Production of a bioherbicide agent in liquid and solid medium and in a biphasic cultivation system

The use of fungi in weeds control programs depends upon the conidia production in large scale. Therefore, this study aimed to evaluate liquid and solid culture media and the cultivation by biphasic system for the conidia production of Bipolaris euphorbiae Muchovej & Carvalho a specific pathogen of Euphorbia heterophylla. The liquid media were obtained from agro-industrial waste or by-products, and the solid media were prepared with mixtures of grains and grain derivatives. The liquid medium made with sugar cane molasses stood out from the others because it provided great sporulation (23 x 10(4) conidia mL-1 of medium), conidial viability (99.7%), and formation of mycelial fungal biomass (1.26 g 100 mL-1 of medium). On solid media conidial production was markedly higher than in liquid media, especially the medium composed by a blend of sorghum grain (40%) and soybean hulls (60%) where the fungus produced 2.3 x 10(7) conidia g-1 of medium. The cultivation of B. euphorbiae in biphasic system not promoted a significant increase in the production of conidia. The solid media were more effective for the mass production of fungus and mixtures of grains and derivatives were effective for increasing conidia production.

Ionic liquid mediated biomass deconstruction: from analysis challenges to fermentable sugars

Ionic liquids, ILs, have recently been studied with accelerating interest to be used for a deconstruction/fractionation, dissolution or pretreatment processing method of lignocellulosic biomass. ILs are usually utilized combined with heat. Regarding lignocellulosic recalcitrance toward fractionation and IL utilization, most of the studies concern IL utilization in the biomass fermentation process prior to the enzymatic hydrolysis step. It has been demonstrated that IL-pretreatment gives more efficient hydrolysis of the biomass polysaccharides than enzymatic hydrolysis alone. Both cellulose (especially cellulose) and lignin are very resistant towards fractionation and even dissolution methods. As an example, it can be mentioned that softwood, hardwood and grass-type plant species have different types of lignin structures leading to the fact that softwood lignin (guaiacyl lignin dominates) is the most difficult to solubilize or chemically disrupt. In addition to the known conventional biomass processing methods, several ILs have also been found to efficiently dissolve either cellulose and/or wood samples – different ILs are suitable for different purposes. An IL treatment of wood usually results in non-fibrous pulp, where lignin is not efficiently separated and wood components are selectively precipitated, as cellulose is not soluble or degradable in ionic liquids under mild conditions. Nevertheless, new ILs capable of rather good fractionation performance have recently emerged. The capability of the IL to dissolve or deconstruct wood or cellulose depends on several factors, (e.g. sample origin, the particle size of the biomass, mechanical treatments as pulverization, initial biomassto-IL ratio, water content of the biomass, possible impurities of IL, reaction conditions, temperature etc). The aim of this study was to obtain (fermentable) saccharides and other valuable chemicals from wood by a combined heat and IL-treatment. Thermal treatments alone contribute to the degradation of polysaccharides (e.g. 150 °C alone is said to cause the degradation of polysaccharides), thus temperatures below that should be used, if the research interest lies on the IL effectiveness. On the other hand, the efficiency of the IL-treatment can also be enhanced to combine other treatment methods, (e.g. microwave heating). The samples of spruce, pine and birch sawdust were treated with either 1-Ethyl-3-methylimidazolium chloride, Emim Cl, or 1-Ethyl-3-methylimidazolium acetate, Emim Ac, (or with ionized water for comparison) at various temperatures (where focus was between 80 and 120 °C). The samples were withdrawn at fixed time intervals (the main interest treatment time area lied between 0 and 100 hours). Double experiments were executed. The selected mono- and disaccharides, as well as their known degradation products, 5-hydroxymethylfurfural, 5-HMF, and furfural were analyzed with capillary electrophoresis, CE, and high-performance liquid chromatography, HPLC. Initially, even GC and GC-MS were utilized. Galactose, glucose, mannose and xylose were the main monosaccharides that were present in the wood samples exposed to ILs at elevated temperatures; in addition, furfural and 5-HMF were detected; moreover, the quantitative amount of the two latter ones were naturally increasing in line with the heating time or the IL:wood ratio.

Genetic engineering of baker's and wine yeasts using formaldehyde hyperresistance-mediating plasmids

Yeast multi-copy vectors carrying the formaldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker's and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use

The Role of Oxidative Stress in Environmental Responses of Fennoscandian Animals

All aerobic organisms have to deal with the toxicity of oxygen. Oxygen enables more efficient energy production compared to anaerobic respiration or fermentation, but at the same time reactive oxygen species (ROS) are being formed. ROS can also be produced by external factors such as UV-radiation and contamination. ROS can cause damage to biomolecules such as DNA, lipids and proteins and organisms try to keep the damage as small as possible by repairing biomolecules and metabolizing ROS. All ROS are not harmful, because they are used as signaling molecules. To cope against ROS organism have an antioxidant (AOX) system which consists both enzymatic and non-enzymatic AOX defense. Some AOX are produced by the organism itself and some are gained via diet. In this thesis I studied environmentally caused changes in the redox regulation of different wild vertebrate animals to gain knowledge on the temporal, spatial and pollution-derived-effects on the AOX systems. As study species I used barn swallow, ringed seal and the Baltic salmon. For the barn swallow the main interest was the seasonal fluctuation in the redox regulation and its connection to migration and breeding. The more contaminated ringed seals of the Baltic Sea were compared to seals from cleaner Svalbard to investigate whether they suffered from contaminant induced oxidative stress. The regional and temporal variation in redox regulation and regional variation in mRNA and protein expressions of Baltic salmon were studied to gain knowledge if the salmon from different areas are equally stressed. As a comparative aspect the redox responses of these different species were investigated to see which parts of the AOX system are substantial in which species. Certain parts of AOX system were connected to breeding and others to migration in barn swallows, there was also differences in biotransformation between birds caught from Africa and Finland. The Baltic ringed seal did not differ much from the seals from Svalbard, despite the difference in contaminant load. A possible explanation to this could be the enhanced AOX mechanisms against dive-associated oxidative stress in diving air-breathing animals, which also helps to cope with ROS derived from other sourses. The Baltic salmon from Gulf of Finland (GoF) showed higher activities in their AOX defense enzymes and more oxidative damage than fish from other areas. Also on mRNA and proteomic level, stress related metabolic changes were most profound in in the fish from GoF. Mainly my findings on species related differences followed the pattern of mammals showing highest activities and least damage and birds showing lower activities and most damage, fish being intermediate. In general, the glutathione recycling-related enzymes and the ratio of oxidized and reduced glutathione seemed to be the most affected parameters in all of the species.

Production, isolation and characterization of bioactive peptides with antihypertensive properties from rapeseed and potato protein

Consumers’ increasing awareness of healthiness and sustainability of food presents a great challenge to food industry to develop healthier, biologically active and sustainable food products. Bioactive peptides derived from food proteins are known to possess various biological activities. Among the activities, the most widely studied are antioxidant activities and angiotensin I converting enzyme (ACE) inhibitory activity related to blood pressure regulation and antihypertensive effects. Meanwhile, vast amounts of byproducts with high protein content are produced in food industry, for example potato and rapeseed industries. The utilization of these by-products could be enhanced by using them as a raw material for bioactive peptides. The objective of the present study was to investigate the production of bioactive peptides with ACE inhibitory and antioxidant properties from rapeseed and potato proteins. Enzymatic hydrolysis and fermentation were utilized for peptide production, ultrafiltration and solid-phase extraction were used to concentrate the active peptides, the peptides were fractionated with liquid chromatographic processes, and the peptides with the highest ACE inhibitory capacities were putified and analyzed with Maldi-Tof/Tof to identify the active peptide sequences. The bioavailability of the ACE inhibitory peptides was elucidated with an in vitro digestion model and the antihypertensive effects in vivo of rapeseed peptide concentrates were investigated with a preventive premise in 2K1C rats. The results showed that rapeseed and potato proteins are rich sources of ACE inhibitory and antioxidant peptides. Enzymatic hydrolysis released the peptides effectively whereas fermentation produced lower activities.The native enzymes of potato were also able to release ACE inhibitory peptides from potato proteins without the addition of exogenous enzymes. The rapeseed peptide concentrate was capable of preventing the development of hypertension in vivo in 2K1C rats, but the quality of rapeseed meal used as raw material was found to affect considerably the antihypertensive effects and the composition of the peptide fraction.

High pressure-sensitive gene expression in Lactobacillus sanfranciscensis

Lactobacillus sanfranciscensis is a Gram-positive lactic acid bacterium used in food biotechnology. It is necessary to investigate many aspects of a model organism to elucidate mechanisms of stress response, to facilitate preparation, application and performance in food fermentation, to understand mechanisms of inactivation, and to identify novel tools for high pressure biotechnology. To investigate the mechanisms of the complex bacterial response to high pressure we have analyzed changes in the proteome and transcriptome by 2-D electrophoresis, and by microarrays and real time PCR, respectively. More than 16 proteins were found to be differentially expressed upon high pressure stress and were compared to those sensitive to other stresses. Except for one apparently high pressure-specific stress protein, no pressure-specific stress proteins were found, and the proteome response to pressure was found to differ from that induced by other stresses. Selected pressure-sensitive proteins were partially sequenced and their genes were identified by reverse genetics. In a transcriptome analysis of a redundancy cleared shot gun library, about 7% of the genes investigated were found to be affected. Most of them appeared to be up-regulated 2- to 4-fold and these results were confirmed by real time PCR. Gene induction was shown for some genes up-regulated at the proteome level (clpL/groEL/rbsK), while the response of others to high hydrostatic pressure at the transcriptome level seemed to differ from that observed at the proteome level. The up-regulation of selected genes supports the view that the cell tries to compensate for pressure-induced impairment of translation and membrane transport.

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

Naudanlanta biokaasulaitosraaka-aineena, käsittely ja logistiikka

Maatalouden lietelannasta saatavien ravinteiden talteenotto ja kierrätys ovat viime aikoina nousseet yhä tärkeämmäksi tavoitteeksi. Ravinteiden tehokas kierrätystapa saadaan ai-kaiseksi käsittelemällä lietelantaa. Lannan käsittelyketjuja on monenlaisia; tässä työssä naudan lannan käsittelyketju muodostuu separoinnista ja mädätyksestä. Tämän diplomityön tarkoituksena oli selvittää naudan lannan mekaanisen separoinnin ja logistiikan kannattavuus märkämädätys- ja kuivamädätyskäsittelyketjussa. Käsittelyketju-jen kannattavuus tai kannattamattomuus on esitetty eri skenaarioissa muodostettujen kus-tannuslaskentojen perusteella. Skenaarioiden kustannuslaskelmat ovat toisiinsa verratta-vissa, jolloin ne voidaan asettaa niin ikään paremmuusjärjestykseen. Viides skenaario ei ole suoraan verrattavissa muihin skenaarioihin, koska siinä lannankäsittelyketjua on tar-kasteltu eri näkökulmasta, keskittämällä suurimmalle maatilalle. Mekaanisista separaattoreista tarkasteltiin työssä ruuvipuristinta, dekantterilinkoa ja ul-koistettua separointipalvelua. Lannan logistiikkakustannuksissa huomioitiin lannan kulje-tusta traktori- ja säiliöautolla tai traktorilla. Separoinnin kustannus liitettiin osaksi logis-tiikkakustannusta. Työn tuloksien perusteella voidaan todeta dekantterilingon separointi-kustannuksen olevan taloudellisesti kannattamattomin johtuen pienestä lantakapasiteetista ja separaattorin korkeasta investointihinnasta. Ravinteiden osalta linko osoittautui tehok-kaimmaksi menetelmäksi erottamaan ravinteet eri jakeisiin. Lannan käsittely separointipalvelulla oli edullisin skenaario, jonka vuosittaiseksi kustan-nukseksi saatiin säiliö- ja kuorma-autolla lantaa kuljetettaessa 58 700 € ja traktorilla kul-jetettaessa 60 400 €. Kallein lannan käsittely oli dekantterilingolla, jonka vuosittaiseksi kustannukseksi saatiin lantaa säiliö- ja kuorma-autolla kuljetettaessa 121 000 € ja trakto-rilla kuljetettaessa 123 300 €. Lannan separointi maatilalla osoittautui kannattavammaksi kuin käsittelemättömän lietteen kuljetus biokaasulaitokselle.

Advanced biofuel production: Engineering metabolic pathways for butanol and propane biosynthesis.

Greenhouse gases emitted from energy production and transportation are dramatically changing the climate of Planet Earth. As a consequence, global warming is affecting the living conditions of numerous plant and animal species, including ours. Thus the development of sustainable and renewable liquid fuels is an essential global challenge in order to combat the climate change. In the past decades many technologies have been developed as alternatives to currently used petroleum fuels, such as bioethanol and biodiesel. However, even with gradually increasing production, the market penetration of these first generation biofuels is still relatively small compared to fossil fuels. Researchers have long ago realized that there is a need for advanced biofuels with improved physical and chemical properties compared to bioethanol and with biomass raw materials not competing with food production. Several target molecules have been identified as potential fuel candidates, such as alkanes, fatty acids, long carbon‐chain alcohols and isoprenoids. The current study focuses on the biosynthesis of butanol and propane as possible biofuels. The scope of this research was to investigate novel heterologous metabolic pathways and to identify bottlenecks for alcohol and alkane generation using Escherichia coli as a model host microorganism. The first theme of the work studied the pathways generating butyraldehyde, the common denominator for butanol and propane biosynthesis. Two ways of generating butyraldehyde were described, one via the bacterial fatty acid elongation machinery and the other via partial overexpression of the acetone‐butanol‐ethanol fermentation pathway found in Clostridium acetobutylicum. The second theme of the experimental work studied the reduction of butyraldehyde to butanol catalysed by various bacterial aldehyde‐reductase enzymes, whereas the final part of the work investigated the in vivo kinetics of the cyanobacterial aldehyde deformylating oxygenase (ADO) for the generation of hydrocarbons. The results showed that the novel butanol pathway, based on fatty acid biosynthesis consisting of an acyl‐ACP thioesterase and a carboxylic acid reductase, is tolerant to oxygen, thus being an efficient alternative to the previous Clostridial pathways. It was also shown that butanol can be produced from acetyl‐CoA using acetoacetyl CoA synthase (NphT7) or acetyl‐CoA acetyltransferase (AtoB) enzymes. The study also demonstrated, for the first time, that bacterial biosynthesis of propane is possible. The efficiency of the system is clearly limited by the poor kinetic properties of the ADO enzyme, and for proper function in vivo, the catalytic machinery requires a coupled electron relay system.

Karboksyylihappojen kromatografinen fraktiointi

Suurin osa alifaattisista karboksyylihapoista tuotetaan nykyään synteettisesti, mutta öljyn hinnan nousu ja ekologisempi ajattelutapa on aiheuttanut kiinnostusta tuottaa näitä karboksyyli- ja hydroksihappoja jatkossa fermentoimalla tai sellun valmistuksen sivuvirtana syntyvästä mustalipeästä. Nykyään mustalipeä poltetaan sellaisenaan soodakattiloissa keittokemikaalien regeneroimiseksi, energiaksi ja sähköksi. Jatkossa mustalipeästä voisi erottaa arvokkaat orgaaniset hapot ennen polttamista. Saadusta happoseoksesta tulisi erottaa yksittäiset alifaattiset karboksyylihapot toisistaan jatkojalostusta varten. Tämän kandidaatintyön tavoitteena oli selvittää, millä kromatografisella erotusmenetelmällä fermentointituotteina ja teollisuuden sivuvirtoina syntyvistä karboksyylihapposeoksista saadaan yksittäiset alifaattiset karboksyylihapot erotettua toisistaan. Mittaukset suoritettiin kolonnilla, jossa hartsipedin halkaisija oli 1,5 cm ja korkeus 15 cm. Kolonnin erototusmateriaaleina kokeiltiin vahvoja ja heikkoja kationinvaihtohartseja, vahvaa anioninvaihtohartsia ja polymeerisiä adsorbentteja. Erotettavaksi happoseokseksi valittiin sitruuna-, viini-, glykoli-, maito- ja etikkahapon seos. Tehokkain erotus saatiin Puroliten valmistamalla Macronet 270:lla, joka on mikrohuokoinen polymeerinen adsorbentti. Macronet 270:lla saatiin erotettua erityisesti viini- ja glykolihappo sitruuna-, maito- ja etikkahaposta. Yksittäisiä happoja ei saatu kuitenkaan kunnolla erotettua. Parhaat koeolosuhteet erotustehokkuuden ja retentioaikojen kannalta saatiin vesieluentin virtausnopeudella 2 mL/min, syöttöpulssin tilavuudella 5 mL ja kolonnin lämpötilassa 75 °C.

Simultaneous allergen inactivation and detoxification of castor bean cake by treatment with calcium compounds

Ricinus communis L. is of great economic importance due to the oil extracted from its seeds. Castor oil has been used for pharmaceutical and industrial applications, as a lubricant or coating agent, as a component of plastic products, as a fungicide or in the synthesis of biodiesel fuels. After oil extraction, a castor cake with a large amount of protein is obtained. However, this by-product cannot be used as animal feed due to the presence of toxic (ricin) and allergenic (2S albumin) proteins. Here, we propose two processes for detoxification and allergen inactivation of the castor cake. In addition, we establish a biological test to detect ricin and validate these detoxification processes. In this test, Vero cells were treated with ricin, and cell death was assessed by cell counting and measurement of lactate dehydrogenase activity. The limit of detection of the Vero cell assay was 10 ng/mL using a concentration of 1.6 x 10(5) cells/well. Solid-state fermentation (SSF) and treatment with calcium compounds were used as cake detoxification processes. For SSF, Aspergillus niger was grown using a castor cake as a substrate, and this cake was analyzed after 24, 48, 72, and 96 h of SSF. Ricin was eliminated after 24 h of SSF treatment. The cake was treated with 4 or 8% Ca(OH)2 or CaO, and both the toxicity and the allergenic properties were entirely abolished. A by-product free of toxicity and allergens was obtained.

Organic acid profile of commercial sour cassava starch

Organic acids are present in sour cassava starch ("polvilho azedo") and contribute with organoleptic and physical characteristics like aroma, flavor and the exclusive baking property, that differentiate this product from the native cassava starch. Samples of commercial sour cassava starch collected in South and Southeast Brazil were prepared for high performance liquid chromatography (HPLC) analysis. The HPLC equipment had a Biorad Aminex HPX-87H column for organic acid analysis and a refractometric detector. Analysis was carried out with 0.005M sulfuric acid as mobile phase, 0.6ml/min flow rate and column temperature of 60° C. The acids quantified were lactic (0.036 to 0.813 g/100g), acetic (0 to 0.068 g/100g), propionic (0 to 0.013 g/100g) and butyric (0 to 0.057 g/100g), that are produced during the natural fermentation of cassava starch. Results showed large variation among samples, even within the same region. Some samples exhibited high acid levels, mainly lactic acid, but in these neither propionic nor butyric acids were detected. Absence of butyric acid was not expected because this is an important component of the sour cassava starch aroma, and the lack of this acid may suggest that such samples were produced without the natural fermentation step.

Effects of nickel on the mineral composition of Fleischmann’s yeast (Saccharomyces cerevisiae)

Sugar cane juice containing 12% (w.w -1) of total reducing sugars and 0.0 to 5.0 mmol of nickel L-1, with pH ranging from 3.5 to 6.5, was inoculated with Fleischmann’s yeast (Saccharomyces cerevisiae) (10% w.w -1). Six hours after fermentation, the yeast’s cellular viability and trehalose content were evaluated. The resulting must was centrifuged and the raw yeast was analyzed by atomic absorption spectroscopy to evaluate the intracellular levels of calcium, copper, iron, magnesium, manganese, nickel, phosphorus, potassium, sulfur and zinc. The intracellular levels of iron, magnesium and calcium were affected and the yeast’s susceptibility to nickel was enhanced by the decrease in pH. The yeast’s growth was not affected by nickel at high pH, but the toxic effects of nickel were potentiated at low pH.

Lactobacillus plantarum AJ2 isolated from naturally fermented sausage and its effects on the technological properties of Milano-type salami

L. plantarum, strain AJ2, was isolated from naturally fermented sausage manufactured in the Southern region of Brazil and inoculated in Milano-type salami. The lactic culture exhibited the ability of growing in the product, decreasing pH in the first seven days of fermentation/maturation and the residual levels of nitrite and nitrate, as well as peroxide and TBARS values. The inoculated sausage had the highest intensity for brightness and red color, but did not present a difference in chemical composition and fatty acid composition, compared to the control.

Lactobacillus plantarum strains isolated from naturally fermented sausages and their technological properties for application as starter cultures

In the present study, technological properties of L. plantarum strains isolated from naturally fermented sausages manufactured in the South region of Brazil were investigated in order to obtain a starter culture. The technological properties evaluated were the following: ability to growth at different pH values, at different temperatures, in different salt concentrations and in the presence of commercial curing salt, fast production of acid, determination of D - and L - lactic acid; nitrate reductase activity; antagonistic activity and stability of the isolated cultures after fermentation, concentration, and freeze-drying process. The isolated strains showed effectiveness to improve technological properties as starter cultures.