Biomass Protein Production Research Articles

Myeloma Cells Addicted to Glutamine for Biomass Production Are Sensitive to Lenalidomide

Introduction We have shown that in myeloma (MM) the immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide, in a CRBN dependent fashion, inhibit thioredoxin reductase and thus increase intracellular oxidative stress as a consequence of peroxide accumulation (Sebastian et al. 2017). Accordingly, we have also shown that CRBN expression alone does not fully correlate with lenalidomide sensitivity, as the cells ability to decompose H2O2 is important - MM cells with lower H2O2 decomposition capacity are more sensitive to IMiDs. We hypothezised that the specific metabolic pathways used by cells for biomass production would influence IMiD sensitivity since they also influence this cellular oxidative capacity. Cellular proteins are key elements of this biomass, and required for the proliferation of MM cells and production of immunoglobulin proteins. Methods We cultured MM cells under various nutrients conditions to understand its effect on cell proliferation and drug responses. Cell viability was assessed by MTT assay, the Luminescent Assay, and direct cell counting. MM cells glycolytic rate was measured using the Seahorse XF analyzer. Western blots were performed to quantify various protein expression levels in MM cells cultured under different nutrients conditions. CRBN CRISPR/Cas9 KO plasmid was used to generate CRBN knockout cells and a lentivirus system was used to over-express CRBN in MM cells. Results MM cells resistant to lenalidomide, but still expressing functional CRBN, show a higher glycolytic pathway use than sensitive cells. We found that the lenalidomide sensitive cell line MM1.S is highly dependent on extracellular glutamine for cell proliferation. In contrast, JJN3, a lenalidomide resistant cells line with wild type CRBN, preferentially consumes glucose. We found that glutamine depletion from culture media completely abolished lenalidomide sensitivity in MM1.S cell line. Western blot analysis revealed that antibody production by MM1.S was primarily dependent on glutamine availability, and that JJN3 antibody production was dependent upon glucose availability. While glutamine depletion from culture medium completely abolished lenalidomide sensitivity in MM1.S cell line, IKZF1, and IKZF3 degradation was unchanged. We further studied the role of CRBN in glutamine dependent cellular biomass protein production using isogenic cell lines (+/- wtCRBN). We found that wtCRBN expressing cells proliferate more and show higher antibody production in the presence of glutamine, over CRBN negative isogenic cell lines. Lenalidomide treatment further increased glutamine dependent antibody production in wtCRBN expressing cells. One possibile explanation is that activation of glutamine catabolism can facilitate protein demethylation via the supplementation α-ketoglutarate (α-KG). Therefore, we tested whether lenalidomide treatment could induce protein demethylation in sensitive cell lines. Western blot probed with mono-methyl lysine antibody showed lenalidomide induced protein demethylation. We further confirmed that protein demethylation, and lenalidomide sensitivity is a consequence of elevated α-KG by treating cells with cell-permeable 5-octyl- α-ketoglutarate which act as a substrate of the lysine demethylases. 5-Octyl-α-ketoglutarate treatment inhibited cell proliferation preferentially in wtCRBN expressing cells, and also enhanced lenalidomide induced sensitivity. Protein demethylation is associated with protein ubiquitination and proteasomal degradation. We thus hypothesize that lenalidomide induced protein demethylation also likely increases proteasome inhibitors sensitivity in MM. Conclusion MM cells with preferential glutamine consumption are likely to be more sensitive to lenalidomide, and extracellular glutamine depletion can induce lenalidomide resistance. MM cells expressing CRBN, but not dependent on glutamine for protein biomass production are more likely to be resistant to lenalidomide. Our study postulates that CRBN protein requirement in lenalidomide sensitivity cells is restricted to high glutamine dependency, and that it is quite likely CRBN has a role in glutamine metabolism. Moreover, MM cells that consume more glutamine are under higher oxidative stress and exhibit less H2O2 decomposition capacity and therefore increasing sensitivity to IMiDs. References Sebastian , S., et al., Blood 2017 129:991-1007 Disclosures Stewart: Ono: Consultancy; Roche: Consultancy; Seattle Genetics: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Bristol Myers-Squibb: Consultancy; Celgene: Consultancy, Research Funding; Ionis: Consultancy; Janssen: Consultancy, Research Funding; Oncopeptides: Consultancy. Bergsagel:Janssen Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy; Celgene: Consultancy. Fonseca:AbbVie, Amgen, Bayer, Celgene, Kite, Janssen, Juno, Merck, Pharmacylics, Sanofi, Takeda: Other: Consultant/Advisor; Prognosticatin of MM based on Genetic Categorization by FISH: Patents & Royalties; Adaptive Biotechnologies: Other: Scientific Advisory Board.

Optimization of process parameters for improved production of biomass protein from Aspergillus niger using banana peel as a substrate.

This study was aimed to optimize the process variables for improved production of biomass protein using Aspergillus niger from banana fruit peel by the use of response surface methodology. A five-level-four factors central composite rotatable design was applied to elucidate the influence of process variables viz. temperature (20-40°C), pH (4-8), substrate concentration (5-25%), and fermentation period (1-5days) on biomass and protein content. The second-order polynomial models were established, which effectively explicated the variation in experimental data and significantly epitomized the appreciable correlation between independent variables and responses. After numerical optimization, the predicted optimum conditions (temperature of 31.02°C, pH of 6.19, substrate concentration of 19.92%, and the fermentation period of 4days) were obtained with biomass of 24.69g/L and protein of 61.23%, which were verified through confirmatory experiments.

Effect of carbon dioxide injection on photosynthetic wastewater treatment using microalgae Chlorella vulgaris and Euglena gracilis

Photosynthetic wastewater treatment of secondary animal wastewater was investigated using two microalgae (Chlorella vulgaris and Euglena gracilis) under both control and CO2-enriched conditions. The addition of CO2 to the microalgae culture contributed to foster nutrient uptake for C. vulgaris, but E. gracilis culture was not affected much. E. gracilis culture under CO2 injected condition showed the highest cell density and C. vulgaris showed the highest specific growth rate in the CO2 enrichment experiment. C. vulgaris cells were 15-times smaller than those of E. gracilis. The noticeably distinguishable sizes affected the separation processes. E. gracilis possessed better filterability and dewaterability. The CO2 injection greatly enhanced biomass protein production in both algae, but reduced lipid and carbohydrate fraction. By adding CO2, lipid profile of C. vulgaris has changed greatly, as opposed to E. gracilis that has remained pretty much the same way.

Optimization of L-Arabinose and Single-Cell Protein Production from Corn Husks Hydrolysate

Glucose, xylose and L-arabinose were released from corn fiber by dilute acid hydrolysis. The ability of a Candida tropicalis strain to ferment glucose and xylose in corn fiber hydrolysates to produce single cell protein has been examined. The crystalline L-arabinose can be prepared from the fermentation supernatant. The L-arabinose retention rate and biomass protein production by the yeast grown on corn fiber hydrolysate were studied under various culture conditions. The optimal volume of the broth, inoculum size, temperature, initial pH and shaker speed for retention rate of L-arabinose and growth of the strain were 30ml broth in 250ml capacity flask, 4% (v/v), 26°C, 4.0 and 180r/min, respectively. Retention rate of L-arabinose was increased by such conditions to 89.26% and cell dry mass to 13.9g/L, respectively. These values corresponded with high biomass protein and crystalline L-arabinose.

A comprehensive pilot plant system for fungal biomass protein production and wastewater reclamation

A comprehensive pilot plant system has been developed for fungal biomass protein (FBP) production and wastewater reclamation from starch processing wastewater (SPW). Consequently, 7.5–9.2 g/l of fungal biomass was produced at 0.65–0.88 g/l per hour in a semi-continuous process by non-aseptic cultivation of Aspergillus oryzae and Rhizopus oligosporus on SPW, without pretreatment and nutrient supplementation. The FBP products contained more than 45% protein and appreciable quantities of essential amino acids, and would be nutritive and edible for animal consumption. The subsequent removal of more than 95% BOD and COD, 75% nitrogen and phosphorus, and total suspended solids make the reclaimed SPW suitable for farm irrigation. The new system appeared to be technically feasible and may be beneficial for food and agricultural industries, with a commercial potential for reclaiming wastewater as a saleable value-added bioproduct.

A bioprocessing mode for simultaneous fungal biomass protein production and wastewater treatment using an external air‐lift bioreactor

AbstractA pilot plant investigation for bioprocessing has been undertaken to develop a simple, non‐aseptic, low‐cost single process for production of fungal biomass protein (FBP) and wastewater treatment using starch processing wastewater. It has been confirmed that the newly developed external air‐lift bioreactor was very suitable for bioconversion of starch materials and FBP production by the microfungi Aspergillus oryzae and Rhizopus arrhizus. Bioproduct yields of 8.5 g dm−3 of FBP that contained 46–50% protein were obtained within a comparatively short retention time. A fungal biomass productivity in a range of 0.85–0.92 g dm−3 h−1 and removals of total suspended solids and 95% COD were achieved in batch, semi‐continuous and continuous processes. The operation modes of the semi‐continuous and continuous processes demonstrated a high biological dynamics in fungal biomass productivity and COD reduction. The semi‐continuous process appeared to be the most practical mode.© 2001 Society of Chemical Industry

Optimization of culture conditions for the production of biomass protein by Aspergillus niger NRRL 567

Optimization of culture conditions for the production of biomass protein by Aspergillus niger NRRL 567

Screening and selection of microfungi for microbial biomass protein production and water reclamation from starch processing wastewater

Thirty strains of microfungi and amylolytic yeasts were screened for production of microbial biomass protein (MBP) and water reclamation from starch processing wastewater (SPW). Three species and six strains of microfungi Aspergillus oryzae, Rhizopus oligosporus and Rhizopus arrhizus showing high enzymatic activities on SWP were selected under non-aseptic growth conditions. In 20 h submerged cultivation the selected strains had a high capacity to enzymatically hydrolyse more than 93% of the starch and produce 4.3–5.6 g dm−3 of dry biomass at a specific biomass growth rate from 0.05 to 0.12 h −2. The fungal biomass contained crude protein ranging from 37.5 to 49.8% of dry biomass. The pellet and flocculated biomass products were easily harvested by simple filtration or sedimentation. After these processes, 76–88% of total organic carbon (TOC), 85%–92% of chemical oxygen demand (COD) and 95% suspended solids in SPW were removed, and the treated water was reusable for farm irrigation. Typical pretreatment processes including hydrolysis, sterilisation and nutrient supplementation were unnecessary. © 1999 Society of Chemical Industry

A Study of Optimum Condition for the Production of Biomass Protein from Maize Stover by Arachniotus sp.

A Study of Optimum Condition for the Production of Biomass Protein from Maize Stover by Arachniotus sp.

Effects of substrate particle size and alkaline pretreatment on protein enrichment by Neurospora sitophila

The effects of solid-substrate characteristics (particle size, pretreatment conditions) on microbial biomass protein production and cellulose utilization by Neurospora sitophila (ATCC 36935) were investigated. Corn stover ground to various particle size fractions (< 1 mm, 1–2 mm, 2–3 mm) was the test substrate. The pretreatment utilized sodium hydroxide (0–0.15 kg/kg substrate, at 121°C for 30 min) for delignification and hemicellulose removal. Cellulose utilization by the fungus and the crude protein production increased with decreasing substrate particle size and with increasing sodium hydroxide concentration in the pretreatment step. Under the best conditions, using < 1 mm substrate particles treated with 0.15 kg NaOH/kg substrate, approx. 90% of the initial cellulose was consumed by the fungus and the crude protein concentration in the dry product exceeded 50% by weight.

Pretreatments of agroellulosic waste for microbial biomass production with a defined mixed culture

Abstract This study describes an attempt to produce microbial protein from the Egyptian sugar can bagasse. An alkali plus gamma irradiation and an acid plus gamma irradiation pretreat‐ment method was applied on the sugar can bagasse. The fermentations were carried out in an aerated 5 L fermentor on the pretreated substrates using a mixed culture of the cellulo‐lytic fungus Trichoderma viride and the yeast Candida utilis. The growth of the tow organisms was followed by production of biomass protein and the in vitro rumen digestibility. The biomass production contained 35.5% crude protein and had an in vitro rumen digestibility of 69.8%. The biomass contained all the essential and nonessential amino acid and compared well with other protein sources. It was concluded that the C.utilis may be grown together with T.viride on the pretreated cane bagasse substrate. The presence of C. utilis dose not cause a more extensive hydrolysis of polysaccharides in the cane bagasse, but it enhances the protein production.

Heterotrophic eubacteria isolated from cultures of the cyanobacterium, spirulina maxima

AbstractThree distinct heterotrophic eubacterial strains were isolated from mixotrophic cultures of the filamentous cyanobacterium, Spirulina maxima (Gom) Geitl. Spirulina spp. are considered to be prime candidates for the phototrophic production of biomass protein, particularly in developing countries. These cyanobacteria are extreme alkaliphiles and halophiles, making their production in arid regions promising.Most previous studies on the eubacteria which live in Spirulina culture systems have focused on determining the possible presence of pathogenic species in biomass protein. Little has been done to understand the symbiotic relationships between the cyanobacterium and its eubacterial cosymbionts. From the perspective of a heterotrophic eubacterium, autotrophic cultural systems of Spirulina have limited carbon and energy resources, being limited to cyanobacterial exudates.In this study, three eubacterial strains were isolated and studied. One strain, a Gram‐negative, non‐sporing, motile rod, grew exceptionally well in a mineral salts medium where only a small amount of a single low molecular weight organic compound (e.g., acetate) was supplied as sole energy source. This strain was also extremely euryresponsive with respect to salinity and alkalinity as well. Two less well‐adapted eubacterial strains are also described.