The Bioprocessing Quick Wins from Avocado Fruit in Uganda

Bioprocessing can help redeem the economic value for avocado (Persea americana) in Uganda. This study reviews the virgin potential of avocado bioprocessing in Uganda. Avocado consists of flesh, seed, and peel. The review indicates that the waste seed and peel are vital for development of high-value products. Both the edible part and waste (peel and seed) can be used for biogas production through a solid-state fermentation process. Biodiesel can be developed using avocado seed oil through a process called transesterification. Avocado oil is a better alternative for biodiesel production compared to waste cooking oil as it requires no further conversions processes for transforming fatty acids to esters. The starch-rich avocado seed is a suitable substrate for bioethanol, pigment and starch production. The high starch content places the avocado fruit as a potential: 1) raw material for the production of bioplastics;2) substrate for bacterial culture media production as opposed to potatoes, cereals, and cassava that double as a staple food. Avocado seeds can also be used for the production of antioxidants relevant for preventing enzymatic browning, thereby increasing product shelf life. Despite the quick wins, there is a need for increased research, financing, personnel training and development of appropriate policies to spur the benefits and untapped potential of avocado bioprocessing in Uganda. The circular economy of avocado waste alone into high-value products could increase gains in the environment and stimulate industrial development, especially the cosmetic, food and pharmaceutical industries in Uganda.

Bioprocessed Black Rice Bran Potentiates the Growth Inhibitory Activity of an Immune Checkpoint Inhibitor against Murine Colon Carcinoma

This study determined the effect of orally fed polysaccharide-rich bioprocessed (fermented) black rice bran produced by culturing with shiitake (Lentinus edodes) mushroom mycelium on CT-26 colon cancer cells in vivo in an intracutaneously transplanted mouse tumor alone and in combination with intraperitoneally administered anti-PD-1 immune checkpoint inhibitor. Analysis of the isolated tumor weights at the end of the study shows that the average tumor size in control mice is 3.78 grams, and the average tumor size in mice treated with anti-PD-1 antibody is 2.16 grams. The average tumor size in mice treated with BRB-F alone is 2.25 grams, and the average tumor size in mice treated with anti-PD-1 antibody BRB-F combination is 1.38 grams. Thus, BRB-F or anti-PD-1 antibody alone each reduce tumor size by 40.5% or 42.9%, whereas the combination of BRB-F and anti-PD-1 antibody reduces tumor size by 63.5%, with their cooperative effect being statistically significant. The observed anti-tumor effects were accompanied by a series of biomarkers associated with cancer formation and inhibition. These results indicate that the reported potentiation of cancer therapy using drug-based medical chemotherapies with added checkpoint inhibitors in human patients are mechanistically similar with the functional food evaluated in the present study. These beneficial effects in mice challenge clinicians to investigate if the black rice bran food product can also protect against human cancer.

Production of palmitoleic acid by oleaginous yeast Scheffersomyces segobiensis DSM 27193 using systematic dissolved oxygen regulation strategy

Palmitoleic acid(POA)can be naturally found only in few oil seeds and has significant applications in pharmaceutical industry.Recently,the isolated oleaginous yeast Scheffersomyces segobiensis DSM 27193 was identified with high content of POA in its intracellular lipid(13.80%).In this study,process optimization focused on dissolved oxygen regulation to improve POA production was conducted.Dynamic agitation was found to do significant enhancement on POA-rich lipid production than aeration regulation.Under the best condition of 1000 r·min^(-1)of agitation and 1 vvm(airvolume/culture volume/min)of aeration,no ethanol was detected during the whole fermentation process,while a dry biomass concentration of 44.80 g·L^(-1)with 13.43 g·L^(-1)of lipid and 2.93 g·L^(-1)of POA was achieved.Transcription analysis revealed that the ethanol synthetic pathway was downregulated under the condition of high agitation,while the expression of the key enzymes responsible for lipid and POA accumulation were enhanced.

Protection of Allergic Asthma in Mice by Black Rice Bran Bioprocessed with Shiitake Mushroom Mycelia

We evaluated the potential of orally fed new food formulations to inhibit biomarkers reported to be involved in the causes of allergic asthma in mice. Asthma, a serious non-communicable disease, affects both adults and children and can be undertreated. New functional foods could provide therapeutic approaches. Here, the anti-asthma mechanism of a new functional food and three isolated fractions produced by bioprocessing black rice bran with shiitake mushroom mycelia was evaluated in mast cells, B cells, and orally fed mice and compared with non-bioprocessed black rice bran. In vitro, the treatments inhibited RBL-2H3 cell degranulation and immunoglobulin E (IgE) production. The in vitro anti-asthma effects were confirmed in orally fed mice following asthma induction by alumina and chicken egg ovalbumin (OVA). The suppression of asthma resulted from the inhibition of inflammation- and immune-related substances, including OVA-specific IgE, thymic stromal lymphopoietin, eotaxin, leukotriene C4, prostaglandin D2, and vascular cell adhesion molecule-1 in bronchoalveolar lavage fluid and serum. The treatment also reversed the thickening of the lung airway wall. The inflammation and asthma inhibition seems to be regulated by the balance of the T-helper cells’ Th1/Th2 immune response and the inhibition of multiple biomarkers associated with the cause of asthma. Future human clinical studies with adults and children should determine the potential therapeutic value of the anti-asthma effects of the new functional foods.

Raman spectroscopy as process analytical tool in downstream processing of biotechnology

Downstream processing or product recovery plays a vital role in the development of bioprocesses.To improve the bioprocess efficiency,some unconventional methods are much required.The continuous manufacturing in downstream processing makes the Process Analytical Technologies(PATs)as an important tool.Monitoring and controlling bioprocess are an essential factor for the principles of PAT and quality by design.Spectroscopic methods can apply to monitor multiple analytes in real-time with less sample processing with significant advancements.Raman spectroscopy is an extensively used technique as an analytical and research tool owing to its modest process form,non-destructive,non-invasive optical molecular spectroscopic imaging with computer-based analysis.Generally,its application is essential for the analysis and characterization of biological samples,and it is easy to operate with minimal sample.The innovation on various types of enhanced Raman spectroscopy was designed to enhance the Raman analytical technique.Raman spectroscopy could couple with chemometrics to provide reliable alternative analysis method of downstream process analysis.Thus,this review aims to provide useful insight on the application of Raman spectroscopy for PAT in downstream processing of biotechnology and Raman data analysis in biological fields.

Switchgrass (<i>Panicum virgatum</i>) Fermentation by <i>Clostridium thermocellum</i>and <i>Clostridium beijerinckii</i>Sequential Culture: Effect of Feedstock Particle Size on Gas Production

Fermentation of cellulosic biomass can be done in a single step with cellulolytic, solventogenic bacteria, such as Clostridium thermocellum. However, the suite of products is limited in consolidated bioprocessing. Fortunately, the thermophilic nature of C. thermocellum can be exploited in sequential culture. Experiments were conducted to determine the effect of feedstock particle size on fermentation by sequential cultures and to demonstrate this effect could be shown by gas production. Dual-temperature sequential cultures were conducted by first culturing with C. thermocellum (63°C, 48 h) before culturing with C. beijerinckii (35°C, 24 h). Switchgrass (2, 5 or 15 mm particle size) was the feedstock in submerged substrate (10% w/v) fermentation. The extent of fermentation was evaluated by gas production and compared by analysis of variance with Tukey’s test post hoc. C. thermocellum alone produced 78 kPa cumulative pressure (approx. 680 mL gas) when the particle size was 2 or 5 mm. The C. thermocellum cultures with 15 mm feedstock particles had a mean cumulative pressure of 15 kPa after 48 h, which was less than the 2 and 5 mm treatments (P °C) and inoculated with C. beijerinckii, and the cumulative pressures were reset to ambient, cumulative pressure values as great as 70 kPa (equivalent to an additional 670 mL gas) were produced in 24 h. Again, the longer (15 mm) particle size produced less gas (P < 0.05). When the substrates were inoculated with C. beijerinckii without previous fermentation by C. thermocellum, the mean cumulative pressures were approximately 10 kPa. These results indicate that biological pretreatment with C. thermocellum increased the availability of switchgrass carbohydrates to C. beijerinckii, and that gas production is suitable method to show the effectiveness of a pretreatment.

Development of Medium and Kinetic Modeling for Enhanced Production of Cutinase from Pseudomonas cepacia NRRL B-2320

The development of medium for the production of cutinase from Pseudomonas cepacia NRRL B 2320 was carried out using Plackett-Burman experimental design followed by central composite design. The medium components were screened by Plackett-Burman experimental design which suggested that cutin, peptone, KCl and MgSO4·7H2O have influenced the cutinase production significantly with very high confidence levels. The concentration levels of these four components were optimized using 24 full factorial central composite design. An optimum combination of 10.06 g·L-1 of cutin, 17.77 g·L-1 of peptone, 0.635 g·L-1 of KCl and 5.455 g·L-1 of MgSO4·7H2O in the medium gave a maximum cutinase activity of 336 U·mL-1. An overall 2 fold increase in the production of cutinase was observed in the optimized medium. Growth and production of cutinase from P. cepacia NRRL B 2320 have been studied in shake flask and batch bioreactor. Time course of cell growth and enzyme production was fitted to the existing kinetic models reported in the literature to estimate the biokinetic parameters. These models suggested that the production of cutinase is growth associated in shake flask and it is a mixed growth type in a batch bioreactor.

Rearing Performance of Juvenile Brown Trout (<i>Salmo trutta</i>) Subjected to Exercise and Dietary Bioprocessed Soybean Meal

This 121-day experiment evaluated the rearing performance of brown trout Salmo trutta fed one of two isonitrogenous and isocaloric diets and reared at velocities of either 2.8 or 16.1 cm/s. Fishmeal was the primary protein source for the reference diet, and bioprocessed soybean meal replaced approximately 67% of the fishmeal in the experimental diet. At the end of the experiment, there were no significant differences in gain, percent gain, feed conversion rates, or specific growth rates between the dietary treatments. There were also no significant differences in intestinal morphology, splenosomatic, hepatosomatic, and viscerosomatic indices related to diet composition. However, gain, percent gain, feed fed, and specific growth rate were all significantly greater in brown trout reared at the higher velocity. No significant differences in any of the other variables measured were observed between the velocity treatments. There were no significant interactions between diet and velocity in any of the variables. Based on the results of this study, bioprocessed soybean meal can replace at least 67% of the fishmeal in brown trout diets, regardless of the rearing velocities used in this study. However, higher rearing velocities are recommended to maximize juvenile brown trout growth rates.

Bioconversion of Fish Hatchery Waste as Feed in the Production of Live Feed

Purple Non-Sulfur Bacteria (PNSB), also known as phototrophic bacteria are widely distributed in both freshwater and marine environment and capable to grow in wide range of substrates. In this study, Bacterium Rhodobacter sphaeroides strain UMS2, a freshwater isolate was used in this study in utilization of fish hatchery waste. This study was conducted to determine the nutritional values of bioprocess product that was grown in fish hatchery waste. Finally, the waste bio-converted product was used as feed supplement to monitor the growth performance of live feed Tubifex spp. Inoculum of Rhodobacter sphaeroides strain UMS2 was developed in 112 synthetic media and 48-h culture of 30% (v/v) inoculum was used in fish hatchery waste during the bioprocess. The nutritional values of bio-converted product, except total ash (%), were not significantly improved with 30% (v/v) inoculum of Rhodobacter sphaeroides, strain UMS2. Feeding trial in bloodworm (Tubifex spp.) with bioconversion product conducted for 15 days to monitor growth (w/v) of live feed. Initial growth 1.42 ± 0.001 g/L of Tubifex spp. was stocked in 15 × 75 × 15 cm plastic tray connected with recirculated system. Tubifex spp. was observed to be comparatively higher (1.55 ± 0.12 g/L) while fed in the product that contained bacterium than the growth (1.44 ± 0.15 g/L) of Tubifex spp. fed in the bioconversion product of without bacterium. The inoculums size (30%) of bacterium not enough to support the growth of Rhodobacter sphaeroides, strain UMS2 in the bioconversion process to improve the nutritional values. However, while used as feed supplement it improved the growth performance of the Tubifex spp. So, bacterium Rhodobacter sphaeroides, strain UMS2 has potentiality to be used as feed supplement in the production of live feed.

A combo technology of autotrophic and heterotrophic denitrification processes for groundwater treatment

In this study,a sequential process(heterotrophic up-flow column and completely mixed membrane bioreactors)was proposed combining advantages of the both processes.The system was operated for 249 days with simulated and real groundwater for nitrate removal at concentrations varying from 25 to 145 mg·L^(-1) NO_(3)^(-)-N.The contribution of heterotrophic process to total nitrate removal in the system was controlled by dozing the ethanol considering the nitrate concentration.By this way,sulfur based autotrophic denitrification rate was decreased and the effluent sulfate concentrations were controlled.The alkalinity requirement in the autotrophic process was produced in the heterotrophic reactor,and the system was operated without alkalinity supplementation.Throughout the study,the chemical oxygen demand in the heterotrophic reactor effluent was(23.7±22)mg L^(-1) and it was further decreased to(7.5±7.2)mg·L^(-1) in the system effluent,corresponding to a 70%reduction.In the last period of the study,the real groundwater containing 145 mg·L^(-1) NO_(3)^(-)-N was completely removed.Membrane was operated without chemical washing in the first 114 days.Between days 115-249 weekly chemical washing was required.

Characterization of Finfish Hatchery Waste for Value Added Product

Commercial fish hatchery generates waste both organic and inorganic;the sources are primarily from uneaten food and fish feces. Conventional methods of treating hatchery wastes will increase the operating cost and become extra burden in production. It is necessary to develop a new research application of this nonconventional resource and reduce the negative impacts of hatchery waste on the environment. The whole project is to utilize hatchery waste through bioprocess for probiotic fortified live feed production. In this study, the chemical composition of hatchery waste was determined to understand the suitability waste to get value-added derived products through bioprocess. Composite samples were collected everyday and dried in an oven at a temperature of 65°C until complete dryness. Dried samples were mixed well and grinded into fine powder. The analytical parameters like total solids, ammonium nitrogen, nitrite, nitrate and phosphate were determined from the freshly collected samples. Total nitrogen, total phosphorus and total potassium were determined from the dry samples. Total solids, ammonium nitrogen, nitrite, nitrate and phosphate-phosphorus were observed in the ranged from 75 - 82 mg/L, 0.25 - 8.5 mg/L, 0.05 - 1.9 mg/L, 0.04 - 6.7 mg/L and 4.1 - 16.7 mg/L respectively. On the other hand, the mean content of 3.75% total nitrogen, 1.80% total phosphorus and 0.15% potassium were determined in dry hatchery wastes. The analytical parameters are useful and demonstrate that the nutrients in both fresh and dry waste will be supportive for the growth of microbes in the bioprocess system.

Nonlinear Control of Bioprocess Using Feedback Linearization, Backstepping, and Luenberger Observers

This paper addresses the analysis, design, and application of observer-based nonlinear controls by combining feedback linearization (FBL) and backstepping (BS) techniques with Luenberger observers. Complete development of observer-based controls is presented for a bioprocess. Controllers using input-output feedback linearization and backstepping techniques are designed first, assuming that all states are available for feedback. Next, the construction of observer in the transformed domain is presented based on input-output feedback linearization. This approach is then extended to observer design based on backstepping approach using the error equation resulted from the backstepping design procedure. Simulation results demonstrating the effectiveness of the techniques developed are presented and compared.

Fast Mineralization of Densely Packed Hydroxyapatite Layers in the Presence of Over-expressed Recombinant Amelogenin

Learning from the process of biominerals formation provides tremendous ideas for developing advanced synthesis techniques. According to the structure formation of tooth enamel, a recombinant amelogenin his-Amel X was designed and constructed. The protein was over-expressed and could be conveniently purified in one-step heat treatment. The mineralization process of hydroxyapatite was initiated by enzyme AP and regulated by the recombinant amelogenin. Effects of solution pH value and mineralization duration were studied. It was demonstrated that his-Amel X could induce the nucleation of apatite and quicken the growth rate at pH 7.0-7.4, while impeded hydroxyapatite growth at pH 6.8. Moreover, a much denser layer of hydroxyapatite was achieved with the addition of his-Amel X. The present study may not only provide insight into the formation of natural biomaterials but also open a new path to prepare materials under environmentally benign conditions.

Sustainable arabitol production by a newly isolated Debaryomyces prosopidis strain cultivated on biodiesel-derived glycerol

The purpose of this research was the bioconversion of biodiesel-derived glycerol to arabitol through the adoption of sustainable bioprocessing.Arabitol production by Debaryomyces prosopidis FMCC Y69 was significantly affected by initial glycerol concentrations.The highest arabitol concentration of 45.5 g/L was achieved in the case of 120 g/L initial glycerol combined with the highest yield(0.379 g arabitol/g initial glycerol).The yeast strain showed a non-typical oleaginous behavior with low accumulation of lipids(up to 20.9%w/w)despite nitrogen-limited condition that prevailed in the fermentation media.Endopolysaccharides(IPs)were considerably high with a yield on the produced dry cell weight(YIPs/X)(=40.9%w/w)when 85 g/L of glycerol was utilized as the starting material.The fermentation efficiency was further improved when the optimal carbon to nitrogen(C/N)ratio was applied.More specifically,arabitol production was maximized(reaching 56.5 g/L)at a C/N ratio of 158 moles/moles while yield and productivity were also enhanced with respective values of 0.48 g arabitol/g consumed glycerol and 0.112 g/L.h.The increase in C/N ratios led to a decrease in cell dry weight(DCW)concentrations,which is rationally associated with decreased nitrogen availability in the growth medium.YIPs/X values were remarkably high(23.1–26.1%w/w)at the end of each fermentation with the highest concentration in absolute values of 7.3 g/L achieved at the lowest C/N ratio employed(i.e.,79 moles/moles).This study demonstrated the efficient arabitol production along with other value-added metabolites,such as IPs and cellular lipids,implementing biotechnology and renewable resources and thus contributing to the development of circular economy technologies.

Ethanol production by a filamentous fungal strain Byssochlamys fulva AM130 under alternating aerobic and oxygen-limited conditions

Byssochlamys fulva AM130,a novel strain of filamentous fungus,could produce ethanol from glucose,xylose,and alkali pretreated rice straw(PRS),while the efficiencies were very low with PRS.Ethanol production of 11.84 g/L was attained by the fungus when grown in glucose,indicating that the limitations while growing on PRS were related to low hydrolytic efficiency.Enzyme profiling of the fungus showed 365 IU/ml of beta-glucosidase and 89 IU/ml of xylanase activity,while endoglucanase and filter paper activity were negligible,which accounts for the low hydrolytic efficiency.The fungus could survive for extended periods under oxygen-limited conditions and produce ethanol.The fungal mycelia could also be used for repeated cycles of anaerobic fermentation,wherein the ethanol yield improved with each consecutive cycle.

Synthetic biology of microbes synthesizing polyhydroxyalkanoates (PHA)

Microbial polyhydroxyalkanoates(PHA)have been produced as bioplastics for various purposes.Under the support of China National Basic Research 973 Project,we developed synthetic biology methods to diversify the PHA structures into homo-,random,block polymers with improved properties to better meet various application requirements.At the same time,various pathways were assembled to produce various PHA from glucose as a simple carbon source.At the end,Halomonas bacteria were reconstructed to produce PHA in changing morphology for low cost production under unsterile and continuous conditions.The synthetic biology will advance the PHA into a bio-and material industry.

Resistance mechanisms and reprogramming of microorganisms for efficient biorefinery under multiple environmental stresses

In the fermentation process of biorefinery,industrial strains are normally subjected to adverse environmental stresses,which leads to their slow growth,yield decline,a substantial increase in energy consumption,and other negative consequences,which ultimately seriously hamper the development of biorefinery.How to minimize the impact of stress on microorganisms is of great significance.This review not only reveals the damaging effects of different environmental stresses on microbial strains but also introduces commonly used strategies to improve microbial tolerance,including adaptive evolution,reprogramming of the industrial host based on genetic circuits,global transcription machinery engineering(gTME)and bioprocess integration.Furthermore,by integrating the advantages of these strategies and reducing the cost of system operation,the tolerance of industrial strains,combined with production efficiency and process stability,will be greatly improved,and the development prospects of biorefinery will be more widespread.

Recent Advances of AIEgens for Targeted Imaging of Subcellular Organelles

Fluorescence imaging based on luminogens with aggregation-induced emission(AIE)effect has drawn great attention in recent two decades,due to their superior advantages to overcome the technical difficulties.Thus,the AIE-active bioprobes with targeted ability at the subcellular level have been widely investigated to visualize the subcellular structures and monitor the biological processes.Considering the very rapid developments and the significance of selective imaging of subcellular structures,we summarize the recent two-year achievements about the AIEgens for targeted imaging of subcellular organelles including nuclei,membranes,lipid droplets(LDs),endoplasmic reticulum(ER),lysosomes,mitochondria and cytoplasm.The designed protocols and advantages of AIEgens,their mechanisms for targeted staining at organelles and the imaging performance are discussed.These AIE bioprobes exhibit great potentials for early diagnosis and therapeutics of diseases that related to subcellular organelles.Finally,the perspectives about AIEgens for these applications are also discussed.

Cell surface protein engineering for high-performance whole-cell catalysts

Cell surface protein engineering facilitated by accumulation of information on genome and protein structure involves heterologous production and modifica- tion of cell surface proteins using genetic engineering, and is important for the development of high-performance whole-cell catalysts. In this field, cell surface display is a major technology by exposing target proteins, such as enzymes, on the cell surface using a cartier protein. The target proteins are fused to the carrier proteins that transport and tether them to the cell surface, as well as to a secretion signal. This paper reviews cell surface display systems for prokaryotic and eukaryotic cells from the perspective of carrier proteins, which determine the number of displayed molecules, and the localization, size, and direction （N- or C-terminal anchoring） of the passengers. We also discuss advanced methods for displaying multiple enzymes and a new method for the immobilization of whole-cell catalysts using adhesive surface proteins.

液体层析系统

Technikrom公司推出的两款中试生物过程台式液体层析系统Pilot Bioprocess LC，一种为500毫升／分种，另一种为1,000毫升／分种，两款系统的特点是符合卫生标准的流动通道设计，通过GMP认证可以用于生物纯化。Adaptive PAT可直接放大到生产规模的商业机型上。这个产品的特点是从生产规模的机型微型化发展出来的适应中试规模的产品，而不是由实验室设备放大而成。每个系统可以根据用户需求而定制。