Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013     

Effect of malic acid on the growth kinetics of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis>

The fermentation kinetics of Lactobacillus plantarum were studied in a specially designed broth formulated from commercially available, dehydrated components (yeast extract, trypticase, ammonium sulfate) in batch and continuous culture. During batch growth in the absence of malic acid, the specific growth rate was 0.20 h^–1. Malic acid in the medium, at 2 mM or 10 mM, increased the specific growth rate of L. plantarum to 0.34 h^–1. An increase in the maximum cell yield due to malic acid also was observed. Malic acid in the medium (12 mM) reduced the non-growth-associated (maintenance energy) coefficient and increased the biomass yield in continuous culture, based on calculations from the Luedeking and Piret model. The biomass yield coefficient was estimated as 27.4 mg or 34.3 mg cells mmol^–1 hexose in the absence or presence of malic acid, respectively. The maintenance coefficient was estimated as 3.5 mmol or 1.5 mmol hexose mg^–1 cell h^–1 in the absence or presence of malic acid. These results clearly demonstrate the energy-sparing effect of malic acid on the growth- and non-growth-associated energy requirements for L. plantarum. The quantitative energy-sparing effect of malic acid on L. plantarum has heretofore not been reported, to our knowledge.     

Effect of carbon dioxide on the rheological behavior of submerged cultures of Chlorella minutissima in stirred tank reactors

The objective of this study was to quantify the effect of algal biomass concentration on the rheology of the algal culture broth. Batch cultivations of Chlorella minutissima were carried out with air and carbon dioxide in a stirred tank bioreactor with a working volume of 1.8 L. The apparent viscosity of the culture broth was significantly affected by the cell mass concentrations in the bioreactor. Culture broth containing 50 g/L cell mass from air fed was found to exhibit an apparent viscosity of 1.52 mPa.s. The apparent viscosity of the carbon‐dioxide‐fed cultivations was found to increase by 20% at a shear rate of 100 s^?1. The flow behavior of the system was adequately described by the Herschel–Bulkley model with a small yield stress.     

Improvement of biomass and juvenile hormone III (JH III) production from <Emphasis Type="Italic">Cyperus aromaticus</Emphasis> cell suspension culture via in situ membrane filtration technology

The incorporation of membrane filtration technology in plant cell suspension culture was to facilitate in situ medium exchange as well as to eliminate possible introduction of contaminant in the process. The study had been carried out in Cyperus aromaticus cell suspension culture, which is known to produce a bioinsecticide known as juvenile hormone III (JH III). The effect of different volume and flow rate of replenishment toward cell biomass and JH III production were studied. The experimental finding indicates that the flow rate of medium replenishment ranging from 1 to 6 ml min⁻¹ did not affect the cellular structure and morphology the cells. Medium replenishment of 75 v/v% through the membrane system leads to higher cell biomass and metabolite production which correlates to a value as high as 64 and 112%, respectively. The reduction in doubling time, extended exponential growth and increment in specific growth rate were also demonstrated. A 50 v/v% medium replenishment with membrane filtration system was identified to be an economical and feasible application for the production of JH III metabolite at a large scale. The results obtained showed the potential of in situ membrane filtration system for the enhancement of cell biomass and metabolite yield in plant cell culture.     

Behaviour of Candida utilis during growth in relation to Mg2+ and K+ concentration in sugar cane molasses

Magnesium and potassium ions are present in sugar cane molasses in a concentration of about 0.3% and 2.5%, respectively, which is high enough to support biomass production from Candida utilis. Culture broth with 40 g/l of total reducing substances supplemented by the addition of 1 ppm of Mg²⁺ leads to a higher yield (Y x/s) in batch fermentation experiments. The subsequent addition of Mg²⁺ up to 10 ppm decreases the yield coefficient from 0.53 to 0.42 without affecting the growth rate. Fermentation media supplemented with 1 to 10 ppm of K⁺ decreased both the yield coefficient and the specific growth rate. A Mg/K ratio of about 0.1 seems to be optimal for yeast biomass propagation.     

NH4 : NO3 nutrition influence on biomass productivity and root respiration of poplar and willow clones

Nitrogen fertilization often improves the yield of intensively managed, short‐rotation coppices. However, information of N nutrition form on the growth of common species and clones used for biomass production is limited. Thus, this study aims at evaluating N form effects on the growth of two Salicaceae clones. Cuttings of the poplar clone Max 4 (Populus maximovizcii × P. nigra) and the willow clone Inger (Salix triandra × S. viminialis) were fertilized in a pot experiment with four ratios of nitrate (NO₃^?) to ammonium (50%, 62.5%, 75% and 87.5% NO₃^? balanced with ammonium (NH₄⁺) to constant total N) for one growing season and under stable soil pH. Plants were harvested for analysis of biomass and morphology of leaves, stem and roots. Respiration of fine and coarse roots (RR) was determined and related to biomass growth. Salix cv. Inger accumulated more total dry matter than Populus cv. Max 4. In both Salicaceae clones, the total biomass was significantly influenced by the nitrate ratio and greatest in plants fertilized with 50% NO₃^? of the total N supply. Both clones possess a different leaf and root morphology, but no significant influence of the NO₃^? ratio on the morphology was found. Fine RR rates differed significantly between clones, with significantly greater fine RR in Max 4; 87.5% NO₃^? fertilization increased the fine RR. Fine RR and total accumulated plant biomass were closely related. Our study is the first to show the tremendous influence of fine root respiration, especially including the carbon‐intensive reduction of NO₃^? to NH₄⁺, on the aboveground growth of Salicaceae clones. Ways to improve yield in SRC are thus to lower the assimilate consumption by fine roots and to match fertilization regimes to the used clones or vice versa.     

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled‐up mist bioreactor

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low‐cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2–3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2–3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day^?1 with biomass yield of 12.75 g DW L^?1. This exceeded that in shake flasks at µ = 0.166 day^?1 and 11.10 g DW L^?1. Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L^?1, which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots. Biotechnol. Bioeng. 2010;107: 802–813. © 2010 Wiley Periodicals, Inc.     

An improved method for cell and spore counts during aerobic cultivation of crystal toxin producingBacillus sphaericus

Summary A simple improved method including sonication treatment is proposed to determine accurate cell and spore counts forBacillus sphaericus, a microorganism which can form cell aggregates. Sonication (10 watt power output) for 40 seconds after dilution of culture broth was effective in dispersing clumps of cells and spores without disruption. This improved method gave approximately 2 times higher cell and spore counts compared with the conventional counting methods (without sonication).     

Effect of various process parameters on morphology, rheology, and polygalacturonase production by Aspergillus sojae in a batch bioreactor

The effects of pH, agitation speed, and dissolved oxygen tension (DOT), significant in common fungal fermentations, on the production of polygalacturonase (PG) enzyme and their relation to morphology and broth rheology were investigated using Aspergillus sojae in a batch bioreactor. All three factors were effective on the response parameters under study. An uncontrolled pH increased biomass and PG activity by 27% and 38%, respectively, compared to controlled pH (pH 6) with an average pellet size of 1.69 +/- 0.48 mm. pH did not significantly affect the broth rheology but created an impact on the pellet morphology. Similarly, at constant agitation speed the maximum biomass obtained at 500 rpm and at 30 h was 3.27 and 3.67 times more than at 200 and 350 rpm, respectively, with an average pellet size of 1.08 +/- 0.42 mm. The maximum enzyme productivity of 0.149 U mL-1 h-1 was obtained at 200 rpm with an average pellet size of 0.71 +/- 0.35 mm. Non-Newtonian and pseudoplastic broth rheology was observed at 500 rpm agitation speed, broth rheology exhibited dilatant behavior at the lower agitation rate (200 rpm), and at the medium agitation speed (350 rpm) the broth was close to Newtonian. Furthermore, a DOT range of 30-50% was essential for maximum biomass formation, whereas only 10% DOT was required for maximum PG synthesis. Non-Newtonian shear thickening behavior (n > 1.0) was depicted at DOT levels of 10% and 30%, whereas non-Newtonian shear thinning behavior (n < 1.0) was dominant at 50% DOT. The overall fermentation duration (50-70 h) was considerably shorter compared to common fungal fermentations, revealing the economic feasibility of this particular process. As a result this study not only introduced a new strain with a potential of producing a highly commercially significant enzyme but also provided certain parameters significant in the design and mathematical modeling of fungal bioprocesses.     

Preliminary studies on the growth of Pseudomonas sp. BA2 and the production of L-aminoacylase

Summary The bacterial strain Pseudomonas sp. BA2 did not develop l-aminoacylase activity in the absence of N-acetyl-dl-alanine. The maximum growth rate and enzyme production were obtained when the acetylated amino acid was used as the sole carbon and nitrogen source. A maximum biomass of A₆₆₀=1.543, after 24 h of cultivation, was obtained. The l-aminoacylase activity reached the maximum value (5.6 U ml^–1 broth) in the stationary growth phase.     

Macronutrients requirements of the dinoflagellate Protoceratium reticulatum

The basal L1 medium was found to be unsatisfactory for culturing the red tide dinoflagellate Protoceratium reticulatum at a high growth rate and biomass yield. The L1 medium enhanced with phosphate to a total concentration of 217 μM supported the highest attainable growth rate and biomass yield. Once the phosphate concentration exceeded 6× L1, phosphate inhibited the dinoflagellate growth and negatively affected cell viability. At the optimal phosphate concentration of 217 μM, an increase in nitrate concentration over the range of 882–8824 μM, did not affect cell growth and yield. Nitrate did not inhibit growth at any of the concentrations used. Clearly, the basal nitrate level in L1 is sufficient for effectively culturing P. reticulatum. At the ranges of phosphate and nitrate concentrations tested, cell volume was not sensitive to the concentration of nutrients but the concentration of phosphate affected both the specific cell number and cell volume growth rates. Elevated levels of nutrients supported their intracellular accumulation. Cell-specific production of yessotoxin was not influenced by concentration of phosphate in the culture medium, but elevated (>1764 μM) nitrate concentration did enhance the yessotoxin level. Phosphate concentration that maximized biomass yield also maximized volumetric production of yessotoxin in the culture broth.     

Changes in morphology of Paecilomyces japonica and their effect on broth rheology during production of exo-biopolymers

The influence of Paecilomyces japonica pellet morphology on fermentation broth rheology and exobiopolymer production was investigated in a 5-1 jar fermenter. Rapid formation of pellets was observed after the first day of fermentation; and these slowly increased in size and roughness. This, together with the increase in biomass concentration, altered the transport characteristics and broth rheology towards a pseudoplastic nature which, in turn, influenced cell growth and exo-biopolymer production. At mild agitation, high aeration and optimum substrate concentration, pellets were the most predominant morphological form, compared with free mycelia. The broth rheology showed pseudoplastic behavior; and the fungal morphology was closely related to the rheological properties.     

A method for the detection of Issatchenkia orientalis in a baker's yeast factory

Summary A method for the detection of Issatchenkia orientalis (Candida krusei), a contaminant of bakers' yeast, is described. Nine different liquid medium types were compared and maximum specific growth rates for the yeast contaminant were determined in each medium. Issatchenkia orientalis grew fastest in malt extract broth (0,37 h^-1) and potato dextrose broth (0,36 h^-1). Five antibiotics were tested for selective inhibition of seven different bakers' yeast strains in malt extract broth. Nystatin was the only antibiotic tested that inhibited the growth of all the bakers' yeast strains used, but did not affect the growth of I. orientalis.     

Changes in morphology of <Emphasis Type="Italic">Rhizopus chinensis</Emphasis> in submerged fermentation and their effect on production of mycelium-bound lipase

In order to control suitable mycelium morphology to obtain high lipase productivity by Rhizopus chinensis in submerged fermentation, the effects of fungal morphology on the lipase production by this strain both in shake flask and fermentor were investigated. Different inoculum level and shear stress were used to develop distinctive morphologies. Analyses and investigations both on micromorphology and macromorphology were performed. Study of micromorphology reveals that micromorphologies for dispersed mycelia and aggregated mycelia are different in cell shape, biosynthetic activity. Macromorphology and broth rheology study in fermentor indicate that pellet formation results in low broth viscosity. Under this condition, the oil can disperse sufficiently in broth which is very important for lipase production. These results indicate that morphology changes affected the lipase production significantly for R. chinensis and the aggregated mycelia were suggested to achieve high lipase production.     

Rheology and Hydrodynamic Properties of Tolypocladium inflatum Fermentation Broth and Its Simulation

A physico-chemical, two phase simulated pseudoplastic fermentation (SPF) broth was investigated in which Solka Floc cellulose fibre was used to simulate the filamentous biomass, and a mixture of 0.1% (w/v) carboxymethyl cellulose (CMC) and 0.15 M aqueous sodium chloride was used to simulate the liquid fraction of the fermentation broth. An investigation of the rheological behaviour and hydrodynamic properties of the SPF broth was carried out, and compared to both a fungal Tolypocladium inflatum fermentation broth and a CMC solution in a 50 L stirred tank bioreactor equipped with conventional Rushton turbines. The experimental data confirmed the ability of the two phase SPF broth to mimic both the T. inflatum broth bulk rheology as well as the mixing and mass transfer behaviour. In contrast, using a homogeneous CMC solution with a similar bulk rheology to simulate the fermentation resulted in a significant underestimation of the mass transfer and mixing times. The presence of the solid phase and its microstructure in the SPF broth appear to play a significant role in gas holdup and bubble size, thus leading to the different behaviours. The SPF broth seems to be a more accurate simulation fluid that can be used to predict the bioreactor mixing and mass transfer performance in filamentous fermentations, in comparison with CMC solutions used in some previous studies.     

Establishment of reed canary grass with perennial legumes or barley and different fertilization treatments: effects on yield,botanical composition and nitrogen fixation

In two field experiments in northern Sweden, we investigated if intercropping reed canary grass (RCG; Phalaris arundinacea L.) with nitrogen‐fixing perennial legumes could reduce N‐fertilizer requirements and also if RCG ash or sewage sludge could be used as a supplement for mineral P and K. We compared biomass production, N uptake and N‐fixation of RCG in monoculture and mixtures of RCG with alsike clover (Trifolium hybridum L.), red clover (Trifolium pratense L.), goat's rue (Galega orientalis Lam.) and kura clover (Trifolium ambiguum M. Bieb.). In one experiment, RCG was also undersown in barley (Hordeum vulgare L.). Three fertilization treatments were applied: 100 kg N ha^?1, 50 kg N ha^?1 and 50 kg N ha^?1 + RCG ash/sewage sludge. We used a delayed harvest method: cutting the biomass in late autumn, leaving it on the field during the winter and harvesting in spring. The legume biomass of the mixtures at the inland experimental site was small and did not affect RCG growth negatively. At the coastal site, competition from higher amount of clover biomass affected RCG growth and spring yield negatively. N‐fixation in red clover and alsike clover mixtures in the first production year approximately covered half of recommended N‐fertilization rate. Goat's rue and kura clover did not establish well at the costal site, but at the inland site goat's rue formed a small but vital undergrowth. RCG undersown in barley gave lower yield, both in autumn and spring, than the other treatments. The high N treatment gave a higher spring yield at the inland site than the low N treatments, but there were no differences due to fertilization treatments at the coastal site. For spring harvest, there were no yield benefits of RCG/legume intercropping compared with RCG monoculture. However, intercropping might be more beneficial in a two‐harvest system.     

Dynamics of ginsenoside biosynthesis in suspension culture of <Emphasis Type="Italic">Panax quinquefolium</Emphasis>

Biosynthesis of six saponins (ginsenosides) in suspension culture of P. quinquefolium Z₅ was investigated. Ginsenoside content in biomass reached the highest level, nearly 30 mg g⁻¹ d.w., between 25 and 30 days of the culture. Saponins were synthesized simultaneously with cell growth but their synthesis rate was not proportional to the growth rate. During the phase of rapid biomass multiplication, after which biomass reached 90% of its maximum yield, only half examined ginsenosides was produced. The second half of the final saponins yield was produced during the slow growth phase, in which only 10% of biomass was grown. During the intensive growth phase the productivity of six saponins examined per biomass (dry weight) unit was 3.4 μg mg⁻¹ d.w. day⁻¹, however, this parameter calculated for slow growth phase reached nearly 30 μg mg⁻¹ d.w. day⁻¹. There were differences in increase of the contents of six saponins determined in biomass, and it was the highest for saponins Re (20(S)-protopanaxatriol-6-[O-α-l-rhamnopyranosyl(1 → 2)-β-d-glucopyranoside]-20-O-β-d-glucopyranoside) and Rg₁ (20(S)-protopanaxatriol-6,20-di-O-β-d-glucoside).     

Biomass yields of Chlorella from iron (Y x/Fe) in iron-limited batch cultures

The maximum biomass in iron-limited photosynthetic batch cultures of chlorella increased as the logarithm of the iron concentration. The growth yield from iron (Y _x/Fe) showed a marked inverse relation to the specific growth rate. The maximum biomass yield, g dry biomass/g iron consumed, was 7.5x10³ with specific growth rate 0.108 h^-1; the minimum was 0.79×10³ with specific growth rate 0.145 h^-1. The maximum specific growth rate in the exponential phase of Fe limited cultures varied as the initial Fe concentration. Fe-limited growth made the cells adhere to a glass surface.Abbreviation O.D. optical density     

The use of a mixed culture of Geotrichum candidum,Candida krusei and Hansenula anomala for microbial protein production from whiskey distillery spent wash

Summary The use of a mixed culture of Geotrichum candidum, Hansenula anomala and Candida krusei for microbial protein production and treatment of whiskey distillery spent wash was investigated in both batch and continuous culture. Although capable of assimilating the same substrate constituents in pure culture the organisms used different constituents for growth in mixed continuous culture, allowing a stable mixed population to be established. The relative proportions of the three organisms in the population was dependent on the dilution rate.The mixed culture did not give superior yields or productivities, but did give proportionally greater COD reduction and would be expected to bemore stable and resistant to contamination.At a dilution rate of 0.10 h^–1 the mixed culture gave a biomass yield of 4.8 g l^–1, containing 55% crude protein, with a COD reduction of 31.5%, while in mixed batch culture a biomass yield of 12.5 g l^–1, containing 48% crude protein, was obtained with a COD reduction of 54.9%.