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Soybean processing generates huge amounts of soy molasses that can support biorefinery but require development of waste-to-value conversion technologies. Here, soy molasses processing by Aspergillus niger enzymes was studied to optimize the conversion of oligosaccharides to monomeric sugars as ready fermentation feedstock. The effects of pH and temperature were first investigated using fixed enzyme composition and loading. pH, in the tested 3.0-6.5 range, significantly affected hydrolysis particularly in galactose release. The hydrolysis was fastest at pH 4.8 and 60 °C although the 48-h sugar (glucose, fructose, and galactose) yields were similar at pH 4.8 and 5.7, and 50 and 60 °C. Study was next made at these favorable pH and temperatures using different enzyme compositions and loadings. Glucose and fructose were effectively released, reaching ∼100 % yields in 24-48 h by most of the enzymes and loadings evaluated. Galactose production was less effective and varied significantly with the pH-temperature condition and enzyme loading and composition. Mechanistic evaluation suggested formation and accumulation of galactose disaccharide, whose slow hydrolysis was rate-limiting in the systems with complete glucose and fructose releases but low galactose yields. Model equations were developed to describe the kinetic sugar-release profiles and make technoeconomic analysis, which showed that a process of lower enzyme loading, while requiring longer duration, is more economical within the analyzed range of 5-50 (U α-galactosidase/g molasses). With 5 (U/g) loading, the total cost is about 30 % lower at 60 °C-pH 5.7 than 50 °C-pH 4.8. The α-galactosidase-to-sucrase ratio plays a less significant role in affecting the overall process cost., Competing Interests: Declaration of Competing Interest We have no conflicts of interest to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Enzyme production is critical and often costly for biorefinery. It is challenging to produce enzymes with not only high titers but also proper combinations of all required activities in a single fermentation. This work aimed at improving productivity and composition of the multiple enzyme activities required for hydrolysis of complex soybean carbohydrate in a single fermentation. A previously selected Aspergillus niger strain was used for its high carbohydrases and low protease production. Strategies of fed-batch substrate addition and programmed pH-decrease rates were evaluated. Cheap soybean hull (SH) was confirmed to induce production of all necessary carbohydrases. Surprisingly, fed-batch SH addition, originally thought to sustain substrate-inducer availability and reduce feedback repression by sugars, did not increase pectinase and cellulase production significantly and even lowered the α-galactosidase production, when compared with batch fermentation having the same total SH amount (all added initially). On the other hand, the pH-decrease rate could be effectively optimized for production of complex enzyme mixtures. The best fermentation was programmed to lower pH from 7 to 4 in 84 h, at a drop rate of .0357 per h. It produced the highest pectinase (19.1 ± .04 U/mL), α-galactosidase (15.7 ± .4 U/mL), and cellulase (.88 ± .06 FPU/mL). Producing these high enzyme activities in a single fermentation significantly improves the effectiveness and economics of enzymatic soy processing, which, e.g., can hydrolyze the 30%-35% carbohydrate in soybean meal to sugars, with minimal protein degradation, to generate high-value protein-rich products and a hydrolysate as fermentation feedstock., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Islam and Ju.)

Phagotrophic algae can consume bacteria that are the predominant microorganisms present in the waste activated sludge (WAS) generated from municipal wastewater treatment processes. In this study, we developed a combined ultrasonication-phagotrophic algal process for WAS conversion. The ultrasonic pretreatment released small volatile solids (VS) including bacteria from WAS flocs. A phagotrophic alga Ochromonas danica then grew by consuming more than 80% of the released VS, with approximately 30% (w/w) algal cell yield. The process reduced the overall WAS VS by 42.4% in 1 day, comparing very favorably with the 27% reduction in 10 days by aerobic digestion. For stabilizing the solids remaining from the ultrasonic step, the total oxygen uptake required was 65%-92% lower than that for the original WAS, indicating substantially reduced aeration cost. Overall, this novel process enhanced the WAS digestion at lower energy requirements and produced microalgae for other potential uses. © 2021 Water Environment Federation PRACTITIONER POINTS: At least 80% of released VS from WAS can be processed by phagotrophic algae. Significant amounts of algae can be produced from WAS. Ultrasonication-phagotrophic algal process can make sludge management more sustainable., (© 2021 Water Environment Federation.)

Soybean is a most promising sustainable protein source for feed and food to help meet the protein demand of the rapidly rising global population. To enrich soy protein, the environment-friendly enzymatic processing requires multiple carbohydrases including cellulase, xylanase, pectinase, α-galactosidase and sucrase. Besides enriched protein, the processing adds value by generating monosaccharides that are ready feedstock for biofuel/bioproducts. Aspergillus could produce the required carbohydrases, but with deficient pectinase and α-galactosidase. Here we address this critical technological gap by focused evaluation of the suboptimal productivity of pectinase and α-galactosidase. A carbohydrases-productive strain A. niger (NRRL 322) was used with soybean hull as inducing substrate. Temperatures at 20 °C, 25 °C and 30 °C were found to affect cell growth on sucrose with an Arrhenius-law activation energy of 28.7 kcal/mol. The 30 °C promoted the fastest cell growth (doubling time = 2.1 h) and earliest enzyme production, but it gave lower final enzyme yield due to earlier carbon-source exhaustion. The 25 °C gave the highest enzyme yield. pH conditions also strongly affected enzyme production. Fermentations made with initial pH of 6 or 7 were most productive, e.g., giving 1.9- to 2.3-fold higher pectinase and 2.2- to 2.3-fold higher α-galactosidase after 72 h, compared to the fermentation with a constant pH 4. Further, pH must be kept above 2.6 to avoid limitation in pectinase production and, in the later substrate-limiting stage, kept below 5.5 to avoid pectinase degradation. α-Galactosidase production always followed the pectinase production with a 16-24 h lag; presumably, the former relied on pectin hydrolysis for inducers generation. Optimal enzyme production requires controlling the transient availability of inducers., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Excessive use of chemical fungicides over the years for plant pathogen control has caused unwanted damage to non-target organisms and resistance buildup in the target organisms. These harmful effects have prompted the industry to look for more sustainable and eco-friendly solutions. Rhamnolipid is a naturally occurring surfactant that is biodegradable, relatively innocuous to non-target species and can effectively lyse zoospores, the life form responsible for the spread of Phytophthora. In this study, rhamnolipid based coatings were developed and evaluated for protection of soybeans from P. sojae zoospores., Results: Pure (acidic) rhamnolipid, when coated on the soybeans, affects the germination negatively. However, sodium and calcium complexed rhamnolipids do not interfere with germination. Seeds coated with 15-20 mg of developed formulation were planted in soil pots and then subjected to P. sojae infection by simulating flooding conditions and zoospore inoculation. Statistical analysis showed that sodium rhamnolipid based coating significantly improved the germination in presence of P. sojae from 42% to 73% (P = 0.017) while the germination of stress-free control was 85% (statistically similar to coated seeds, P = 1)., Conclusion: Neutralized rhamnolipid can protect soybeans from P. sojae without any negative effect on germination. This work illustrates the strategy to use rhamnolipid as effective fungicide. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Grease balls collected from a municipal wastewater treatment plant were melt-screened and used for cultivation of microalga Ochromonas danica, which could phagocytize droplets and particles as food. After autoclaving, the waste grease (WG) separated into two (upper and lower) phases. O. danica grew well on both, accumulating 48-79% (w/w) intracellular lipids. Initial WG contained approximately 50:50 triglycerides and free fatty acids (FFAs); over time, almost only FFAs remained in the extracellular WG presumably due to hydrolysis by algal lipase. PUFAs, mainly C18:2n6, C18:3n3, C18:3n6, C20:4n6, and C22:5n6, were synthesized and enriched to up to 67% of intracellular FAs, from the original 15% PUFA content in WG. The study showed feasibility of converting wastewater-originated WG to PUFA-rich O. danica algae culture, possibly as aquaculture/animal feed. WG dispersion was identified as a major processing factor to further improve for optimal WG conversion rate and cell and FA yields.

A substantial amount of organic matter is wasted in current wastewater treatment processes. To reclaim the value of organic matter, a two-stage continuous-flow open process has been developed by utilizing the capability of phagotrophic algae in ingesting bacterial cells. In this process, wastewater is first pumped into a bacteria tank to grow bacterial cells, and then the effluent containing grown bacteria cells is fed to an algae tank to grow phagotrophic algae. The operation conditions such as dilution rate, pH, and dissolved oxygen level were comprehensively investigated and optimized with long-term tests. Results show that phagotrophic algae can be stably cultivated with wastewater organics through this open process without costly chemical/physical sterilization. The produced phagotrophic algae had high lipid content and can be potentially used as biofuel feedstock.

In this study waste activated sludge (WAS) was sonicated to release bacteria-sized volatile solids (VS) from flocs, after initial pH adjustment to 10 for higher energy efficiency. The released VS supported growth of phagotrophic alga Ochromonas danica. Initial-rate growth experiments confirmed the Monod-type kinetics but the specific cell growth rate, μ, correlated with the prey-to-predator ratio, i.e., the ratio of (fed VS concentration)-to-(initial O. danica concentration), significantly better than with the VS alone, as the typical Monod dependency on soluble substrates. The best-fit kinetics had the following parameters: μ max  = 0.198 h -1 and K M  = 1.056 (g-VS/g-algae). Post-sonication reflocculation could render particles too large to ingest by O. danica; therefore, pH and VS effects on reflocculation were investigated. Batch cultivations were then conducted in fermentors at pH 5, under nonsterile conditions. Algae number reached 8.86 × 10 10  L -1 after 20 h, corresponding to ∼2.3 g/L dry-weight and volumetric algae productivity of 2.8 g/L-day. VS reduction was 38%, giving an O. danica VS yield of 44.5%. The ultrasonication-algae process can be used to produce algae while achieving at least partial WAS treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Wastewater treatment generates large amounts of waste activated sludge (WAS) that contains concentrated bacteria and particulate organics and requires costly treatment prior to disposal. This study develops an approach to harness the unique capability of oleaginous phagotrophic microalgae for treating WAS and producing algal biomass and lipids. WAS ultrasonication is studied for releasing particulates and bacteria suitable for direct ingestion by phagotrophic microalgae, without bacterial destruction/lysis, and thus minimizing energy requirement. Particle release into supernatant was followed by optical density at 610 nm (OD610) and volatile solid concentration (VS); OD610 correlated well with micron-size particle count rates measured by dynamic light scattering. Microalgae (Ochromonas danica) grew with a 7.6-h doubling time in sonication-generated WAS supernatant alone, giving approximately 66% (w/w) cell yield from consumed VS and ∼30% intracellular lipids. Effects of sonication power (P in W), WAS volume (V in mL) and sonication duration (t in s) were studied with a 3 × 3 × 6 factorial design. Supernatant OD610 increased with increasing P and t and decreasing V. Multiple linear regression gave the following equation with only significant terms: OD610TS=-0.0536+0.000592P-0.000213t+0.000003P×t+0.000274P×tV (R 2  = 0.94). Sonicating 500-mL WAS at 180 W for 240 s was selected for giving high particulate release (∼29% VS) with maximal energy efficiency, corresponding to a specific energy input of 4320 kJ (kg TS) -1 , which was much lower than the range (15,000-250,000 kJ (kg TS) -1 ) reported previously for WAS ultrasonication. The results supported development of new ultrasonication-phagotrophic algae processes for WAS treatment and algae production., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Defatted soybean meal has 30-35% oligo-/polymeric carbohydrates and approximately 50% proteins. Enzymatic carbohydrate monomerization enables easy separation to enrich protein content, reduces indigestibility concerns, and facilitates use of carbohydrate as fermentation feedstock. Among soybean carbohydrates, pectin and glucan are more recalcitrant to hydrolyze. To destabilize Ca 2+ -bridged junctures in pectin, effects of 3 chelators ethylenediaminetetraacetic acid (EDTA), sodium hexametaphosphate (HMP) and citric acid under 2-h 90 °C pretreatments were investigated here. Citric acid was the most effective while EDTA decreased enzymatic hydrolysis. In a 3-factor 2-level factorial study, heat (90 °C, 2 h) and citric acid (10 g/L) pretreatments and cellulase supplementation (10 FPU/g) were found to increase yields of all monosaccharides, to 86.8 ± 5.2% glucose, 98.1 ± 1.6% xylose, 87.5 ± 5.2% galactose, 83.6 ± 1.6% arabinose, and 91.4 ± 3.1% fructose + mannose. The largest percentage improvements were for arabinose (382%), mannose (113%) and glucose (51%). Achieving high monosaccharide yields greatly increases value of soybean carbohydrate as fermentation feedstock., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Rhamnolipids are well-known microbial surfactants with many potential applications. Their production cost, however, remains high due to the severe foaming tendency in aerobic fermentation and the relatively low productivity and yield. In this study, we assessed the boundaries set by these constraints after optimization of basic parameters such as dissolved oxygen concentration (DO), pH and carbon sources. DO 10% and pH 5.5-5.7 were found optimal; cell growth and/or rhamnolipid production were slower at lower DO (5%) or pH (5.0) while foaming became hard to control at higher DO (30%) or pH (6.0 and 6.5). Although the Pseudomonas aeruginosa strain used was selected for its high rhamnolipid production from glycerol as substrate, soybean oil was still found to be a better substrate that increased specific rhamnolipid productivity to 25.8mg/g cells-h from the glycerol-supported maximum of 8.9mg/g cells-h. In addition, the foam volume was approximately halved by using soybean oil instead of glycerol as substrate. Analysis by liquid chromatography coupled with mass spectrometry revealed that rhamnolipid compositions from the two carbon sources were also very different, with primarily (82%) monorhamnolipids from soybean oil and more (64%) dirhamnolipids from glycerol. The optimized fermentation produced 42g/l rhamnolipids at a yield of approximately 47% and a volumetric productivity of 220mg/l-h. These values are among the highest reported., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Guayule rubber production leaves >80% biomass as ground bagasse, which can be hydrolyzed to release sugars but also fermentation inhibitors. Here inhibitor generation and sugar conversion by the CO 2 -H 2 O pretreatment and enzyme hydrolysis were studied. Different pretreatment conditions: 550-4900 psi, 160-195 °C, 10-60 min and fixed 66.7% water, generated widely varying amounts of inhibitors (per dry-bagasse mass): 0.014-0.252% hydroxymethylfurfural, 0.012-0.794% furfural and 0.17-8.02% acetic acid. The condition (195 °C/3400 psi/30 min) giving highest reducing sugar (86.9 ± 1.5%) and cellulose (99.2 ± 1.3%) conversions generated more inhibitors. Kluyveromyces marxianus fermentation showed complete growth and ethanol production inhibition at ≥14 g/L combined inhibitors. Considering both sugars and inhibitors, the optimum condition was 180 °C, 1800 psi and 30 min, enabling 82.8 ± 2.8% reducing sugar, 74.8 ± 4.8% cellulose and 88.5 ± 6.9% hemicellulose conversions with low levels of hydroxymethylfurfural (0.07%), furfural (0.25%) and acetic acid (3.0%). The optimized CO 2 -H 2 O pretreatment gave much lower inhibitor formation and higher sugar conversion than other pretreatment methods., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Arabitol is a low-calorie sugar alcohol with anti-cariogenic properties. Enzymatic hydrolysate of soybean flour is a new renewable biorefinery feedstock containing hexose, pentose, and organic nitrogen sources. Arabitol production by Debaryomyces hansenii using soybean flour hydrolysate was investigated. Effects of medium composition, operating conditions, and culture stage (growing or stationary phase) were studied. Production was also compared at different culture volumes to understand the effect of dissolved oxygen concentration (DO). Main factors examined for medium composition effects were the carbon to nitrogen concentration ratio (C/N), inorganic (ammonium) to organic nitrogen ratio (I/O-N), and sugar composition. Arabitol yield increased with increasing C/N ratio and a high I/O-N (0.8-1.0), suggesting higher yield at stationary phase of low pH (3.5-4.5). Catabolite repression was observed, with the following order of consumption: glucose > fructose > galactose > xylose > arabinose. Arabitol production also favored hexoses and, among hexoses, glucose. DO condition was of critical importance to arabitol production and cell metabolism. The yeast consumed pentoses (xylose and arabinose) only at more favorable DO conditions. Finally, arabitol was produced in fermentors using mixed hydrolysates of soy flour and hulls. The process gave an arabitol yield of 54%, volumetric productivity of 0.90 g/L-h, and specific productivity of 0.031 g/g-h.

Soybean is well known for its high-value oil and protein. Carbohydrate is, however, an underutilized major component, representing almost 26-30% (w/w) of the dried bean. The complex soybean carbohydrate is not easily hydrolyzable and can cause indigestibility when included in food and feed. Enzymes can be used to hydrolyze the carbohydrate for improving soybean processing and value of soybean products. Here the enzyme-based processing developed for the following purposes is reviewed: hydrolysis of different carbohydrate-rich by/products from soybean processing, improvement of soybean oil extraction, and increase of nutritional value of soybean-based food and animal feed. Once hydrolyzed into fermentable sugars, soybean carbohydrate can find more value-added applications and further improve the overall economics of soybean processing., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The high carbohydrate content of soybean hull makes it an attractive biorefinery resource. But hydrolyzing its complex structure requires concerted enzyme activities, at least cellulase, xylanase, pectinase and α-galactosidase. Effective pretreatment that generates minimal inhibitory products is important to facilitate enzymatic hydrolysis. Combined CO 2 -H 2 O pretreatment and enzymatic hydrolysis by Aspergillus niger and Trichoderma reesei enzyme broths was studied here. The pretreatment was evaluated at 80°C-180°C temperature and 750psi-1800psi pressure, with fixed moisture content (66.7%) and pretreatment time (30min). Ground hulls without and with different pretreatments were hydrolyzed by enzyme at 50°C and pH 4.8 and compared for glucose, xylose, galactose, arabinose, mannose and total reducing sugar release. CO 2 -H 2 O pretreatment at 1250psi and 130°C was found to be optimal. Compared to the unpretreated hulls hydrolyzed with 2.5-fold more enzyme, this pretreatment improved glucose, xylose, galactose, arabinose and mannose releases by 55%, 35%, 105%, 683% and 52%, respectively. Conversions of 97% for glucose, 98% for xylose, 41% for galactose, 59% for arabinose, 87% for mannose and 89% for total reducing sugar were achieved with Spezyme CP at 18FPU/g hull. Monomerization of all carbohydrate types was demonstrated. At the optimum pretreatment condition, generation of inhibitors acetic acid, furfural and hydroxymethylfurfural (HMF) was negligible, 1.5mg/g hull in total. The results confirmed the effective CO 2 -H 2 O pretreatment of soybean hulls at much lower pressure and temperature than those reported for biomass of higher lignin contents. The lower pressure requirement reduces the reactor cost and makes this new pretreatment method more practical and economical., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Despite having high protein and carbohydrate, soybean flour utilization is limited to partial replacement of animal feed to date. Enzymatic process can be exploited to increase its value by enriching protein content and separating carbohydrate for utilization as fermentation feedstock. Enzyme hydrolysis with fed-batch and recycle designs were evaluated here for achieving this goal with high productivities. Fed-batch process improved carbohydrate conversion, particularly at high substrate loadings of 250-375g/L. In recycle process, hydrolysate retained a significant portion of the limiting enzyme α-galactosidase to accelerate carbohydrate monomerization rate. At single-pass retention time of 6h and recycle rate of 62.5%, reducing sugar concentration reached up to 120g/L using 4ml/g enzyme. When compared with batch and fed-batch processes, the recycle process increased the volumetric productivity of reducing sugar by 36% (vs. fed-batch) to 57% (vs. batch) and that of protein product by 280% (vs. fed-batch) to 300% (vs. batch)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Soybean hull consists mainly of three major plant carbohydrates, i.e., cellulose, hemicellulose and pectin. It is inexpensive and a good potential substrate for carbohydrase production because it is capable of inducing a complete spectrum of activities to hydrolyze complex biomass. Aspergillus is known for carbohydrase production but no studies have evaluated and compared, among Aspergillus species and strains, the soybean hull induced production of various carbohydrases. In this study, A. aculeatus, A. cinnamomeus, A. foetidus, A. phoenicis and 11 A. niger strains were examined together with T. reesei Rut C30, another known carbohydrase producer. The carbohydrases evaluated included pectinase, polygalacturonase, xylanase, cellulase, α-galactosidase and sucrase. Growth morphology and pH profiles were also followed. Among Aspergillus strains, morphology was found to correlate with both carbohydrase production and pH decrease profile. Filamentous strains gave higher carbohydrase production while causing slower pH decrease. The enzyme broths produced were also tested for separation of soy flour carbohydrate and protein. Defatted soy flour contains about 53% protein and 32% carbohydrate. The enzymatic treatment can increase protein content and remove indigestible oligo-/poly-saccharides, and improve use of soy flour in feed and food. Protease production by different strains was therefore also compared for minimizing protein degradation. A. niger NRRL 322 and A. foetidus NRRL 341 were found to be the most potent strains that produced maximal carbohydrases and minimal protease under soybean hull induction., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Soybean carbohydrate is often found to limit the use of protein in soy flour as food and animal feed due to its indigestibility to monogastric animal. In the current study, an enzymatic process was developed to produce not only soy protein concentrate and soy protein isolate without indigestible carbohydrate but also soluble reducing sugar as potential fermentation feedstock. For increasing protein content in the product and maximizing protein recovery, the process was optimized to include the following steps: hydrolysis of soy flour using an Aspergillus niger enzyme system; separation of the solid and liquid by centrifugation (10 min at 7500×g); an optional step of washing to remove entrapped hydrolysate from the protein-rich wet solid stream by ethanol (at an ethanol-to-wet-solid ratio (v/w) of 10, resulting in a liquid phase of approximately 60 % ethanol); and a final precipitation of residual protein from the sugar-rich liquid stream by heat treatment (30 min at 95 °C). Starting from 100 g soy flour, this process would produce approximately 54 g soy protein concentrate with 70 % protein (or, including the optional solid wash, 43 g with 80 % protein), 9 g soy protein isolate with 89 % protein, and 280 ml syrup of 60 g/l reducing sugar. The amino acid composition of the soy protein concentrate produced was comparable to that of the starting soy flour. Enzymes produced by three fungal species, A. niger, Trichoderma reesei, and Aspergillus aculeatus, were also evaluated for effectiveness to use in this process.

A unique oleaginous phagotrophic microalga Ochromonas danica is poised for effective lipid production from waste. Cell harvesting and dewatering are major costs in making algae-based products. In this work an effective additive-free harvesting method was developed, taking advantage of O. danica's comparatively more hydrophobic surface and larger size. The algal cells' partitioning to oil/water interface was evaluated. Recovery by flotation with waste cooking oil was optimized using an L-9 Taguchi orthogonal-array design. Further, additive-free cell collection and concentrating by air flotation was studied for the effects of both physical factors (column dimension, air-stone pore size, sample-to-column volume ratio) and culture properties (pH, culture growth stage, cell concentration, and pure versus impure cultures). The optimized process consistently achieved >90 % recovery in a single stage. 98+ % recovery could be achieved when starting concentrations were >10(8) cells/ml, or potentially using a two- or multi-stage process for diluter cultures.

In aqueous solutions, rhamnolipids effectively kill the motile zoospores responsible for spreading many pathogens, including soy-infecting Phytophthora sojae. For use in soil, adsorption properties need to be considered. Having low critical micelle concentrations, rhamnolipids tend to form micelles/aggregates with unknown effects on soil adsorption. Effects of soil pH, rhamnolipid congener structure, and concentration were examined. Congeners were identified and each quantitated for adsorptive partitioning. The adsorption isotherm at pH 6.5 showed a multi-stage profile plateauing at 1700 μg/g of soil. Less hydrophilic congeners adsorbed preferentially: R-C10-C12 > R-C10-C12:1 > RR-C10-C12:1 > RR-C10-C12 > R-C10-C10 > RR-C10-C10 > R-C8-C10 > RR-C8-C10 (where R is rhamnose and C# is the carbon number of β-hydroxy fatty acid). Adsorptive selectivity among congeners was very clear in dilute solutions but diminished with increasing concentrations. Results were interpreted with aggregate formation in solutions and on the soil surface. The cost estimate made accordingly supported the economic feasibility of rhamnolipid antizoosporic uses in soil.

Soy protein is a well-known nutritional supplement in proteinaceous food and animal feed. However, soybeans contain complex carbohydrate. Selective carbohydrate removal by enzymes could increase the protein content and remove the indigestibility of soy products for inclusion in animal feed. Complete hydrolysis of soy flour carbohydrates is challenging due to the presence of proteins and different types of non-structural polysaccharides. This study is designed to guide complex enzyme mixture required for hydrolysis of all types of soy flour carbohydrates. Enzyme broths from Aspergillus niger, Aspergillus aculeatus and Trichoderma reesei fermentations were evaluated in this study for soy carbohydrate hydrolysis. The resultant hydrolysate was measured for solubilized carbohydrate by both total carbohydrate and reducing sugar analyses. Conversion data attained after 48h hydrolysis were first fitted with models to determine the maximum fractions of carbohydrate hydrolyzable by each enzyme group, i.e., cellulase, xylanase, pectinase and α-galactosidase. Kinetic models were then developed to describe the increasing conversions over time under different enzyme activities and process conditions. The models showed high fidelity in predicting soy carbohydrate hydrolysis over broad ranges of soy flour loading (5-25%) and enzyme activities: per g soy flour, cellulase, 0.04-30 FPU; xylanase, 3.5-618U; pectinase, 0.03-120U; and α-galactosidase, 0.01-60U. The models are valuable in guiding the development and production of optimal enzyme mixtures toward hydrolysis of all types of carbohydrates present in soy flour and in optimizing the design and operation of hydrolysis reactor and process., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Substantial energy is reserved in waste activated sludge (WAS) organics but much of it is difficult to recover because the solid organics require long time to solubilize. In this work we introduced the new approach of recovering WAS organics into the biomass of phagotrophic algae. Phagotrophic algae have the unique ability to grow by ingesting insoluble organic particles including microbial cells. This phagotrophic ability renders the solubilization of WAS organics unnecessary and makes this approach remarkably fast. The approach consists of two stages: a short anaerobic digestion treatment followed by the algal growth on treated WAS. The short anaerobic digestion was exploited to release discrete bacteria from WAS flocs. Phagotrophic algae could then grow rapidly with the released bacteria as well as the solubilized nutrients in the treated WAS. The results showed that WAS organics could be quickly consumed by phagotrophic algae. Among all studied conditions the highest WAS volatile solids (VS) reduction was achieved with 72 h anaerobic digestion and 24 h algal growth. In this optimal process, 28% of WAS VS was reduced, and 41% and 20% of the reduced VS were converted into algal biomass and lipids, respectively. In comparison, only 18% WAS VS were reduced after the same time of aerobic digestion without algae addition. Through this approach, the amount of WAS organics requiring further treatment for final disposal is significantly reduced. With the production of significant amounts of algal biomass and lipids, WAS treatment is expected to be more economical and sustainable in material recycling., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This work describes the use of nutrient limitations with Trichoderma reesei Rut C-30 to obtain a prolonged stationary phase cellulase production. This period of non-growth may allow for dependable cellulase production, extended fermentation periods, and the possibility to use pellet morphology for easy product separation. Phosphorus limitation was successful in halting growth and had a corresponding specific cellulase production of 5±2 FPU/g-h. Combined with the addition of Triton X-100 for fungal pellet formation and low shear conditions, a stationary phase cellulase production period in excess of 300 h was achieved, with a constant enzyme production rate of 7±1 FPU/g-h. While nitrogen limitation was also effective as a growth limiter, it, however, also prevented cellulase production., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The aim of this study was to compare arabitol with its better studied isomer xylitol for their inhibitory effects on cell growth and acid production of oral bacteria. Streptococcus mutans, Streptococcus salivarius and Streptococcus sobrinus were used as representatives of oral streptococci and Lactobacillus acidophilus and Lactobacillus fermentum were used for oral lactobacilli. Growth was followed by measuring the absorbance at 660nm, acid production by pH change. Sensitivity of these oral bacteria to arabitol and xylitol was first compared at 1% (65mM) additive concentration with glucose as sugar substrate. For all bacteria tested, the inhibitory effects of the two polyols were comparable; both were significantly stronger on streptococci (with 20-60% inhibition) than on lactobacilli (with 5-10% inhibition). Effects of arabitol and xylitol were also compared for S. mutans and S. salivarius in media with 1% of different sugar substrates: glucose (55mM), fructose (55mM), galactose (55mM) and sucrose (30mM). Inhibition occurred for all sugars: stronger on glucose and galactose (60-65%) than on fructose and sucrose (40-45%). Inhibition dependency on the arabitol/xylitol concentration from 0.01% (0.65mM) to 2% (130mM) was further determined for S. mutans and S. salivarius. Regardless of the concentration, sugar substrate and bacterial species tested, arabitol showed very similar inhibition effects to its isomer xylitol., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Polygalacturonase and pectinase activities reported in the literature were measured by several different procedures. These procedures do not give comparable results, partly owing to the complexity of the substrates involved. This work was aimed at developing consistent and efficient assays for polygalacturonase and pectinase activities, using polygalacturonic acid and citrus pectin, respectively, as the substrate. Different enzyme mixtures produced by Aspergillus niger and Trichoderma reesei with different inducing carbon sources were used for the method development. A series of experiments were conducted to evaluate the incubation time, substrate concentration, and enzyme dilution. Accordingly, for both assays the recommended (optimal) hydrolysis time is 30min and substrate concentration is 5g/L. For polygalacturonase, the sample should be adjusted to have 0.3-0.8U/mL polygalacturonase activity, because in this range the assay outcomes were consistent (independent of dilution factors). Such a range did not exist for the pectinase assay. The recommended procedure is to assay the sample at multiple (at least 2) dilution factors and determine, by linear interpolation, the dilution factor that would release reducing sugar equivalent to 0.4g/L d-galacturonic acid, and then calculate the activity of the sample accordingly (dilution factor×0.687U/mL). Validation experiments showed consistent results using these assays. Effects of substrate preparation methods were also examined., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Rhamnolipid biosurfactants have potential applications in the control of zoosporic plant pathogens. However, rhamnolipids have not been closely investigated for the anti-zoospore mechanism or for developing new anti-zoospore chemicals. In this study, RhL-1 and RhL-3 groups of rhamnolipids were used to generate the corresponding RhL-2 and RhL-4 groups and the free diacids. Conversion of RhL-3 to RhL-1 was also accomplished in vitro with cellobiase as the catalyst. The anti-zoospore effects of RhL-1-RhL-4 and the diacids were investigated with zoospores of Phytophthora sojae. For RhL-1-RhL-4, approximately 20, 30, 40, and 40 mg/L, respectively, were found to be the lowest concentrations required to stop movement of all zoospores, which indicates that the anti-zoospore effect remains strong even after RhL-1 and RhL-3 are hydrolyzed into RhL-2 and RhL-4. The free diacids required a significantly higher critical concentration of about 125 mg/L. Rhamnose can be obtained as a co-product.

Initial bacterial attachment, likely affected by local shear, could influence biofilm formation. However, there are contradictory reports for the shear effects on attachment of different bacteria onto different surfaces. In this study, four bacteria, Staphylococcus epidermidis, Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli, were examined for their attachment to glass and octadecyltrichlorosilane (OTS) modified glass under different shears. Polystyrene particles were used to verify that their shear dependent attachment on glass and OTS could be interpreted using an analysis based on the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. In particular, the critical shear force (F(c-shear)) could correlate with the maximum attractive force (F(MAX)(XDLVO)) toward the secondary energy minimum as F(c-shear)=cF(MAX)(XDLVO). For these particles, c of ~1 was obtained, the value was within the coefficient range (0.1-1) of substances sliding over glass. For S. epidermidis, E. coli and P. aeruginosa on glass, c was 0.3, <0.6 and 0.2, respectively. When considering potential protein adsorption on OTS during bacterial attachment, c of these species on OTS was slightly above 1. A greatly enhanced attachment of P. aeruginosa on OTS was also observed, probably due to the presence of flagella. For P. putida, the attachment first decreased slightly or maintained with shear and then increased. Such behaviors were probably caused by the increased secretion of extracellular polymeric substances (EPS) at higher shears by P. putida. The results from this study suggested that, without complications from surface features/EPS, the analysis based on the XDLVO theory could provide a basis for understanding shear effect on initial bacterial attachment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rhamnolipids are high-value effective biosurfactants produced by Pseudomonas aeruginosa. Large-scale production of rhamnolipids is still challenging especially under free-cell aerobic conditions in which the highly foaming nature of the culture broth reduces the productivity of the process. Immobilized systems relying on oxygen as electron acceptor have been previously investigated but oxygen transfer limitation presents difficulties for continuous rhamnolipid production. A coupled system using immobilized cells and nitrate instead of oxygen as electron acceptor taking advantage of the ability of P. aeruginosa to perform nitrate respiration was evaluated. This denitrification-based immobilized approach based on a hollow-fiber setup eliminated the transfer limitation problems and was found suitable for continuous rhamnolipid production in a period longer than 1,500 h. It completely eliminated the foaming difficulties related to aerobic systems with a comparable specific productivity of 0.017 g/(g dry cells)-h and allowed easy recovery of rhamnolipids from the cell-free medium., (Copyright © 2013 American Institute of Chemical Engineers.)

Bacterial attachment on solid surfaces has various implications in environmental, industrial and medical applications. In this study, the effects of rhamnolipid biosurfactants on initial attachment of bacteria on hydrophilic glass and hydrophobic octadecyltrichlorosilane (OTS) modified glass were evaluated under continuous-flow conditions. The bacteria investigated were three Gram-negative species Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli, and two Gram-positive species Staphylcoccus epidermidis and Bacillus subtilis. Rhamnolipids, at 10 and 200 mg/l, significantly reduced the attachment of all but S. epidermidis on both glass and OTS-modified glass. For S. epidermidis rhamnolipids reduced the attachment on OTS-modified glass but not on glass. Studies were further done to identify the mechanism(s) by which rhamnolipids reduced the cell attachment. The following potential properties of rhamnolipids were investigated: inhibition of microbial growth, change of cell surface hydrophobicity, easier detachment of cells already attached to substratum, and modification of substratum surface properties. Results showed that rhamnolipids were ineffective for the latter two effects. Rhamnolipids, up to 200mg/l, inhibited the growth of B. subtilis, S. epidermidis and P. aeruginosa PAO1 but not the growth of E. coli, P. putida and P. aeruginosa E0340. Also, rhamnolipids tended to increase the hydrophobicity of P. aeruginosa PAO1 and E. coli, decrease the hydrophobicity of P. putida and S. epidermidis, and have no clear effect on the hydrophobicity of B. subtillis. These trends however did not correlate with the observed trend of cell attachment reduction. The responsible mechanism(s) remained unknown., (Copyright © 2012 Elsevier B.V. All rights reserved.)

It is desirable to modify the normally filamentous Trichoderma reesei Rut C-30 to a pellet form, for easy biomass separation from the fermentation medium containing soluble products (e.g., cellulase). It was found in this study that this morphological modification could be successfully achieved by addition of the biosurfactant rhamnolipid (at ≥ 0.3g/L) and the synthetic Triton X-100 (at ≥ 0.1g/L) to the fermentation broth before the cells started to grow actively. Thirteen other surfactants tested were not as effective. Furthermore, the added rhamnolipid and Triton X-100 increased the maximum cellulase activity (Filter Paper Units) produced in the fungal fermentation; the increase was 68 ± 7.8% for rhamnolipid and 73 ± 12% for Triton X-100. At the concentrations required for pellet formation, rhamnolipid had negative effect on the cell growth: with increasing rhamnolipid concentrations, the growth rate decreased and the lag-phase duration increased linearly. Triton X-100 caused no significant differences in growth rate or lag phase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bacterial biofilm can have significant effects on the behaviors and/or performance of natural and man-made systems. Understanding the factors governing initial bacterial attachment is critical to biofilm management. In this study, the initial attachment of three bacteria, Pseudomonas aeruginosa, Escherichia coli and Pseudomonas putida, on two substrates, glass and octadecyltrichlorosilane (OTS) modified glass, was examined in flow chambers. The flow chambers were designed and operated to mimic slow moving water bodies and minimize the gravitational settlement of cells. The hydrophobicity of bacterial surface was evaluated by partitioning of cells to the water-hexadecane interface and the liquid contact angles on cell layers collected on filter papers. On the more hydrophilic glass surface, the attachment trend was found to be E. coli>P. putida>P. aeruginosa, while the opposite trend was observed on the hydrophobic, OTS modified surface. The attachment trend on glass could be explained by the magnitude of the negative interaction energy at secondary minima, as predicted by the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. The much higher attachments of P. aeruginosa and P. putida on the OTS-modified substrate, on the other hand, suggested that these cells could overcome the energy barrier between the primary and secondary minima of interaction energy to become attached to the primary minimum. The extent of primary-minimum attachment appeared to correlate with the scale of the energy barrier, with higher attachments in the bacteria-substrate combinations of lower energy barriers. The study generated important insights into the effects of cell and substrate surface properties on initial bacterial attachment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Glycerol is a major by-product from biodiesel production, and developing new uses for glycerol is imperative to overall economics and sustainability of the biodiesel industry. With the aim of producing xylitol and/or arabitol as the value-added products from glycerol, 214 yeast strains, many osmotolerant, were first screened in this study. No strains were found to produce large amounts of xylitol as the dominant metabolite. Some produced polyol mixtures that might present difficulties to downstream separation and purification. Several Debaryomyces hansenii strains produced arabitol as the predominant metabolite with high yields, and D. hansenii strain SBP-1 (NRRL Y-7483) was chosen for further study on the effects of several growth conditions. The optimal temperature was found to be 30°C. Very low dissolved oxygen concentrations or anaerobic conditions inhibited polyol yields. Arabitol yield improved with increasing initial glycerol concentrations, reaching approximately 50% (w/w) with 150 g/L initial glycerol. However, the osmotic stress created by high salt concentrations (≥50 g/L) negatively affected arabitol production. Addition of glucose and xylose improved arabitol production while addition of sorbitol reduced production. Results from this work show that arabitol is a promising value-added product from glycerol using D. hansenii SBP-1 as the producing strain.

Selective and effective separation can potentially be achieved with affinity foam fractionation using simple foaming setup and operation. In this study the use of affinity foam fractionation for selective collection and enrichment of β-glucosidase from a cellulase enzyme mixture was evaluated. Rhamnolipids, a group of glycolipids produced most commonly by Pseudomonas aeruginosa, were used as the affinity foaming agent, because of their foaming property and the presence of dirhamnose moiety (a potential substrate analog for β-glucosidase) in some rhamnolipids. The effects of aeration rate, medium pH, cellulase concentration and rhamnolipid concentration on the foam fractionation performance were examined. Among the pH studied (3.1, 5.0, 7.0 and 9.0), pH 5 was clearly the optimal for selective enrichment of β-glucosidase, presumably corresponding to the high binding affinity between the enzyme and the substrate analog (dirhamnose). With adequate rhamnolipid concentrations (≥0.1 g/L), the aeration rate of 0.1L/min (i.e., 2VVM) for 50 ml test samples was found to give the highest enrichment; higher aeration rates produced wetter foam and, thus, lower (diluted) enzyme activity in the foamate. The enrichment increased with the increasing rhamnolipids-to-cellulase ratio, in the range of 0-2 (w/w) investigated in this study. The finding indicated that rhamnolipids were the limiting compounds in these systems so that the amount of surfactant-enzyme complexes formed and removed into the foam phase would increase when more rhamnolipids were added. At the rhamnolipids-to-cellulase ratio of 2, the β-glucosidase activity in the foamate was about 9 times as high as the activity in the original sample of cellulase mixture and about 17 times the activity in the remaining solution (after foaming). The overall FPU (filter paper unit, a measurement of total cellulase activity) and the activities of endo- and exo-glucanases were only enriched 70-150%. The feasibility of affinity foam fractionation was demonstrated., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Guayule, a desert shrub harvested for commercial production of hypoallergenic latex and resins constitutes <20% of the biomass. Converting the remaining bagasse to biorefinery feedstock for value-added products is an optimal economic option. A supercritical CO(2)-based process had been developed previously for resin extraction. In this study, the feasibility of including a supercritical CO(2)-based bagasse pretreatment method was evaluated. The pretreatment involved: adding water to the bagasse, raising system temperature, pressurizing using supercritical CO(2), holding the system for a period of time, and exploding the bagasse. The pretreated biomass was subjected to enzyme hydrolysis. The yields of released sugars were used as pretreatment effectiveness indicators. Supercritical method outperformed other methods and gave much higher overall sugar yields for guayule (as high as 77% for glucose and 86% for total reducing sugars through both pretreatment and hydrolysis, as compared to 50% for glucose and 52% for total sugars with the dilute-acid pretreatment and 36% for glucose and 52% for total sugars with the delignification pretreatment). The enzymatic hydrolyzates were tested on the cellulase-producing fungus Trichoderma reesei Rut C-30. No inhibitory/toxic effects were apparent in terms of cell growth, sugar consumption, and cellulase and xylanase production. The supercritical CO(2)-based method was found to be very promising for pretreatment of waste biomass as the feedstock for subsequent enzymatic hydrolysis and fermentation to produce value-added bioproducts., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background, Aim, and Scope: Solid surfaces in contact with water have been found to be biofouled due to the attachment of various organisms. For better understanding of the biofilm formation, the important initial stage of bacterial attachment was investigated with Pseudomonas aeruginosa PAO1 as a model microorganism. Effects of the biosurfactant rhamnolipids and the shear conditions were particularly examined., Materials and Methods: A highly reproducible procedure was employed. The procedure involved monitoring and counting the number of attached cells on glass walls of the flow chambers, through which a PAO1 suspension was circulated and, subsequently, a saline solution was passed for washing. The experiments were made under different circulation rates (exerting different shear on the bacteria) and rhamnolipid concentrations., Results and Discussions: Reproducibility of the procedure was confirmed. The velocity profiles near the flow chamber wall were determined. Rhamnolipids, even at a very low concentration of 13 mg/l, were found to deter the bacterial attachment substantially. Prewashing the cells with a 100 mg/l rhamnolipid solution, however, did not affect the attachment significantly. As for the effect of shear, the PAO1 attachment showed an increasing-then-decreasing trend in the range investigated, i.e., 1.0 to 26 mN/m(2) shear stresses at the chamber wall. The diffusion-limited transport of cells to the chamber wall might have contributed to, but could not fully explain, the increasing attachment observed in the very low shear range (up to 3.5-5.0 mN/m(2))., Conclusions: As compared to static systems, the flow chamber systems significantly improved the reproducibility of initial attachment results. Flow chamber systems were more suitable for experimental investigations of bacterial attachment to surfaces. Rhamnolipids were found to be potent antifoulants for PAO1 attachment on glass. The initial cell attachment increased with increasing shear at the very low shear range (up to 3.5-5.0 mN/m(2)), but the attachment could be minimized with further increase of the shear.

An acid hydrolysate was prepared by a procedure chosen for retaining more oligosaccharides to improve the cellulase-inducing capability when used as substrate in the fungal fermentation for cellulase production. The effect was evaluated with continuous culture of Trichoderma reesei Rut C30 at the dilution rates of 0.03-0.08 h(-1). The specific cellulase production rates were found to be relatively constant at 8.9+/-0.3 (FPU/g dry cells-h), except for the lower rate, i.e., 7.2 (FPU/g-h), at the lowest dilution rate investigated (0.03 h(-1)). The former value was slightly higher than the rate obtained with a lactose-based medium, i.e., 8.2 (FPU/g-h). The maximum specific cell growth rate supported by the hydrolysate-based medium was 0.096 (h(-1)) and the apparent cell yield increased from 0.44 to 0.57 (g dry cells)/(g consumed reducing sugars) with increasing dilution rates. The best-fit maximum/ideal cell yield (without endogenous metabolism) was 0.68 (g/g), the endogenous substrate consumption rate was 0.023 (g reducing sugars)/(g dry cells-h), and the specific cell death rate was 0.016 h(-1).

Rhamnolipids, produced predominantly by Pseudomonas aeruginosa, are biosurfactants with important applications. For efficient culture screening according to rhamnolipid productivity, the method using agar plates containing methylene blue (MB) and cetyl trimethylammonium bromide (CTAB) was re-examined. An alternative set-up using a fixed underneath light source and image analysis software improved the detection of the circles formed due to complexation between anionic rhamnolipids and cationic MB/CTAB. The roles and effects of MB and CTAB concentrations and pH on the complexation phenomena are reported.

In situ affinity foam fractionation is a potential powerful tool for continuous, selective removal of products from bioprocesses. When evaluating its applicability to cellulase production by Trichoderma reesei fermentation, we encountered the difficulty of significant removal of fungal mycelia along with the cellulase. To solve this problem, cell immobilization using cut pieces of hydrophilic polyurethane (PU) foam was evaluated. Five commercial PU foams with different pore sizes and porosities were tested. Two were found to support good cell growth, cellulase production, and cell loading (about 0.6 g dry cells per g PU). The PU-immobilized mycelia were successfully retained in the foaming process.

Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Current methods for rhamnolipid quantification involve the use of strong hazardous acids/chemicals, indirect measurement of the concentration of sugar moiety, or require the availability of expensive equipment (HPLC-MS). A safer, easier method that measures the whole rhamnolipid molecules would significantly enhance strain selection, metabolic engineering, and process development for economical rhamnolipid production. A semi-quantitative method was reported earlier to differentiate between the rhamnolipid-producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB). In this study, a rapid and simple method for rhamnolipid analysis was developed by systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB. The method relies on measuring the absorbance (at 638 nm) of the rhamnolipid-methylene blue complex that partitions into the chloroform phase. With P. aeruginosa fermentation samples, the applicability of this method was verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique.

Simultaneous nitrification and denitrification (SND or SNdN) may occur at low dissolved oxygen concentrations. In this study, bench-scale (approximately 6 L) bioreactors treating a continuous feed of synthetic wastewater were used to evaluate the effects of solids retention time and low dissolved oxygen concentration, under cyclic aeration, on the removal of organics, nitrogen, and phosphorus. The cyclic aeration was carried out with repeated cycles of 1 hour at a higher dissolved oxygen concentration (HDO) and 30 minutes at a lower (or zero) dissolved oxygen concentration (LDO). Compared with aeration at constant dissolved oxygen concentrations, the cyclic aeration, when operated with proper combinations of HDO and LDO, produced better-settling sludge and more complete nitrogen and phosphorus removal. For nitrogen removal, the advantage resulted from the more readily available nitrate and nitrite (generated by nitrification during the HDO period) for denitrification (during the LDO period). For phosphorus removal, the advantage of cyclic aeration came from the development of a higher population of polyphosphate-accumulating organisms, as indicated by the higher phosphorus contents in the sludge solids of the cyclically aerated systems. Nitrite shunt was also observed to occur in the LDO systems. Higher ratios of nitrite to nitrate were found in the systems of lower HDO (and, to less dependency, higher LDO), suggesting that the nitrite shunt took place mainly because of the disrupted nitrification at lower HDO. The study results indicated that the HDO used should be kept reasonably high (approximately 0.8 mg/L) or the HDO period prolonged, to promote adequate nitrification, and the LDO kept low (< or =0.2 mg/L), to achieve more complete denitrification and higher phosphorus removal. The above findings in the laboratory systems find strong support from the results obtained in full-scale plant implementation. Two plant case studies using the cyclic low-dissolved-oxygen aeration for creating and maintaining SND are also presented.

Biological nitrogen removal via simultaneous nitrification and denitrification (SND) may be achieved in the single-tank bioreactors operated at low dissolved oxygen concentrations (DO). The continuous-stirred tank reactor (CSTR) configuration and the low DO environments employed are; however, more conducive to growth of filamentous bacteria and, thus, poor sludge settling in clarifiers. In this work, a synthetic wastewater was treated in bench-scale (approximately 6L) bioreactors under either cyclic or constant-rate aeration, at various sludge retention times (SRT) and DO. The objective was to evaluate the effects of these factors on the sludge settling indicated by sludge volume index. The cyclic aeration was carried out by alternating the aeration between a higher rate for 1h and a lower (or zero) rate for 30 min. In different experiments, the DO during the period of higher aeration (HDO) was 0.4, 0.6, 0.8, or 2.0 mg/L and the DO during lower aeration (LDO) was 0.0 or 0.2mg/L. The sludge established under the cyclic aeration was found to settle better than that under constant-rate aeration. Shortening SRT also improved the sludge settling significantly. NAD(P)H fluorescence profiles in these bioreactors were monitored using an online fluorometer. A procedure was developed to quantitatively describe the metabolic state of sludge's heterotrophic population on a 0-1 scale using the fluorescence profile, with "0" corresponding to the fully anoxic-denitrifying state and "1" to the fully aerobic state.

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.

Heterocyst differentiation is a unique feature of nitrogen-fixing cyanobacteria, potentially important for photobiological hydrogen production. Despite the significant advances in genetic investigation on heterocyst differentiation, there were no quantitative culture-level models that describe the effects of cellular activities and cultivation conditions on the heterocyst differentiation. Such a model was developed in this study, incorporating photosynthetic growth of vegetative cells, heterocyst differentiation, self-shading effect on light penetration, and nitrogen fixation. The model parameters were determined by fitting experimental results from the growth of the heterocystous cyanobacterium Anabaena flos-aquae CCAP 1403/13f in media without and with different nitrate concentrations and under continuous illumination of white light at different light intensities (2, 5, 10, 17, 20 and 50 microE m-2 s-1). The model describes the experimental profiles well and gives reasonable predictions even for the transition of growth from that on external N source to that via nitrogen fixation, responding to the change in external N concentrations. The significance and implications of the best-fit values of the model parameters are discussed.

The gas vesicles isolated from the cells of filamentous cyanobacterium Anabaena flos-aquae were treated and sterilized with glutaraldehyde and then evaluated for their effectiveness as gas carriers in cell culture. Anchorage-dependent Vero cells were grown in a packed bed of microcarrier beads under the perfusion of Dulbecco's Modified Eagle's Medium with 1% serum. The culture medium supplemented with 1.8% (v/v) gas vesicles was found to support a 30% higher maximum glucose utilization rate than the same medium without gas vesicles. The gas vesicle suspension was confirmed to have no apparent effects on cell metabolism in T-flask cultures. The study results indicated that the gas vesicles, with high oxygen carrying capacity, can be used to increase the oxygen supply in cell culture systems.

Continuous culture of P. aeruginosa was conducted with nitrate-containing media under the dilution rates (D) of 0.026, 0.06, and 0.13/h and the dissolved oxygen concentrations (DO) of 0-2.2 mg/L. The bacterium performed simultaneous O(2) and nitrate respiration in all of the systems studied. For each D, the (apparent) cell yield from glucose (Y(X/S)) was lower at zero DO, but did not change substantially with non-zero DO. In non-zero DO systems, Y(X/S) increased with increasing D, and when fit with a model considering cell death, gave the following parameters: maximum cell yield Y(X/S) (m) = 0.49, maintenance coefficient M(S) = 0.029 (/h), and cell decay constant k(d) = 0.014/h. The same model failed to describe the behaviors of zero-DO systems, where neither glucose nor nitrate was limiting and the limiting factor(s) remained unknown. The cell yield from accepted electron (Y(X/e)) was however relatively constant in all systems, and the energy yield per electron accepted via denitrification was estimated at approximately 69% of that via O(2) respiration. A closer examination revealed that increasing DO enhanced O(2) respiration only at extremely low DO ( <0.05 mg/L), beyond which the increasing DO only slightly increased its weak inhibition on denitrification. While O(2) was the preferred electron acceptor, the fraction of electrons accepted via denitrification increased with increasing D.

Cellulase could not be selectively collected from fermentation broth by simple foam fractionation, because of the presence of other more surface-active compounds. A new approach of affinity foam fractionation was investigated for improvement. A hardwood hydrolysate (containing cellulose oligomers, substrates to cellulase) and two substrate analogs, i.e., carboxymethyl cellulose (CMC) and xylan hydrolysate, were added before the foaming process. The substrates and substrate analogs were indeed found to bind the cellulase selectively and form more hydrophobic complexes that partition more readily onto bubble surfaces. In this study, the effects of the type and concentration of substrate/analog as well as the presence of cells at different growth stages were examined. The foam fractionation properties evaluated included foaming speed, foam stability, foamate volume, and enrichment of filter paper unit (FPU) and individual cellulase components (i.e., endoglucanases, exoglucanases, and beta-glucosidases). Depending on the broth and substrate/analog employed, the foamate FPU could be more than fourfold higher than the starting broth FPU. Addition of substrate/analog also deterred the enrichment of other extracellular proteins, resulting in the desired cellulase purification in the foamate. The value of E/P (enzyme activity-FPU/g/L of proteins) in the foamate reached as high as 18, from a lactose-based fermentation broth with original E/P of 5.6. Among cellulase components, exoglucanases were enriched the most and beta-glucosidases the least. The study with CMC of different molecular weights (MW) and degrees of substitution (DS) indicated that the CMC with low DS and high MW performed better in cellulase foam fractionation.

Defined as the transition conditions in which the organism(s) performs simultaneous aerobic and anaerobic respiration or fermentation, microaerobic conditions are commonly present in the nature. Microaerobic metabolism of microorganisms is however poorly characterized. Being extremely sensitive to the change in cellular electron-accepting mechanisms, NAD(P)H fluorescence provides a useful ways for online monitoring of microaerobic metabolism. Its application to studies of microbial nitrate respiration and particularly, denitrification of Pseudomonas aeruginosa is reviewed here, centering on four topics: (1) online monitoring of anaerobic nitrate respiration by NAD(P)H fluorescence, (2) effects of denitrification on P. aeruginosa phenotypes, (3) microaerobic denitrification of P. aeruginosa in continuous culture, and (4) correlation between NAD(P)H fluorescence and denitrification-to-respiration ratio. Online NAD(P)H fluorescence is shown to sensitively detect the changes of cellular metabolism. For example, it revealed the intermediate nitrite accumulation in C-limited Escherichia coli performing anaerobic nitrate respiration via dissimilative ammonification, by exhibiting two-stage profiles with intriguing fluorescence oscillation. When applied to continuous culture studies of P. aeruginosa (ATCC 9027), the online fluorescence helped to identify that the bacterium conducted denitrification even at DO > 1 mg/l. In addition, the fluorescence profile showed a unique correlation with the fraction of electrons accepted by denitrification (out of all the electrons accepted by aerobic and anaerobic respiration). The applicability of online NAD(P)H fluorescence in monitoring and quantitatively describing the sensitive microaerobic state of microorganisms is clearly demonstrated.

The objectives of this study are to examine the phosphorus release in aerobic sludge digestion and to better understand its governing mechanisms. In this study, phosphorus release was examined using the secondary sludge from both conventional and biological nutrient removal processes. The experiments were carried out at room temperature (22 +/- 2 degrees C), with or without automatic control of pH (4.5 to 7.8), and under three aeration schemes: fully aerobic (dissolved oxygen [DO] at 3 to 4 mg/L), low DO (0.2 to 0.8 mg/L), and cyclic (with alternate on/off aeration). The released phosphorus concentrations were 20 to 80 mg/L for the conventional sludge and 60 to 130 mg/L for the biophosphorus sludge. Higher phosphorus release also occurred at low pH (<6.0). As for the effect of DO, fully aerobic digestion caused higher phosphorus release than the low-DO and cyclic operations. For better understanding, the solid phosphorus in sludge was conceptually categorized into three forms: inorganic phosphorus precipitates, organic cellular phosphorus, and polyphosphate (poly-P) in polyphosphate-accumulating organisms. Dissolution of inorganic phosphorus precipitates is controlled by physical and chemical conditions, with pH being the most important in this study. Lowering the pH to 4 to 6 clearly promoted the release of inorganic phosphorus. Polyphosphate hydrolysis, on the other hand, was found to be regulated biologically (sensitive to occurrence of anaerobic conditions) and was insignificant in the glutaraldehyde-fixed sludge. Phosphorus release from organic phosphorus should correlate with the volatile solid (VS) digestion, which lyses the cells and frees the phosphorus covalently bonded with the organic matters. The amounts of phosphorus released per unit VS digested (deltaP/deltaVS) were therefore calculated for experiments with long periods of constant pH (to minimize interferences from dissolution/precipitation of inorganic phosphorus). The results suggested that some poly-P was hydrolyzed and released accompanying the aerobic VS digestion, but at rates far lower than those under anaerobic conditions.

The quorum-sensing (las and rhl) systems play critical roles in the pathogenicity of Pseudomonas aeruginosa and its synthesis of the important biosurfactants, rhamnolipids. In this work, P. aeruginosa PAO1 and its rhlI and rhlR null mutants were used to study the degradation and synthesis kinetics of the rhl system's autoinducer PAI2 (N-butanoyl-homoserine lactone). The two mutants, lacking the ability of synthesizing PAI2 or RhlR protein, produced insignificant amounts of rhamnolipids while having similar growth profiles as the wild-type culture. The regulatory RhlR:PAI2 complex is thus essential to rhamnolipid synthesis. In batch culture of the wild-type PAO1, the autoinducer PAI2 concentration increased along cell growth, especially during the transition from exponential-growth phase to stationary phase, and began to decrease after entering the stationary phase. The decrease in the stationary phase resulted from a faster PAI2 degradation than its synthesis. The degradation kinetics was studied using PAI2-containing supernatants (from centrifuged broth of wild-type culture) with and without the rhlI(-) mutant cells incapable of PAI2 synthesis. Being insignificant in the cell-free systems, PAI2 degradation was found predominantly cell-associated and could be described empirically by the first-order, exponential decay kinetics with the best-fit degradation constant (k(d)) of 0.195 h(-1). When similarly modeled with a first-order kinetics, PAI2 synthesis in stationary-phase wild-type culture was derived to have a synthesis constant (k(s)) of 0.189 h(-1). The PAI2 concentration in batch cultivation of the rhlR(-) mutant also showed an increase-then-decrease profile. However, the maximum PAI2 concentration was about one third of that from the wild-type culture. The constitutive rate of PAI2 synthesis was therefore significantly lower than the rate attainable with active auto-induction by RhlR-PAI2 complex.

With sludge samples from two wastewater treatment plants, batch experiments of aerobic sludge digestion were conducted under different dissolved oxygen (DO) and solids concentrations. A fluorometer capable of online excitation and emission scanning was used to monitor the digestion process. Three major fluorescence peaks were observed. The peak at excitation/emission maxima of 290/350 nm was attributed to the fluorescence of proteinaceous materials in the sludge, with tryptophan residues being the primary contributor. The sources for the other two peaks (at 370/430 nm and 430/510 nm) remain unknown. The well-known biological fluorescence from reduced nicotinamide adenine dinucleotides (NADH and NADPH), at excitation/emission maxima of 340/460 nm, was found very weak in the aerobic digestion systems studied. It was buried under the broad peak at 370/430 nm and was detectable only in the early stage of the experiment that had the highest solids loading (at 4.8%) and was operated under low DO (0.2-1.0mg/L) conditions. On the other hand, the profile of the protein fluorescence (PF) correlated well with that of the volatile solids (VS) reduction in all the experiments. A semi-empirical exponential decay function was developed, which described well the profiles of both normalized VS and normalized PF. The feasibility of following the real-time performance of aerobic sludge digestion by monitoring PF was clearly demonstrated.