indicated that FF contained 1.44 mg/kg flavonoids and 0.5 mg/kg silybin, which significantly increased by 2.42 and 1.63 times, respectively than those in unfermented substrates. FF recorded reduced molecular weight of proteins from 20.1 to 44.3 kDa to below 14.3 kDa. The results of feeding trial of FF replacement with soybean meal in broilers diets for 8 weeks showed that FF significantly improved carcass characteristics including abdominal fat rate, serum biochemical parameters including aspartate transaminase, blood urea nitrogen and high density lipoprotein cholesterol, and immune responses of broilers. A potential feed quality improvement was achieved through mixed strains SSF of industrial solid wastes of S. marianum fruits.     

Effects of diets differing in protein source and technical treatment on digestibility,performance and visceral and biochemical parameters of fattening pigs

The aim of the experiment on 100 cross-bred barrows was to compare commercial diets for fattening pigs based on either soya bean meal (SBM) imported from non-European countries with diets based on a mixture of locally produced rape seed meal, distillers’ dried grains with solubles and soya beans as main protein sources. In addition, these both types of diets were processed by two different technical feed treatments, i.e. coarse grinding without hydrothermal treatment or fine grinding and pelleting. With only few exceptions, nutrients of the diet without SBM were more digestible (p < 0.05) resulting in a higher metabolisable energy (ME) content. Fine grinding and pelleting increased also the ME content and the nutrient digestibility with the exception of crude fibre. Higher feed intake of animals that fed diets without SBM (p < 0.01) resulted in higher average daily gain (p < 0.01). However feeding this diet, the higher digestibility was not reflected in a decreased feed-to-gain ratio (FGR), but fine grinding and pelleting reduced FGR (p < 0.001). A higher pH value and a lower DM content of caecal chymus were detected in animals that received coarsely ground feed (p < 0.05). Animals that fed finely ground and pelleted feed had higher slaughter and relative liver weights and higher blood cholesterol concentrations (p = 0.040). The urea concentrations of blood were lower (p = 0.019) after feeding diets without SBM. In conclusion, SBM imported from non-European countries can be replaced by alternative local protein sources without compromising digestibility or performances of animals. Although fine grinding and thermal treatment particularly seemed to be advantageous for digestibility and performance, the possible risk of development of stomach lesions should be considered.     

Bioethanol Production from Horticultural Waste Using Crude Fungal Enzyme Mixtures Produced by Solid State Fermentation

It was desired to study efficient and simplified methods to convert organosolv-pretreated horticultural waste (HW) to ethanol fuel using cellulase produced under solid-state fermentation (SSF). The unprocessed cellulase crude (72.2 %) showed better reducing sugar yield using filter paper than the commercial enzyme blend (68.7 %). Enzymatic hydrolysis of organosolv-pretreated HW using the crude cellulase with 20 % solid content, enzyme loading of 15 FPU/g HW at 50 °C, and pH 5.5 resulted in a HW hydrolysate containing 25.06 g/L glucose after 72 h. Fermentation of the hydrolysate medium produced 12.39 g/L ethanol with 0.49 g/g yield from glucose and 0.062 g/g yield from HW at 8 h using Saccharomyces cerevisiae. This study proved that crude cellulase complex produced under SSF and organosolv pretreatment can efficiently convert woody biomass to ethanol without any commercial cellulase usage.     

Effects of feeding high-quality hay with graded amounts of concentrate on feed intake,performance and blood metabolites of cows in early lactation

Dairy cows are commonly fed energy-dense diets with high proportions of concentrate feedstuffs to meet the increased energy needs of early lactation. However, feeding large amounts of concentrates may cause rumen acidosis and impact cow health. The hypothesis tested was that the energy supply and metabolic health of early-lactation Simmental cows can be maintained when high-quality hay rich in water-soluble carbohydrates (WSC) and crude protein (CP) is fed, despite the proportion of concentrates in the diet being reduced or even excluded. Twenty-four Simmental cows were allocated to one of four feeding groups beginning 10 d before the expected calving date, until 28 d thereafter. The feeding groups were 60CH (60% conventional fibre-rich hay plus 40% concentrate feed), 60HQH (60% high-quality hay plus 40% concentrate feed), 75HQH (75% high-quality hay plus 25% concentrate feed) and 100HQH (100% high-quality hay). The fibre-rich hay and high-quality hay differed in WSC content (110 g vs. 198 g of dry matter (DM)), neutral detergent fibre (646 g vs. 423 g of DM) and CP (65 g vs. 223 g of DM). Individual feed intake and milk production were monitored daily, and blood samples were collected weekly. Dry matter intake (DMI) and milk yield increased post partum, but 4 weeks post partum, the DMI of cows fed 100HQH only reached a daily mean DMI of 18.6 kg, whereas the DMI of the other groups averaged 21.9 kg (p < 0.046). The negative energy balance was less pronounced in cows fed 75HQH since they showed similar milk yields to the cows fed 60CH and 100HQH, but their energy intake was higher. Concentrations of milk components were similar across rations 60CH, 60HQH and 75HQH, as were most of blood parameters. Cows fed 100HQH responded to the energy deficit post partum with a higher ratio of non-esterified fatty acids to cholesterol and a higher concentration of ß-hydroxybutyrate (significant in comparison to cows fed 75HQH, p < 0.05). In conclusion, feeding high-quality hay with a WSC content of 20% in DM has the potential to decrease the proportion of concentrates in dairy cow feeding in early lactation, but cannot fully replace their supplementation due to a limited rumen capacity for forage intake.