Roland Schneider

Fermentative utilization of coffee mucilage using Bacillus coagulans and investigation of down-stream processing of fermentation broth for optically pure l(+)-lactic acid production.

In this study, mucilage, a residue from coffee production, was investigated as substrate in fermentative l(+)-lactic acid production. Mucilage was provided as liquid suspension consisting glucose, galactose, fructose, xylose and sucrose as free sugars (up to 60gL(-1)), and used directly as medium in Bacillus coagulans batch fermentations carried out at 2 and 50L scales. Using mucilage and 5gL(-1) yeast extract as additional nitrogen source, more than 40gL(-1) lactic acid was obtained. Productivity and yield were 4-5gL(-1)h(-1) and 0.70-0.77g lactic acid per g of free sugars, respectively, irrespective the scale. Similar yield was found when no yeast extract was supplied, the productivity, however, was 1.5gL(-1)h(-1). Down-stream processing of culture broth, including filtration, electrodialysis, ion exchange chromatography and distillation, resulted in a pure lactic acid formulation containing 930gL(-1)l(+)-lactic acid. Optical purity was 99.8%.

Fermentative lactic acid production from coffee pulp hydrolysate using Bacillus coagulans at laboratory and pilot scales.

In this study, the lignocellulosic residue coffee pulp was used as carbon source in fermentative l(+)-lactic acid production using Bacillus coagulans. After thermo-chemical treatment at 121°C for 30min in presence of 0.18molL(-1) H2SO4 and following an enzymatic digestion using Accellerase 1500 carbon-rich hydrolysates were obtained. Two different coffee pulp materials with comparable biomass composition were used, but sugar concentrations in hydrolysates showed variations. The primary sugars were (gL(-1)) glucose (20-30), xylose (15-25), sucrose (5-11) and arabinose (0.7-10). Fermentations were carried out at laboratory (2L) and pilot (50L) scales in presence of 10gL(-1) yeast extract. At pilot scale carbon utilization and lactic acid yield per gram of sugar consumed were 94.65% and 0.78gg(-1), respectively. The productivity was 4.02gL(-1)h(-1). Downstream processing resulted in a pure formulation containing 937gL(-1)l(+)-lactic acid with an optical purity of 99.7%.

Investigation of food waste valorization through sequential lactic acid fermentative production and anaerobic digestion of fermentation residues

This work concerns the investigation of the sequential production of lactic acid (LA) and biogas from food waste (FW). LA was produced from FW using a Streptococcus sp. strain via simultaneous saccharification and fermentation (SSF) and separate enzymatic hydrolysis and fermentation (SHF). Via SHF a yield of 0.33gLA/gFW (productivity 3.38gLA/L·h) and via SSF 0.29gLA/gFW (productivity 2.08gLA/L·h) was obtained. Fermentation residues and FW underwent anaerobic digestion (3wt% TS). Biogas yields were 0.71, 0.74 and 0.90Nm3/kgVS for FW and residues from SSF and SHF respectively. The innovation of the approach is considering the conversion of FW into two different products through a biorefinery concept, therefore making economically feasible LA production and valorising its fermentative residues. Finally, a mass balance of three different outlines with the aim to assess the amount of LA and biogas that may be generated within different scenarios is presented.

From lignin to nylon: Cascaded chemical and biochemical conversion using metabolically engineered Pseudomonas putida

Cis,cis-muconic acid (MA) is a chemical that is recognized for its industrial value and is synthetically accessible from aromatic compounds. This feature provides the attractive possibility of producing MA from mixtures of aromatics found in depolymerized lignin, the most underutilized lignocellulosic biopolymer. Based on the metabolic pathway, the catechol (1,2-dihydroxybenzene) node is the central element of this type of production process: (i) all upper catabolic pathways of aromatics converge at catechol as the central intermediate, (ii) catechol itself is frequently generated during lignin pre-processing, and (iii) catechol is directly converted to the target product MA by catechol 1,2-dioxygenase. However, catechol is highly toxic, which poses a challenge for the bio-production of MA. In this study, the soil bacterium Pseudomonas putida KT2440 was upgraded to a fully genome-based host for the production of MA from catechol and upstream aromatics. At the core of the cell factories created was a designed synthetic pathway module, comprising both native catechol 1,2-dioxygenases, catA and catA2, under the control of the Pcat promoter. The pathway module increased catechol tolerance, catechol 1,2-dioxygenase levels, and catechol conversion rates. MA, the formed product, acted as an inducer of the module, triggering continuous expression. Cellular energy level and ATP yield were identified as critical parameters during catechol-based production. The engineered MA-6 strain achieved an MA titer of 64.2 g L-1 from catechol in a fed-batch process, which repeatedly regenerated the energy levels via specific feed pauses. The developed process was successfully transferred to the pilot scale to produce kilograms of MA at 97.9% purity. The MA-9 strain, equipped with a phenol hydroxylase, used phenol to produce MA and additionally converted o-cresol, m-cresol, and p-cresol to specific methylated variants of MA. This strain was used to demonstrate the entire value chain. Following hydrothermal depolymerization of softwood lignin to catechol, phenol and cresols, MA-9 accumulated 13 g L-1 MA and small amounts of 3-methyl MA, which were hydrogenated to adipic acid and its methylated derivative to polymerize nylon from lignin for the first time.

Biosurfactant production by Aureobasidium pullulans in stirred tank bioreactor: New approach to understand the influence of important variables in the process

Surfactants are amphiphilic molecules with large industrial applications produced currently by chemical routes mainly derived from oil industry. However, biotechnological process, aimed to develop new sustainable process configurations by using favorable microorganisms, already requires investigations in more details. Thus, we present a novel approach for biosurfactant production using the promising yeast Aureobasidium pullulans LB 83, in stirred tank reactor. A central composite face-centered design was carried out to evaluate the effect of the aeration rate (0.1-1.1min-1) and sucrose concentration (20-80g.L-1) in the biosurfactant maximum tensoactivity and productivity. Statistical analysis showed that the use of variables at high levels enhanced tensoactivity, showing 8.05cm in the oil spread test and productivity of 0.0838cm.h-1. Also, unprecedented investigation of aeration rate and sucrose concentration relevance in biosurfactant production by A. pullulans in stirred tank reactor was detailed, demonstrating the importance to establish adequate conditions in bioreactors, aimed to scale-up process.

Leguminose green juice as an efficient nutrient for l(+)-lactic acid production.

Lactic acid is one of the most important building blocks for the production of bioplastic. Many investigations have been conducted to reduce the lactic acid production costs. In this work, the focus was put on the application of legume pressed juice or green juice as nutrient source. The pressed juice was utilized directly without prior pre-treatment and sterilization. Using two different alfalfa green juices and a clover green juice from two different harvest years as sole nutrients, non-sterile fermentations were performed at 52°C and pH 6.0 with a thermotolerant strain Bacillus coagulans AT107. The results showed that alfalfa green juices generally were more suitable for high lactic acid production than clover green juices, presumably due to the higher nitrogen content. A final titer of 98.8g/L after 30h with l(+)-lactic acid purity of >99% was obtained.

Membrane Technologies for Lactic Acid Separation from Fermentation Broths Derived from Renewable Resources

Lactic acid (LA) was produced on a pilot scale using a defined medium with glucose, acid whey, sugar bread and crust bread. The fermentation broths were then subjected to micro- and nanofiltration. Microfiltration efficiently separated the microbial cells. The highest average permeate flow flux was achieved for the defined medium (263.3 L/m2/h) and the lowest for the crust bread-based medium (103.8 L/m2/h). No LA losses were observed during microfiltration of the acid whey, whilst the highest retention of LA was 21.5% for crust bread. Nanofiltration led to high rejections of residual sugars, proteins and ions (sulphate, magnesium, calcium), with a low retention of LA. Unconverted sugar rejections were 100% and 63% for crust bread and sugar bread media respectively, with corresponding LA losses of 22.4% and 2.5%. The membrane retained more than 50% of the ions and proteins present in all media and more than 60% of phosphorus. The average flux was highly affected by the nature of the medium as well as by the final concentration of LA and sugars. The results of this study indicate that micro- and nanofiltration could be industrially employed as primary separation steps for the biotechnologically produced LA.