Improvement of intracellular β-galactosidase production in fed-batch culture of Kluyveromyces fragilis

Four fed-batch control strategies were evaluated to improve the specific lactase activity of Kluyveromyces fragilis. Control strategies tested included DO-stat control, exponential feeding, exponential feeding with manual feedback control and corrected feed-forward control. Each was implemented with standard sensors (i.e., temperature, dissolved oxygen and pH sensors) commonly installed in fermenters. The highest specific activity was obtained using the corrected feed-forward control strategy, a strategy incorporating a novel method for on-line estimation of specific growth rate. The control strategy was able to operate effectively to a final cell density of 69 g dry wt l^–1 with a specific lactase activity of 2 U mg^–1 cell dry wt.     

Studies on mobilization of chromium with reference to its plant availability – Role of organic acids

Metal-organic acid interactions with special reference to their plant availability have been studied. The role of organic acids in the mobilization of Cr by converting it into labile organically bound form, enhancing its availability, is highlighted. Pot experiments are conducted to investigate the effect of various organic acids on the uptake and translocation of root absorbed trivalent and hexavalent chromium by maize (z. mays) plants grown in sand and soil culture. Statistically significant increases in chromium accumulation from CrIII-treated plants in the presence of increasing concentrations of organic acid suggest the existence of CrIII – organic acid interactions in the soil plant system. In order to support the above mentioned hypothesis of formation of organically bound CrIII in the presence of various organic acids (carboxylic and amino acids), separate experiments have been performed to synthesize and estimate its respective organically bound forms. Amendments with organic acids, however, do not appear to markedly affect chromium accumulation from CrVI treatment. The results are discussed on the basis of the potential of organic acids to form complexes with CrIII.     

Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture

Human granulocyte–macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal α peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h⁻¹ for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y _P/X) was ∼2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l⁻¹ h⁻¹.     

Submerged culture production of chitinase by Trichoderma harzianum in stirred tank bioreactors – the influence of agitator speed

The chitinase fermentation process utilizing chitin as the sole carbon source was investigated in a stirred tank bioreactor. Agitator speed of 224 rpm was found to be most suitable for cell growth as well as for chitinase production. Chitinase yield decreased rapidly at higher agitator speed, while decrease in cell yield at higher agitator speed was not rapid. Probably, mass transfer limitation was predominant in the fermentation process at lower agitator speed. Higher agitator speed appears to reduce chitinase production.     

Enhanced production of α-ketoglutarate by fed-batch culture in the metabolically engineered strains of Corynebacterium glutamicum

The fed-batch culture system was employed to enhance production of α-ketoglutarate (α-KG) by the strainsof Corynebacterium glutamicum, whose genes encoding the key enzymes responsible for the biosynthesis of L-glutamate from α-KG were deleted. In a shake flask fermentation, C. glutamicum JH110 in which the 3 genes, gdh (encoding glutamate dehydrogenase), gltB (encoding glutamate synthase), and aceA (encoding isocitrate lyase) were disrupted showed the highest production of α-KG (12.4 g/L) compared to the strains JH102 (gdh mutant), JH103 (gltB mutant), and JH107 (gdh gltB double mutant). In the fed-batch cultures using a 5 L-jar fermenter, the strain JH107 produced more α-KG (19.5 g/L), but less glutamic acid (23.3 g/L) than those produced by the parent strain HH109, as well as JH102. The production of α-KG was significantly enhanced and the accumulation of glutamicacid was minimized by the ammonium-limited fed-batch cultures employing C. glutamicum JH107. Further improvement of α-KG production by the strain JH107 was achieved through the ammonium-limited fed-batch culture with the feeding of molasses, and the levels of α-KG and glutamic acid produced were 51.1 and 0.01 g/L, respectively.     

The use of charcoal in in vitro culture – A review

Activated charcoal is commonly used in tissue culture media. Its addition to culture medium may promote or inhibit in vitro growth, depending on species and tissues used. The effects of activated charcoal may be attributed to establishing a darkened environment; adsorption of undesirable/inhibitory substances; adsorption of growth regulators and other organic compounds, or the release of growth promoting substances present in or adsorbed by activated charcoal.     

Production of γ-linolenic acid by the fungus Mucor rouxii in fed-batch and continuous culture

Summary The production of -linolenic acid (GLA) and lipid was studied in Mucor rouxii CBS 416.77. In a fed-batch culture productivities of 39.4 mg/l per hour for GLA and 99 mg/l per hour for the total amount of lipid were determined at 18 h of cultivation. At this point the highest value of GLA in lipid (39.7%, w/w) was also reached. Production of GLA was also studied in a series of continuous cultures. It was observed that, in addition to growth rate, the nitrogen concentration of the input medium was of great importance for high productivities. The highest productivity values for GLA (37 mg/l per hour) and for lipid (95 mg/l per hour) were reached at a dilution rate of 0.10 h^-1 with a concentration of 4.5g/l NH₄Cl in the input medium.     

Production of recombinant β-galactosidase in bioreactors by fed-batch culture using DO-stat and linear control

β-Galactosidase enzymes continue to play an important role in food and pharmaceutical industries. These enzymes hydrolyze lactose in its constituent monosaccharides, glucose and galactose. The industrial use of enzymes presents an increase in process costs reflecting in higher final product value. An alternative to enhance processes’ productivity and yield would be the use of recombinant enzymes and their large-scale fed-batch production. The overexpression of recombinant β-galactosidase from Kluyveromyces sp. was carried out in 2-L bioreactors using Escherichia coli strain BL21 (DE3) as host. Effect of induction time on recombinant enzyme expression was studied by adding 1?mM isopropyl thiogalactoside (IPTG) at 12?h, 18?h and 24?h of cultivation. Glucose feeding strategies were compared employing feedback-controlled DO-stat and ascendant linear pump feeding in bioreactor fed-batch cultivations. Linear feeding strategy with IPTG addition at 18?h of cultivation resulted in approximately 20?g/L and 17,745?U/L of biomass and β-galactosidase activity, respectively. On the other hand, although the feedback-controlled DO-stat feeding strategy induced at 12?h of cultivation led to lower final biomass of 18?g/L, it presented an approximately 2.5 increase in enzymatic activity, resulting in 42,367?U/L, and most importantly it led to the most prominent specific enzymatic activity of approximately 40?U/mg_protein. Comparing to previous results, these results suggest that the DO-stat feeding is a promising strategy for recombinant β-galactosidase enzyme production.     

Production of ethanol,organic acids and hydrogen: an opportunity for mixed culture biotechnology?

Anaerobic fermentation of biodegradable organic materials is usually carried out to obtain the final product, methane, a valuable energy source. However, it is also well known that various intermediates are produced in this process, e.g. ethanol, volatile organic acids and hydrogen. All these species have applications and value as fuels or chemicals. This paper shows a critical analysis of the potential of using anaerobic fermentation by mixed cultures to produce intermediates, e.g. ethanol, acetic, lactic and butyric acid and hydrogen, rather than methane. This paper discusses the current processes to produce these chemicals and compares them with the alternative approach of using open mixed cultures to produce them simultaneously via fermentation from renewable resources. None of these chemicals is currently produced via mixed culture fermentation: ethanol and lactic acid are usually produced in pure culture fermentation using food crops, e.g. corn or sugar cane, as starting materials; hydrogen, acetic and butyric acids are mainly produced via chemical synthesis from fossil fuel derived starting materials. A possible flow-sheet for the production of these chemicals from organic waste using mixed culture fermentation is proposed and the advantages and disadvantages of this process compared to current processes are critically discussed. The paper also discusses the research challenges which need to be addressed to make this process feasible.     

MYCOMON-monitoring project of toxigenic fungi in food in years 1999 – 2001

An occurrence of the toxigenic fungi producing aflatoxins and ochratoxin A in food was investigated in the study (“MYCOMON”) in years 1999–2001. Twenty five commodities were purchased from retail in twelve collection places in the Czech Republic (300 food samples together). The presence of potentially toxigenic fungiAspergillus flavus was observed in 28% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 1999, in 17% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 2000 and in 18% of the sampled food (black pepper, black tea, caraway seeds, fine flour, fruit tea, oat flakes) in the year 2001.Aspergillus tamarii (aflatoxins producer) was found in black pepper samples (25%) in year 1999, in black pepper samples and black tea (25%) in year 2000 and in black pepper samples and black tea (21%) in year 2001.Aspergillus parasiticus andAspergillus nomius were not isolated.Aspergillus sectionNigri (potential producer of ochratoxin A) was detected in some food (black pepper, black tea, caraway seeds, fine flour, fruit tea, raisins, sweet red pepper).Penicillium verrucosum and Aspergillus ochraceus were not isolated from the tested food.     

Enhanced cadmium accumulation in maize roots—the impact of organic acids

Low molecular weight organic acids are important components of root exudates and therefore, knowledge regarding the mechanisms of cadmium (Cd) uptake and distribution within plants under the influence of organic acids, is necessary for a better understanding of Cd behavior in the plant–soil system. In this study, acetic and malic acids increased the uptake of Cd by maize (Zea mays L. cv. TY2) roots and enhanced Cd accumulation in shoots under hydroponic conditions. Concentration-dependent net Cd influx in the presence and absence of organic acids could be resolved into linear and saturable components. The saturable component followed Michaelis–Menten kinetics, which indicated that Cd uptake across the plasma membrane was transporter-mediated. While the K _m values were similar, the V _max values in the presence of acetic and malic acids were respectively 6.0 and 3.0 times that of the control. Zinc transporters were the most probable pathways for Cd accumulation. It was hypothesized that Cd(II)–organic acid complexes associated with the root zone, could decompose and liberate Cd²⁺ for subsequent absorption by maize roots; and that in the layer of the roots or within the root free space, depletion of Cd²⁺ was buffered by the presence of Cd(II)–organic acid complexes. Plant response to elevated Cd levels involved overproduction of organic acids in maize roots as a resistance mechanism to alleviate Cd toxicity.     

Evaluation of agronomic traits in tissue culture – derived progeny of bird's-foot trefoil

Two newly selected Lotus corniculatus cultivars (Bokor and Zora), introduced recently as pasture legumes, were studied with respect to their in vitro regeneration capacity and field performance of R₁ regenerates. Multiple buds were induced in callus tissues derived from apical buds, petioles, leaf and cotyledon explants. The rooted regenerates, acclimated in a greenhouse, were fertile. Seeds from the R₁ generation were sown in the field and the plants found to be superior to controls in stem height and green mass production.     

Food waste posing a serious threat to sustainability in the Kingdom of Saudi Arabia – A systematic review

Worldwide, food waste is one of the prime issues threatening food security and the Kingdom of Saudi Arabia (KSA) is not an exception. With 427 kg of food wasted per capita per year, the country ranks among the top food wasters. Ironically, the Kingdom has limited arable lands and scarce water resources to support mass-scale agriculture and to feed its increasing population, KSA relies heavily on imports and subsidized food to meet needs. Yet, food is wasted at restaurants, caterers, cafeterias and, especially, by households such that food waste is the single-largest component of the landfills. The review article is based on the grey and scientific literature published in the English and Arabic languages on the issue of food waste in Saudi Arabia. Information sources like Web of knowledge, online resources and the databases available through the King Saud University, Saudi Arabia were accessed and used to collect information on food waste, its social, cultural, economic and environmental impacts and related topics. Since food items and groceries are abundantly available to all living in KSA and they are highly subsidized, the residents take food for granted. According to a recent survey, about 78% of food purchased in KSA is discarded each week in order to make room for new groceries. The factors responsible for food waste include: lack of awareness; and insufficient and inappropriate planning when shopping. Food waste in restaurants, celebrations, social events and occasions are enormous. Waste is common in festivals and special events where the customs is to provide more food than required. There is a need to change society’s food culture, particularly among the women and the youth, as they are largest segment of the society and the prime food wasters. The analysis of the factors responsible for food waste, identified in this article suggests a “Stop Wasting Food” campaign should be launched. It is also recommended to determine and activate the role of extension education to reduce food waste in the KSA through vibrant capacity building programs for youth and women, in particular, and society in general.     

The time aspect of bioenergy – climate impacts of solid biofuels due to carbon dynamics

The climate impacts from bioenergy involve an important time aspect. Using forest residues for energy may result in high initial emissions, but net emissions are reduced over time since, if the residues were left on the ground, they would decompose and release CO₂ to the atmosphere. This article investigates the climate impacts from bioenergy with special focus on the time aspects. More specifically, we analyze the climate impacts of forest residues and stumps where combustion related emissions are compensated by avoided emissions from leaving them on the ground to decompose. These biofuels are compared with fossil gas and coal. Net emissions are defined as emissions from utilizing the fuel minus emissions from a reference case of no utilization. Climate impacts are estimated using the measures radiative forcing and global average surface temperature. We find that the climate impacts from using forest residues and stumps depend on the decomposition rates and the time perspective over which the analysis is done. Over a 100 year perspective, branches and tops have lower climate impacts than stumps which in turn have lower impacts than fossil gas and coal. Over a 20 year time perspective, branches and tops have lower climate impacts than all other fuels but the relative difference is smaller. However, stumps have slightly higher climate impacts over 20 years than fossil gas but lower impacts than coal. Regarding metrics for climate impacts, over shorter time scales, approximately 30 years or less, radiative forcing overestimates the climate impacts compared with impacts expressed by global surface temperature change, which is due to the inertia of the climate system. We also find that establishing willow on earlier crop land may reduce atmospheric CO₂, provided new land is available. However, these results are inconclusive since we haven't considered the effects of producing the agricultural crops elsewhere.     

On the inhibition of α-oxobutyrate utilization by fatty acids in rat liver mitochondria

It has been found that free fatty acids and acylcarnitine inhibit α-oxobutyrate utilization in rat liver mitochondria. It has been recognized that the intramitochondrial accumulation of acetyl-CoA, produced by the β-oxidation of activated fatty acids, is responsible for such inhibition. In fact acetyl-CoA is shown to inhibit α-oxobutyrate dehydrogenase (α-oxoglutarate: lipoate oxidoreductase (acceptor acylating) EC 1.2.4.2)     

Contamination of livestock due to the operation of a small waste incinerator: a case incident in Skutulsfjörður,Iceland, in 2010

Background

Declining fertility is a major concern for dairy farmers today. One explanation is shorter and weaker expression of oestrus in dairy cows making it difficult to determine optimal time for artificial insemination (AI). Chemical communication is of interest in the search for tools to detect oestrus or to synchronise or enhance oestrous periods. Pheromones, used in chemical communication within species, can influence reproduction in different ways. The aim here was to investigate whether oestrous cycle length, and duration and intensity of oestrous expression in dairy heifers could be manipulated through exposure to pheromones in oestrual substances from other females.

Methods

Beginning on day 16 of two consecutive control oestrous cycles, ten heifers of the Swedish Red Breed (SRB) were exposed to water. During the two following cycles the heifers were exposed to urine and vaginal mucus, obtained from cows in oestrus. Cyclicity parameters were monitored through hormone measurements, oestrus detection and ultrasonographic examination.

Results

We found no difference in cycle length or in duration of standing oestrus between control and treatment. We did, however, find a tendency of interaction between type of exposure (control or treatment) and cycle number within type of exposure for cycle length (p = 0.068), with the length differing less between the treatment cycles. We also found a tendency of effect of type of exposure on maximal concentration (p = 0.073) and sum of concentrations (p = 0.063) of LH during the LH surge, with values being higher for the control cycles. There were also significant differences in when the different signs of oestrus occurred and in the intensity of oestrous expression. The score for oedema and hyperaemia of external genitalia was significantly higher (p = 0.004) for the control cycles and there was also a significant interaction between type of exposure and time period for restlessness (p = 0.011), with maximum score occurring earlier for treatment cycles.

Conclusions

No evidence of altered oestrous cycle length or duration of oestrus after exposure of females to oestrous substances from other females was found. Expression of oestrus, and maybe also LH secretion, however, seemed influenced by the exposure, with the effect of treatment being suppressive rather than enhancing.     

d-Amino acids in molecular evolution in space – Absolute asymmetric photolysis and synthesis of amino acids by circularly polarized light

Living organisms on the Earth almost exclusively use l-amino acids for the molecular architecture of proteins. The biological occurrence of d-amino acids is rare, although their functions in various organisms are being gradually understood. A possible explanation for the origin of biomolecular homochirality is the delivery of enantioenriched molecules via extraterrestrial bodies, such as asteroids and comets on early Earth. For the asymmetric formation of amino acids and their precursor molecules in interstellar environments, the interaction with circularly polarized photons is considered to have played a potential role in causing chiral asymmetry. In this review, we summarize recent progress in the investigation of chirality transfer from chiral photons to amino acids involving the two major processes of asymmetric photolysis and asymmetric synthesis. We will discuss analytical data on cometary and meteoritic amino acids and their potential impact delivery to the early Earth. The ongoing and future ambitious space missions, Hayabusa2, OSIRIS-REx, ExoMars 2020, and MMX, are scheduled to provide new insights into the chirality of extraterrestrial organic molecules and their potential relation to the terrestrial homochirality. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.     

Sequence stability of the T-DNA – plant junctions in tissue culture in Arabidopsis transgenic lines

The stability of the inserted transgenes and particularly the junction regions of transgenic events is a concern of food labeling, traceability and post release monitoring, as these regions are used for development of event-specific DNA-based detection methods. During the standard agricultural breeding practices, the transgenic lines can be exposed to completely different conditions than those in the laboratory environment. Some of these conditions have the potential to affect the stability of the transgenic locus and the surrounding DNA. As tissue culture is recognized as a stressful and mutagenic factor, we have analyzed the effect of this process on the stability of the junction regions at nucleotide level in five Arabidopsis thaliana transgenic lines in comparison with the respective integration loci in ColO and C24 ecotypes. No indication of any kind of alteration at nucleotide level of the junctions was found. The relevance of the stability of the plant–T-DNA junction regions for application of the DNA-based methods in commercial transgenic plants is discussed.