De nombreux problèmes ont été identifiés lors des tentatives d’incorporation de protéines végétales dans nos aliments. En particulier, les faux goûts, le goût et la texture ont été mis en évidence comme de véritables obstacles à l’acceptabilité des produits végétaux par les consommateurs. Le consommateur lui-même est aussi un déterminant important en ce qui concerne le terme « acceptabilité ». Donc, dans le but de développer un nouveau produit fermenté à base de protéines de pois deux volets ont été étudié : le produit et le consommateur. Afin de comprendre la physico-chimie et l’acceptabilité de produits fermentés à base de protéines de pois, plusieurs facteurs ont été étudiés dans des étapes successives tels que la culture, les allégations positives sur la santé et l’environnement, les cocktails de souches lactiques et les procédés de préparation. Dès les premiers tests sensoriels, il était clair qu’il ne serait pas facile de convaincre les consommateurs de consommer des produits fermentés à base de pois : les consommateurs français n’acceptent pas les produits même à une faible concentration de pois (10 %). Ni la familiarité pour un produit proche de nos produits étudiés, ni l’encouragement des consommateurs à accepter ce type d’aliment en passant des messages positifs sur les protéines végétales n’étaient assez efficaces. Ainsi, une deuxième série d’études a été réalisée afin d’optimiser les propriétés rhéologiques et organoleptiques de ces produits. La meilleure combinaison, 1- cocktail bactérien (S. thermophilus + Lb. bulgaricus) 2- matière première (globuline de pois isolée dans notre laboratoire) 3- paramètres de préparation (mélange des deux laits avant le traitement thermique à 90 °C) a été sélectionnée afin d’optimiser les produits fermentés en ce qui concerne l’acidité, la fermeté, les profils volatils et peptidiques. Enfin, du point de vue sensoriel, une légère amélioration de l’acceptabilité a été remarquée. 20 % de protéines de pois a donné un produit accepté par la plupart des consommateurs, et 40 % de protéines de pois a été évaluée positivement par certains consommateurs et négativement par d'autres. / Many problems have been identified following the attempts to incorporate vegetal proteins in our food. In particular, off-flavor, taste or texture have been highlighted as real barriers to the acceptability of plant products by consumers. The consumer himself is also an important determinant regarding the term "acceptability." So, in order to develop a new fermented product based on pea, two issues were studied: the product and the consumer. To understand the physico-chemical properties and the acceptability of pea protein-based fermented products, several factors have been investigated in successive stages such as culture, positive health and environmental claims, lactic acid bacteria strains and preparation processes. From the first sensory tests, it was clear that it will not be easy to convince consumers to buy the pea-based fermented product: French consumers did not accept products even in a lower concentration of pea (10%). Neither the familiarity to close products nor encouraging consumers to accept this type of food by transmitting positive messages about vegetal protein were efficient enough. Thus, a second series of studies was carried out in order to optimize the rheologic and organoleptic properties of these products. Best combination of 1- starter culture (S. thermophilus + Lb. bulgaricus) 2- raw material (pea globulin isolated in our laboratory) 3- preparation parameters (mixing both milk before heat treatment at 90 ° C) were selected to optimize the fermented products in terms of volatile compounds and peptide profiles, acidity and firmness. Finally, from the sensory point of view, a slight improvement in the acceptability was noticed. 20% of pea protein gave a product accepted by most of the consumers, and 40% of pea protein was assessed positively by some consumers and negatively by others

Les bactéries lactiques sont utilisées pour la fermentation d'aliments dans le but d'augmenter leur durée de conservation et d'améliorer leurs propriétés organoleptiques et nutritionnelles. Dans le but de diversifier l'offre d'aliments fonctionnels et de limiter les pertes en produit frais, nous proposons des produits fermentés à base de fruits ou légumes, riches en antioxydants et plaisants pour le consommateur. Pour cela, mes travaux de thèse ont été menés en deux étapes : caractériser la flore bactérienne lactique présente à la surface de fruits et de légumes cultivés à La Réunion, puis sélectionner des bactéries autochtones possédant certaines propriétés fonctionnelles. Ainsi, 77 bactéries lactiques isolées de papayes, de tomates et d'achards de la Réunion, appartenant aux genres Leuconostoc, Lactococcus, Weissella, Lactobacillus et Fructobacillus ont été caractérisées génétiquement et phénotypiquement. Parmi la grande diversité observée, certains isolats ont présenté des caractéristiques technologiques (conditions et vitesse de croissance, résistance aux stress environnementaux) et fonctionnelles (production d'exo-polysaccharides) intéressantes pour l'élaboration d'aliments fermentés, en particulier les isolats Weissellla cibaria 64 et 30, Leuconostoc pseudomesenteroides 60 et Lactobacillus plantarum 75. A partir de ces données, des étapes complémentaires de criblage ont été réalisées sur différents substrats (mangue, papaye, ananas, thé vert, thé noir) fermentés, en examinant leurs propriétés organoleptiques et leurs activités antioxydantes. Deux isolats, Lc. pseudomesenteroides 12b et W. cibaria 64, ont augmenté significativement la teneur en composés phénoliques et l'activité antioxydante de jus d'ananas Victoria au cours d'une fermentation de 48h. Le produit fermenté se conserve 16 jours au froid sans aucune altération et conserve l'ensemble de ses bénéfices nutritionnels. Une odeur et des goûts typiques ont été détectés dans les boissons fermentées obtenues. L'augmentation de l'activité antioxydante observée au cours de la fermentation résulte probablement du métabolisme des composés phénoliques par les bactéries. L'identification des molécules produites et des enzymes impliquées est nécessaire afin de comprendre les mécanismes mis en jeu. / Lactic acid bacteria are used for the production of large variety of fermented foods in order to enhance shelf-life together with improved organoleptic and nutritional properties. In order to create new functional foods and to reduce fresh product waste, the objectives of my PhD were to develop and characterize fruit or vegetables fermented foods, rich in antioxidants and pleasant for consumers. To reach these goals, my PhD work was split in two steps: 1- characterization of lactic bacteria present on fruits and vegetables grown at Reunion Island and 2- selection of autochthonous bacteria with functional properties. Thus, 77 lactic bacteria from genera Leuconostoc, Lactococcus, Weissella, Lactobacillus and Fructobacillus were isolated from papaya, tomato and sliced cabbage from Reunion Island. They were genetically and phenotypically characterized. A huge diversity in term of genetic and phenotypic characteristics was determined. Furthermore, several isolates exhibiting specific technological and functional properties (growth rate, resistance to environmental stress, production of exopolysaccharides) were identified. These isolates, potentially useful for the production of fermented foods, were Weissella cibaria 64 and 30, Leuconostoc pseudomesenteroides 60 and Lactobacillus plantarum 75. Then, further screening steps were performed on different food substrates (pineapple, mango, papaya, tea infusions) in order to select isolates able to improve antioxidant and organoleptic properties. Two isolates, W. cibaria 64 and Lc. pseudomesenteroides 12b were shown to significantly enhance the phenolic content and the antioxidant activity of Victoria pineapple juice after a 48h-fermentation. The fermented products exhibited a good shelf-life of 16 days without alteration and preserved nutritional benefits. Characteristic odour and tastes were detected in the fermented drinks. The increase in antioxidant activity observed during fermentation was probably the consequence of a depolymerization of phenolic compounds. Further works are required to identify the composition changes over fermentation and to reach a better understanding of the mechanisms involved.

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第20479号 / エネ博第348号 / 新制||エネ||69(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー社会・環境科学専攻 / (主査)教授 坂 志朗, 教授 梅澤 俊明, 准教授 河本 晴雄 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

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Soft sensors have been suggested as potent tools for on-line estimations of critical bioprocess variables to be able to control the biological process in an as high extent as possible. The formation of inhibitory by-products in the form of organic acids, caused by an overflow of glucose, is a problem in most bioprocesses expressing recombinant proteins.    In this project a new method of controlling the glucose feeding in an Escherichia coli fed-batch cultivation expressing the green fluorescent protein (GFP) was investigated. The new controller system implemented in the software controlled the feed rate based on on-line HPLC measurements of the concentration of organic acids.      The results showed that the controller managed to down-regulate the inhibitory organic acids to a low level as it tried to keep the glucose uptake rate at an optimum for maximum cell growth. The results suggested that the controller could be a powerful tool to create a more secure reproducibility and to generate high product yields in recombinant protein productions.

<p>Pivski trop čini približno 85% od ukupnih sporednih proizvoda proizvodnje piva, i dostupan je po veoma niskim cenama tokom čitave godine. Pivski trop ima veliku perspektivu za primenu u biotehnologiji i proizvodnji visoko vrednih proizvoda. Jedna od veoma ekolo&scaron;ki i ekonomski isplativih alternativa je upotreba pivskog tropa u proizvodnji mlečne kiseline, jer se poslednjih par decenija uočava intenzivan rast potražnje za mlečnom kiselinom. Mlečna kiselina je najvažnija hidroksikarbonska kiselina &scaron;iroko rasprostranjena u prirodi, sa velikom primenom u prehrambenoj, farmaceutskoj, tekstilnoj i hemijskoj industriji i industriji prerade kože.<br />Cilj istraživanja ove doktorske disertacije je ispitivanje primene pivskog tropa u proizvodnji mlečne kiseline. Prvo je izvr&scaron;ena optimizacija enzimske hidrolize pivskog tropa u cilju dobijanja &scaron;to je moguće veće koncentacije redukujućih &scaron;ećera neophodne za mlečno-kiselu fermentaciju. Hidrolizat pivskog tropa je dobijen enzimskom hidrolizom dodatkom komercijalnih enzima za razgradnju skroba i celuloze. Parametri čiji je uticaj na efikasnost enzimske hidrolize ispitanu su: pH vrednost, temperatura hidrolize i količina dodatih enzima. Nakon &scaron;to su određeni najbolji uslovi razgranje pivskog tropa, dobijeni postupak hidrolize je primenjen u proizvodnji hidrolizata pivskog tropa koji je kori&scaron;ćen u mlečno-kiselim fermentacijama.<br />Nakon toga je ispitana mlečno-kisela fermentacija sa dva proizvodna mikoorganizma. Kao proizvodni mikroorganizmi u mlečno-kiselim fermentacijama primenjena su dva soja bakterija mlečne kiseline: Lactobacillus fermentum PL-1 i Lactobacillus rhamnosus ATCC 7469. Ispitan je uticaj dodatka različitih koncentracija ekstrakta kvasca (0,5-5,0%) uz korekciju pH vrednosti tokom fermentacije sa dodatkom kalcijum-karbonata. U zavisnosti od udela L-(+)- i D-(-)-mlečne kiseline koje nastaju tokom fermentacije izabran je proizvodni mikroorganizam koji proizvodi vi&scaron;e L-(+)-mlečne kiseline.<br />U daljim ispitivanjima je ispitan uticaj korekcije pH pomoću natrijum-hidroksida kao i dodatak različitih koncentracija ekstrakta kvasca (0,5-5,0%) i redukujućih &scaron;ećera (2,7; 5,4 i 8,1%) u hidrolizatu pivskog tropa na mlečno-kiselu fermentaciju pomoću odabranog soja bakterija mlečne kiseline. Na osnovu dobijenih rezultata izabrana je najbolja koncentracija redukujućih &scaron;ećera i ekstrakta kvasca koji će se koristiti u daljim istraživanjima.<br />Takođe je ispitana i mogućnost zamene skupog ekstrakta kvasca i glukoze sa obnovljivim sirovinama, kao &scaron;to su pivski kvasac, džibra i bistra džibra.<br />Ispitan je uticaj dodatka različitih koncentracija pivskog kvasca (0,5-5,0%), džibre (5-20%) i bistre džibre (5-50%) pre fermentacije kao i dodatak bistre džibre u dolivnoj fermentaciji, na mlečno-kiselu fermentaciju hidrolizata pivskog tropa.<br />Ispitan je i dolivni postupak fermentacije hidrolizata pivskog tropa dodatkom glukoze, glukoze i ekstrakta kvasca i sladovine. Takođe je ispitana mogućnost izvođenja vi&scaron;e uzastopnih fermentacija sa imobilisanim ćelijama odabranog soja bakterija mlečne kiseline u kalcijum-alginatu.<br />Na osnovu eksperimentalnih rezultata zaključujeno je da je dodatak kalcijum-karbonata imao pozitivan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. Sa dodatkom kalcijum-karbonata povećali su se utro&scaron;ak redukujućih &scaron;ećera, koncentracija i prinos mlečne kiseline i vijabilnost ćelija L. fermentum i L. rhamnosus. Ekstrakt kvasca i kalcijum-karbonat su imali značajan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. U fermentacijama sa L. fermentum najveći prinos ukupne mlečne kiseline (44%) je postignut sa dodatkom 5,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. U fermentacijama sa L. rhamnosus najveći prinos ukupne mlečne kiseline (98%) i L-(+)-mlečne kiseline (96%) je ostvaren u fermentaciji sa dodatkom 2,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. Na osnovu rezultata odlučeno je da se u daljim ispitivanjima mlečno-kisele fermentacije hidrolizata pivskog tropa kao proizvodni mikoorganizam koristi L. rhamnosus.<br />Primenom natrijum-hidroksida za korekciju pH je skratila fermentaciju za 48 sati a ostvareno je i značajno povećanje zapreminske produktivnosti L-(+)-mlečne kiseline (za 200%, povećanje sa 0,21 na 0,63 g/l&middot;h-1). Korekcija pH u svim daljim istraživanjima je vr&scaron;ena sa dodatkom natrijum-hidroksida.<br />U mlečno-kiselim fermentacijama sa različitim početnim koncentracijama redukujućih &scaron;ećera (2,7; 5,4 i 8,1%) i sa dodatkom različitih koncentracija ekstrakta kvasca (0,5-5,0%), najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 91,29% i 1,69 g/l&middot;h-1, kao i vijabilnost ćelija L. rhamnosus od 9,7&middot;109 CFU/ml ostvareni su u fermentaciji sa početniom koncentracijom redukujućih &scaron;ećera od 5,4% i dodatkom 5,0% ekstrakta kvasca.<br />Na osnovu ostvarenih rezultata u istraživanjima sa dodatkom džibre i dodacima tokom fermentacije kao i u fermentacijama sa imobilisanim ćelijama je kori&scaron;ćen hidrolizat pivskog tropa sa početnom koncentracijom redukujućih &scaron;ećera od 5,4%.<br />U mlečno kiseloj fermentaciji sa dodatkom pivskog kvasca najveći prinos L-(+)-mlečne kiseline (89,01%) i zapreminska produktivnost (0,89 g/l&middot;h-1) L-(+)-mlečne kiseline su ostvareni u fermentaciji sa dodatkom 5,0% pivskog kvasca i korekcijom početne koncentracije redukujućih &scaron;ećera na 5,0%. Na osnovu rezultata utvrđeno je da se može izvr&scaron;iti delimična ili potpuna zamena ekstrakta kvasca pivskim kvascem uz značajno smanjenje cene podloge za mlečno-kiselu fermentaciju, bez značajnog smanjenja efikasnosti mlečno-kisele fermentacije.<br />U mlečno-kiseloj fermentaciji sa dodatkom džibre i bistre džibre najveć koncetracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline od 31,03 g/l, 86,15% i 0,93 g/l&middot;h-1, ostvareni su u fermentaciji sa dodatkom 50% bistre džibre. Najvi&scaron;a koncentracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline ostvareni u dolivnoj fermentaciji sa dodatkom glukoze i bistre džibre tokom mlečno-kisele fermentacije su iznosili su 48,02 g/l, 87,82% i 0,96 g/l&middot;h-1.<br />U fermentacijama sa dodatkom nutritijenata tokom mlečno-kisele fermentacije najveća vrednost koncetracije, prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline od 116,08 g/l, 93,32% i 2,04 g/L&middot;h-1, su ostvarene u fermentaciji sa dodatkom glukoze i ekstrakta kvasca tokom fermentacije. Na osnovu rezultata utvrđeno je da se dolivni postupak fermentacije može koristiti u cilju povećanja efikasnosti mlečno-kisele fermentacije.<br />Izvr&scaron;ena je imobilizacija ćelija L. rhamnosus u kalcijum-alginatu uz izuzetno visoku vijabilnost (1010 CFU/ml). Imobilisane ćelije L. rhamnosus su uspe&scaron;no kori&scaron;ćene u tri mlečno-kisele fermentacije. Prinos L-(+)-mlečne kiseline i zapreminska produktivnost su u sve tri fermentacije bili izuzetno visoki, pri čemu su najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 95,2% i 1,76 g/l&middot;h-1, ostvareni u drugoj fermentaciji. Upotrebom imobilisanih ćelija L. rhamnosus je osim povećanja prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline skraćena fermentacija za 12 sati u poređenju sa &scaron;aržnim fermentacijama.</p> / <p>Brewers spent grain represents (BSG) about 85% of the total by-products from brewing process and is available at low price during the whole year. Due to its chemical composition BSG has great potential use in biotechnology and production of high-value products. One of very eco-friendly and economical alternative uses of BSG is in production of lactic acid (LA), since in the last few decades the demand for the LA has significantly risen, mostly because of development of biodegradable lactic polymers, which are eco-friendly and nontoxic.<br />Lactic acid is the most important hydrocarboxylic acid with an asymmetrical carbon atom, widely distributed in nature, and it has shown great potential in fields of food, pharmaceutical, textile, leather and chemical industries.<br />The aim of this doctoral thesis was to investigate the application of BSG in lactic acid production. First, the optimization of enzymatic hydrolysis of BSG was conducted, with the goal to achieve high reducing sugar concentrations, as much as possible, that are necessary on LA fermentation. BSG hydrolysis was conducted by usage of commercial enzymes for degradation of starch and cellulose. Effect of pH value, temperature and enzyme dosage on BSG hydrolysis efficiency was investigated. After the best conditions for BSG hydrolysis were determined, the optimized procedure for BSG hydrolysis was used for the production of BSG hydrolysate that will be used in LA fermentations.<br />After optimization of BSG hydrolysis, LA fermentation by two LA producing microorganisms was investigated. The strains investigated were two LA bacteria strains: Lactobacillus fermentum PL-1 and Lactobacillus rhamnosus ATCC 7469. The effect of yeast extract (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%) addition in BSG hydrolysate, with the correction of pH value during LA fermentation by the addition of calcium-carbonate, on LA fermentation was investigated. Based on the results achieved for L-(+)- and D-(-)-LA ratio the LAB strains that produced more L-(+)-LA was chosen for further research.<br />In further research the effect of pH correction (with addition of NaOH), yeast extract (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0%) addition and reducing sugar concentration (2.7; 5.4 and 8.1%) in BSG hydrolysate on LA fermentation was investigated. Based on the results achieved the best yeast extract and reducing sugars concentrations was determined and used in further analysis or research. Also the possible replacement of expensive yeast extract and glucose with cheap alternatives, like brewer`s spent grain and stillage was investigated. The effect of brewer`s spent grain (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%), whole stillage (5, 10, 15 i 20%) and thin stillage (5, 10, 15, 20, 30, 40, 50%) addition before fermentation as well as thin stillage addition in fed-batch fermentation in BSG hydrolysate on LA fermentation were investigated.<br />Also fed-batch fermentation procedure (addition of glucose, glucose and yeast extract and wort during fermentation) was investigated. The possible application of cells immobilized in Ca-alginate for LA fermentation of BSG hydrolysate was also investigated.<br />Based on the results it was concluded that BSG can be successfully utilized as a raw material in production of LA, after optimization of hydrolysis and addition of nitrogen source.<br />According to the results of chemical composition before and after optimized hydrolysis 78.6% of total cellulose was hydrolyzed.<br />Addition of calcium-carbonate had positive effect on LA production by L. fermentum i L. rhamnosus. With the addition of calcium-carbonate reducing sugar utilization, LA yield and concentration and cell viability (both L. fermentum i L. rhamnosus) increased. Addition of calcium-carbonate and yeast extract had a positive effect on LA fermentation by L. fermentum and L. rhamnosus. In LA fermentation by L. fermentum the highest LA yield (44%) was achieved with addition of 5.0% of yeast extract and 2.0% of calcium-carbonate. In L. rhamnosus fermentations the highest total LA yield (98%) and L-(+)-LA yield (96%) was reached when 2.0% of yeast extract and 2.0% of calcium-carbonate were added.<br />Based on the results achieved it was concluded that BSG hydrolysate, with the addition of yeast extract, is a good fermentation media for LA fermentation with L. rhamnosus, and it was decided that L. rhamnosus will be used in further research of LA fermentation on BSG hydrolysate.<br />Addition of NaOH instead of calcium-carbonate for the pH correction shortened the fermentation time by 48 h and increased the L-(+)-LA volumetric productivity (by 200%, from 0.21 to 0.63 g/L&middot;h-1). Based on this results pH correction in further experiments was done by addition of NaOH.<br />In LA fermentation with different reducing sugar (2.7, 5.4 and 8.1%) and yeast extract concentrations (0.5-5.0%), the highest L-(+)-LA yield and volumetric productivity of 91.29%, and 1.69 g/L&middot;h-1, respectively, as well as L. rhamnosus cell viability (9.67 log CFU/mL), were achieved with the reducing sugar content of 5.4% and yeast extract content of 5.0%.<br />Based on this results in further experiment with the addition of stillage, in fed-batch fermentation and fermentation with immobilized cell BSG hydrolysate with 5.4% of reducing sugars and 5.0% yeast extract was used.<br />In fermentation with the addition of brewer&rsquo;s spent yeast the highest L-(+)-LA yield (89.01%) and volumetric productivity (0.89 g/L&middot;h-1) were achieved in the fermentation of BSG hydrolysate with 5.0% of reducing sugar and 5.0% of brewer&rsquo;s yeast. Based on the results achieved it was concluded that yeast extract can be partial or complete replaced by brewer&rsquo;s spent yeast with significant decrease of media cost, without the decrease in LA fermentation efficiency.<br />In fermentation with the addition of thin stillage the highest L-(+)-LA concentration, yield, and volumetric productivity of 31.03 g/L, 86.15%, and 0.93 g/L&middot;h-1, respectively, was obtained in fermentation with the addition of 50% of thin stillage. The highest L-(+)-LA concentration, yield, and volumetric productivity achieved in fed-batch fermentation with the addition of glucose and thin stillage during fermentation, were 48,02 g/L, 87,82% i 0,96 g/L&middot;h-1.<br />In fed-batch fermentation the highest L-(+)-LA concentration, yield, and volumetric productivity of 116.08 g/L, 93.32%, and, 2.04 g/L h-1, respectively, were achieved in fermentation with glucose and yeast extract addition during fermentation. The results showerd that fed-batch fermentation could be used to increase L-(+)-LA fermentation efficiency<br />Immobilization of L. rhamnosus cells with high viability (1010 CFU/mL) in Ca-alginate was conducted. Immobilized cells we successfully utilized in three repeated batch fermentation. L-(+)-LA yield and volumetric productivity were very high in all three batch fermentation, with the highest results achieved (95.20% and 1.76 g/L&middot;h-1, respectively) in second fermentation. Application of immobilized L. rhamnosus cells increased L-(+)-LA yield and volumetric productivity and shortened the fermentation time for 12 h in comparison with batch fermentation.</p>

Using mixed-culture fermentation, the carboxylate platform produces carboxylic acids, which are chemically converted into chemicals and fuels. To optimize the mixed-acid fermentation, different bioreactor configurations and operating modes were investigated. Intermittent air exposure did not affect fermentation performance and bacterial profiles, but reduced the high-molecular-weight carboxylic acids. The microbial flora contained strict and facultative microbes, suggesting the presence of a facultative anaerobic community existing in a biofilm. Compared to countercurrent trains, propagated fixed-bed fermentations have similar selectivity and acid distribution, but lower yield, conversion, productivity, and acid concentration. One- to six-stage countercurrent fermentations were operated with similar conditions. Fewer stages increased conversion, whereas more stages increased acid concentration and selectivity. One to four stages achieved similar yield, and four to six stages achieved similar maximum acid concentration. Maximum conversion was achieved with a single stage. Recycling residual biomass retained microorganisms and nutrients and increased yield and productivity. Relative to lower biomass reflux, higher reflux increased conversion, decreased selectivity, and did not affect yield. The recommended carbon-nitrogen ratio is ~24 g carbon/g nitrogen. In four-stage fermentations, recycle to the second fermentor and in parallel to the first three fermentors was optimal. Fermentations with excess or insufficient nitrogen had higher selectivity, but decreased yield and conversion. The glucose-utilization assay is a rapid and repeatable method for determining the amount of microbial activity in a sample. This method determined ~25% efficiency of a new cell separation method. In continuous fermentation, compared to no cell recycle, recycling cellular biomass increased selectivity and yield, but decreased conversion. Compared to lower cell reflux, higher reflux increased productivity, yield, and conversion, but decreased selectivity. Compared to residual biomass recycle, cell recycle had increased selectivity and yield, but decreased conversion. A new method to screen and rank inoculum sources from natural environments was successfully developed and tested.

Manure from animal farms and sewage sludge contain pathogens and opportunistic organisms in various concentrations depending on the health of the herds and human sources. Other than for the presence of pathogens, these waste substances are excellent nutrient sources and constitute a preferred organic fertilizer. However, because of the pathogens, the risks of infection of animals or humans increase with the indiscriminate use of manure, especially liquid manure or sludge, for agriculture. This potential problem can increase with the global connectedness of animal herds fed imported feed grown on fields fertilized with local manures. This paper describes a simple, easy-to-use, low-tech hygienization method which conserves nutrients and does not require large investments in infrastructure. The proposed method uses the microbiotic shift during mesophilic fermentation of cow manure or sewage sludge during which gram-negative bacteria, enterococci and yeasts were inactivated below the detection limit of 3 log10 cfu/g while lactobacilli increased up to a thousand fold. Pathogens like Salmonella, Listeria monocytogenes, Staphylococcus aureus, E. coli EHEC O:157 and vegetative Clostridium perfringens were inactivated within 3 days of fermentation. In addition, ECBO-viruses and eggs of Ascaris suum were inactivated within 7 and 56 days, respectively. Compared to the mass lost through composting (15–57%), the loss of mass during fermentation (< 2.45%) is very low and provides strong economic and ecological benefits for this process. This method might be an acceptable hygienization method for developed as well as undeveloped countries, and could play a key role in public and animal health while safely closing the nutrient cycle by reducing the necessity of using energy-inefficient inorganic fertilizer for crop production.

No description available.