Influence of medium composition on penicillin V production in a stirred tank reactor.

Penicillin production with a high-producing strain Penicillium chrysogenum was investigated under well-controlled conditions in a stirred tank reactor with complex media containing lard oil and lactose on the one hand, and lactose on the other hand. With lard oil, cell growth and product formation rates were higher, and the production time was shorter by 40 h than without lard oil. On account of the longer production time without lard oil, the amount of beta-lactam compounds was higher (29.93 g l-1), but the mole fraction of the decomposed products (penicilloic acid and penilloic acid) was larger (0.282) than the amount of penicillin V (23.25 g l-1) and the decomposed mole fraction (0.0747) with lard oil. The final product concentrations were about the same (20.86 g l-1 or 35,462 IU ml-1 with lard oil, and 20.43 g l-1 or 34510 IU ml-1 without lard oil). The mole fractions of the by-product (p-OH-penicillin V) were 0.0365 and 0.066. The substitution of lard oil with lactose is possible without a considerable reduction of process performance.     

Production and Analysis of Fast Pyrolysis Oils from Proteinaceous Biomass

Fast pyrolysis of lignocellulosic biomass is a facile method for producing high yields of liquid fuel intermediates. However, because most fast pyrolysis oils are highly oxygenated, acidic, and unstable, identification of feedstocks that produce higher quality pyrolysis liquids is desirable. Therefore, the effect of feedstock protein content was studied by performing fast pyrolysis experiments on biomass with varying protein content. The feedstocks ranged from low-protein content, ??5% up to feedstocks with >40 wt.% protein content. Protein content was not a major factor in the yield of pyrolysis oil or the distribution of biomass carbon into the pyrolysis products. However, elevated levels of protein did cause a deoxygenation effect in the pyrolysis process with more of the oxygen rejected from the biomass as water. The deoxygenation caused the pyrolysis oil from the higher protein containing biomass to have higher energy content. Furthermore, the concentration of basic nitrogen groups caused the pyrolysis oil from the higher protein biomass to shift to a more neutral pH and lower total acid number than has been measured typically for lignocelluloic biomass pyrolysis oils. Some of the pyrolysis oils, particularly those from the mustard seed family presscakes exhibited better thermal stability than low-protein pyrolysis oils.     

Efficient concomitant production of lipids and carotenoids by oleaginous red yeast <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> cultured in palm oil mill effluent and application of lipids for biodiesel production

Rhodotorula glutinis TISTR 5159 is oleaginous red yeast that accumulates both lipids and carotenoids. It was cultured in palm oil mill effluent (POME) with only the addition of ammonium sulfate and Tween 20 as a suitable nitrogen source and surfactant, respectively. Response surface methodology (RSM) was applied to optimize initial chemical oxygen demand (COD) in POME, C/N ratio, and Tween 20 concentration for concomitant production of lipids and carotenoids. Among three investigated factors, C/N ratio contributed a significant effect upon lipid and carotenoids production. Analysis of response surface plots revealed that the optimum C/N ratio for the biomass was 140, while that for lipid content and carotenoids were higher at 180 and 170, respectively. The high level of the nitrogen source (with a low C/N ratio) enhanced the biomass, making the accumulation of lipids and carotenoids less preferable. Hence, the two-stage process was attempted as an optimal way for cell growth in the first stage and product accumulation in the second stage. The lipid yield and carotenoid production obtained in the two-stage process were higher than those in the one-stage process. In the semi-continuous fermentation, R. glutinis TISTR 5159 accumulated high lipid content and produced a considerably high concentration of carotenoids during long-term cultivation. Additionally, efficient COD removal by R. glutinis TISTR 5159 was observed. The biodiesel produced from yeast lipids was composed mainly of oleic and palmitic acids, similar to those from plant oil.     

海藻糖对脂肪酶的保护机理及酶失活动力学

采用自制的磁性固定化酶(MIE),考察了高温下二糖类对酶的保护作用。结果显示:海藻糖对悬浮于水溶液中的MIE没有保护作用;而在高温干燥后,对酶的保护作用效果依次为:海藻糖>乳糖>蔗糖,支持‘玻璃态学说’;此外,采用两步失活动力学模型能够较好的拟合酶的失活过程,并且得到酶的失活速率常数k和半衰期t1/2,加入海藻糖和乳糖之后,MIE的半衰期分别增长了31和23倍。     

From selection to complementarity: shifts in the causes of biodiversity-productivity relationships in a long-term biodiversity experiment

In a 10-year (1996-2005) biodiversity experiment, the mechanisms underlying the increasingly positive effect of biodiversity on plant biomass production shifted from sampling to complementarity over time. The effect of diversity on plant biomass was associated primarily with the accumulation of higher total plant nitrogen pools (N g m-2) and secondarily with more efficient N use at higher diversity. The accumulation of N in living plant biomass was significantly increased by the presence of legumes, C4 grasses, and their combined presence. Thus, these results provide clear evidence for the increasing effects of complementarity through time and suggest a mechanism whereby diversity increases complementarity through the increased input and retention of N, a commonly limiting nutrient.     

Optimization of Banana Juice Fermentation for the Production of Microbial Oil

Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D), a lipid-accumulating yeast, was grown in banana juice. The optimum conditions for biomass production in shake flasks were 30°C growth temperature, efficient aeration, a juice concentration of 25%, and preliminary heat treatment at less than sterilization conditions. Under controlled conditions in a fermentor, 20% banana juice was optimum. High concentrations of yeast extract (0.3%) increased biomass production by 40% but decreased oil production by 30%. A lower yeast extract concentration (0.05%) increased biomass production by 2% and oil production by 25%. The best growth and oil production were observed when asparagine (1.4 g/liter) and mineral salts were added to the banana juice. The addition of minerals seemed to improve the utilization of carbon. Growth inhibition was observed when the fermentor was aerated with pure oxygen, even when additional nutrients were present. A fed-batch process permitted the juice concentration to be increased from 15 to 82%; biomass accumulation was three times higher than in batch fermentations. However, the cellular lipid content was only 30% of dry weight, and chemical oxygen demand reduction was slow and inefficient.     

Cadmium tolerance and its phytoremediation by two oil yielding plants Ricinus communis (L.) and Brassica juncea (L.) from the contaminated soil

The effect of increasing level of cadmium in soil was investigated on biomass production, antioxidants, Cd bioaccumulation and translocation in Ricinus communis vis-à-vis a commonly studied oil crop Brassica juncea. The plants were exposed to 25, 50, 75, 100, and 150 mg Cd/Kg soil for up to 60 days. It was found that R. communis produced higher biomass at all the contamination levels than that of B. juncea. Proline and malondialdehyde in the leaves increased with increase in Cd level in both the species, whereas soluble protein decreased. The bioaccumulation of Cd was higher in B. juncea on the basis of the per unit biomass, total metal accumulation per plant was higher in R. communis. The translocation of Cdfrom roots to shoot was also higher in B. juncea at all Cd concentrations. R. communis appeared more tolerant and capable to clean Cd contaminated soil for longer period in one sowing than B. juncea and the former can grow in wasteland soil also in which later cannot be cultivated.     

Biomass and lipids from lactose or whey by Trichosporon beigelii

The yeast Trichosporon beigelii produced the highest amount of biomass when grown in chemical-defined medium with a ratio of carbon source to nitrogen source of 30:1. On the other hand, carbon-limited medium (C-N ratio 2:1) enhanced unsaturated fatty acids synthesis. The yeast efficiently converted unsalted whey lactose to biomass, while sodium chloride in whey raised lactose assimilation to single-cell oil (SCO).     

Modeling of olive oil degradation and oleic acid inhibition during chemostat and batch cultivation of Bacillus thermoleovorans IHI-91

Olive oil degradation by the thermophilic lipolytic strain Bacillus thermoleovorans IHI-91 in chemostat and batch culture was modeled to obtain a general understanding of the underlying principles and limitations of the process and to quantify its stoichiometry. Chemostat experiments with olive oil as the sole carbon source were successfully described using the Monod chemostat model extended by terms for maintenance requirements and wall growth. Maintenance requirements and biomass yield coefficients were in the range reported for mesophiles. For a chemostat experiment at D = 0.3 h(-1) the model was validated up to an olive oil feed concentration of about 3.0 g L(-1) above which an inhibitory effect occurred. Further analysis showed that the liberated oleic acid is the main cause for this inhibition. Using steady-state oleic acid concentrations measured in chemostat experiments with olive oil as substrate it was possible to derive a kinetic expression for oleic acid utilization, showing that a concentration of 430 mg L(-1) leads to a complete growth inhibition. Oleic acid accumulation observed during batch fermentations can be predicted using a model involving growth-associated lipase production and olive oil hydrolysis. Simulations confirmed that this accumulation is the cause for the sudden growth cessation occurring in batch fermentations with higher olive oil start concentrations. Further, an oscillatory behavior, as observed in some chemostat experiments, can also be predicted using the latter model. This work clearly demonstrates that thermophilic lipid degradation by Bacillus thermoleovorans IHI-91 is limited by long-chain fatty acid beta-oxidation rather than oil hydrolysis.     

Relationships between hydrodynamics and rheology of flocculating yeast suspensions in a high-cell-density airlift bioreactor

In this article a hydrodynamic and rheological analysis of a continuous airlift bioreactor with high-cell-density system is presented. A highly flocculating recombinant strain of Sacharomyces cerevisiae containing genes for lactose transport (lactose permease) and hydrolysis (beta-galactosidase) was exploited to ferment lactose from cheese whey to ethanol. The magnetic particle-tracer method was used to assess the effect of operational conditions (air-flow rate, biomass concentration) on hydrodynamic behavior of an airlift bioreactor during the fermentation process. Measurements of liquid circulation velocity showed the existence of a critical value of biomass concentration at which a dramatic deceleration of net liquid flow appeared with increasing biomass quantity. Rheological analysis revealed exponential increase of viscosity of the yeast floc suspension at the same biomass concentration of about 73 g/dm3 corresponding to 42.8% v/v of solid fraction. These facts have a particular importance for the successful processing of a high-cell-density airlift bioreactor as only a circulated flow regime will be favorable to keep the solid particles in suspension state and evenly distributed throughout the bioreactor.     

Fermentation efficiency of thermally dried kefir

Three thermal drying methods (conventional, vacuum and convective) were used for drying of kefir biomass and their effect on cell viability, fermentation rate and other kinetic parameters of lactose and whey fermentation were studied. Convective drying rate was higher than conventional and even higher than vacuum at each studied temperature (28, 33 and 38 degrees C). After that, fermentations were performed by kefir biomass dried by the three drying methods. Ethanol concentration, ethanol productivity and ethanol yield are higher in whey fermentations performed by kefir biomass dried with convective drying method. Regarding lactic acid production, fermentation performed by kefir biomass dried with conventional drying method gave higher concentrations, compared to other drying methods. Storage of kefir biomass convectively dried at 33 degrees C for 4months, without any precaution decreases its fermentability and thus reduces ethanol (31%) and lactic acid productivity (20%), but remains a promising technology, since a significant part of its initial fermentative activity is retained.     

High-productivity lipid production using mixed trophic state cultivation of <Emphasis Type="Italic">Auxenochlorella</Emphasis> (<Emphasis Type="Italic">Chlorella</Emphasis>) <Emphasis Type="Italic">protothecoides</Emphasis>

A mixed trophic state production process for algal lipids for use as feedstock for renewable biofuel production was developed and deployed at subpilot scale using a green microalga, Auxenochlorella (Chlorella) protothecoides. The process is composed of two separate stages: (1) the photoautotrophic stage, focused on biomass production in open ponds, and (2) the heterotrophic stage focused on lipid production and accumulation in aerobic bioreactors using fixed carbon substrates (e.g., sugar). The process achieved biomass and lipid productivities of 0.5 and 0.27 g/L/h that were, respectively, over 250 and 670 times higher than those obtained from the photoautotrophic cultivation stage. The biomass oil content (over 60 % w/DCW) following the two-stage process was predominantly monounsaturated fatty acids (~82 %) and largely free of contaminating pigments that is more suitable for biodiesel production than photosynthetically generated lipid. Similar process performances were obtained using cassava hydrolysate as an alternative feedstock to glucose.     

Inhibition of autophagic-lysosomal delivery and autophagic lactolysis by asparagine

Overall autophagy was measured in isolated hepatocytes as the sequestration and lysosomal hydrolysis of electroinjected [14C]lactose, using HPLC to separate the degradation product [14C]glucose from undegraded lactose. In addition, the sequestration step was measured separately as the transfer from cytosol to sedimentable cell structures of electroinjected [3H]raffinose or endogenous lactate dehydrogenase (LDH; in the presence of leupeptin to inhibit lysosomal proteolysis). Inhibitor effects at postsequestrational steps could be detected as the accumulation of autophaged lactose (which otherwise is degraded intralysosomally), or of LDH in the absence of leupeptin. Asparagine, previously shown to inhibit autophagic but not endocytic protein breakdown, strongly suppressed the autophagic hydrolysis of electroinjected lactose. Vinblastine, which inhibits both types of degradation, likewise suppressed lactose hydrolysis. Asparagine had little or no effect on sequestration, but caused an accumulation of autophaged LDH and lactose, indicating inhibition at a postsequestrational step. Neither asparagine nor vinblastine affected the degradation of intralysosomal lactose preaccumulated in the presence of the reversible lysosome inhibitor propylamine. However, if lactose was preaccumulated in the presence of asparagine, both asparagine and vinblastine suppressed its subsequent degradation. The data thus indicate that autophagic-lysosomal delivery, i.e., the transfer of autophaged material from prelysosomal vacuoles to lysosomes, is inhibited selectively by asparagine and non-selectively by vinblastine.     

Comparison of lactose uptake in resting and energized Escherichia coli cells: high rates of respiration inactivate the lac carrier

The transport of lactose by Escherichia coli cells was radically different in the absence and in the presence of an exogenous energy source: in the former case, the time course of lactose accumulation was monotonous; in the latter case, lactose accumulation reached a maximum and then decreased to a final steady-state level lower than that observed in the absence of an energy source. We show that this "overshoot" is the result of a decrease in the influx rate and of an increase in the rate constant of efflux as lactose accumulates. These phenomena were irreversible. The extent of the overshoot was dependent upon the experimental conditions: it was maximal at alkaline pH, for low external potassium concentrations, and for relatively high external lactose concentrations (around or above the KT of uptake). The addition of an energy source to resting E. coli cells resulted in an increase in both the electrochemical gradient of protons and in the rate of respiration. We demonstrate that the overshoot is the result of the latter and unrelated to the former. We observed an irreversible decrease in the membrane potential as lactose accumulated in the presence of an exogenous energy source. We discuss the whole of our data in terms of an irreversible inactivation of the lactose carrier as a result of a possible interaction with the respiratory chain.     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

The effects of palm oil breakdown products on lipid turnover and morphology of fungi

The oleaginous fungi Cryptococcus curvatus and Mucor circinelloides were used to determine the effect of palm oil breakdown products, measured as polymerized triglycerides (PTGs), on lipid turnover and on fungal growth and morphology. In M. circinelloides, we found after 7 days of growth, a decrease in biomass and in lipid utilization and accumulation at increased PTG levels, both at low and neutral pH. In C.?curvatus, there was also a decrease in lipid utilization and biomass production at increased PTG levels, at both low and neutral pH. However, an increase in oil accumulation was observed at low pH while it remained similar at neutral pH for all PTG levels tested. Hairy and warty protuberances on the cell surface were observed when C. curvatus was grown on oils with 15% and 45% PTGs, respectively. Using nano scanning Auger microscopy, we found no evidence to suggest a difference in elemental composition of the surfaces of the warty protuberances compared with the rest of the cell wall surface. We conclude that the warty protuberances are outgrowths of cell walls and that the changes observed in lipid turnover in both fungi are due to the presence of palm oil breakdown products.     

Impact of nitrogen limitation on biomass,photosynthesis, and lipid accumulation in <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Induction of oil accumulation in algae for biofuel production is often achieved by withholding nitrogen. However, withholding nitrogen often reduces total biomass yield. In this report, it is demonstrated that Chlorella sorokiniana will not only accumulate substantial quantities of neutral lipids when grown in the absence of nitrogen but will also exhibit unimpeded growth rates for up to 2 weeks. To determine the physiological basis for the observed increase in oil and biomass accumulation, we compared photosynthetic and respiration rates and chlorophyll, lipid, and total energy content under ammonia replete and deplete conditions. Under N-depleted growth conditions, there was a 64 % increase in total energy density and a ～20-fold increase in oil accumulation relative to N-replete growth leading to a 1.6-fold greater total energy yield in N-depleted than in N-replete cultures. We propose that the higher energy accumulation in N-depleted cultures is due to enhanced photosynthetic energy capture and conversion associated with reduced chlorophyll levels and reduced self-shading as well as a shift in metabolism leading to the accumulation of oils.     

Influence of pH and impeller tip speed on the cultivation of<Emphasis Type="Italic">Bifidobacterium pseudocatenulatum</Emphasis> G4 in a milk-based medium

Biomass production ofBifidobacterium pseudocatenulatum G4 in a milk-based medium was carried out in a 2- and 10-L stirred tank fermenters. The effects of impeller tip speed (0.28, 0.56, and 0.83 m/s) and pH control (6.0, 6.5, and 7.0) on the biomass production were investigated. The growth performance in the 2-L fermenter was significantly improved when the impeller tip speed was held constant at 0.56 m/s and the pH was controlled at 6.5. These conditions yielded a maximum biomass of 1.687×10⁹ cfu/mL, a maximum specific growth rate of 0.504 h⁻¹, a biomass productivity of 9.240×10⁷ cfu/mL·h, and a biomass yield of 9.791×10¹⁰ cfu/g lactose. The consumption of milk lactose resulted in the accumulation of 7.353 g/L acetic acid and 6.515 g/L lactic acid, with an acetic:lactic ratio of 1.129. Scale-up of the fermentation process to a 10-L fermenter based on a constant impeller tip speed of 0.56 m/s yielded reproducible results with respect to biomass production and cell viability.     

Factors affecting the growth and the oil accumulation of marine microalgae, Tetraselmis suecica

Biomass production and oil productivity in microalgae culture are the most important key factors for algal biodiesel production. However, proper culture condition for the biomass production of microalgae is different from that for the oil production of microalgae. A study on the biomass production of Tetraselmis suecica using various light intensities and nitrate concentrations as growth factors was carried out to evaluate proper culture conditions in 20-L batch culture. The effect of nitrate depletion on the oil accumulation was also evaluated with two-stage culture. It took 5 days to reach the stationary phase for the cultures of T. suecica on the light intensities of 108.9 and 133.1 μmol m(-2 )s(-1) with biomass of 0.89 and 0.88 g dcw L(-1), respectively. Biomass productions of 1.07 and 1.00 g dcw L(-1) were obtained with the nitrate concentrations of 18.6 and 24.7 mg L(-1), respectively. The two-stage culture increased oil contents from 7.6 to 17.3% (w/w) and contents of C(16)-C(18) fatty acids from 540.2 to 720.5 mg g(-1) oil. The predominant fatty acid was palmitic acid (C(16:0)) in nitrate depletion group, however, oleic acid (C(18:1)) was predominated in nitrate added groups. The two-stage culture enhanced overall oil productivity of 18.7 mg g(-1) day(-1) which is higher than that of 12.2 mg g(-1) day(-1) in single-stage culture.