• Energy Research

Fermentation by filamentous fungi in Erlenmeyer flasks is a favoured method for comparing different fermentation conditions. However, significant inter-flask variation often occurs when using Erlenmeyer flasks, which makes the comparison of fermentation product levels less reliable. We have investigated the use of a 24-well plate method for citric acid, ethanol and glycerol batch fermentation using the filamentous fungi Aspergillus carbonarius and compared the relative standard deviation (RSD) from sextuplicates obtained using Erlenmeyer flasks and 24-well plates. The production levels using the Erlenmeyer flasks showed a combined average RSD of 29%, which is two and a half-fold higher than what was measured using the 24-well plates showing an average RSD of 12%. We conclude that fermentation in 24-well plates is a more reliable screening method for metabolite production by filamentous fungi and possibly for screening metabolites in general.Fermentation studies with filamentous fungi and especially screening experiments often struggle with high inter-vessel variations in metabolite production. This study compares two different types of frequently used screening methods namely batch fermentation in Erlenmeyer flasks with batch fermentation in 24-well plates. The results demonstrate that the variance potentially can be reduced two and a half-fold using 24-well plates leading to improved resolution when testing the impact of varying fermentation parameters on product formation.

Nowadays, the organic farming sector is growing at a fast pace in Europe while needs to face the lack of organic protein sources and in particular, feeding monogastric animals is becoming more and more urgent. Green biorefinery concepts might become the suitable solution for the production of organic protein-rich feeds from green crops. In this context, red clover, clover grass, alfalfa and oilseed radish were studied as possible feedstocks for the development of an organic biorefinery system in Northern Europe. For this purpose, the green crops were processed into a nitrogen-rich protein concentrate, a fiber-rich press cake, and a residual stream of soluble nutrients (brown juice). The process, which involved a novel protein refining technique using lactic acid fermentation, yielded between 6 and 13 kg of dry organic protein product per ton of fresh crop. The protein products of the different crops presented balanced amino acid composition compared to soybeans, which are commonly used in organic farming. Moreover, methionine contents between 7.8 and 9.1 g/kg DM were obtained in the protein products, which is more than the typical concentration found in animal feeds (5.2 g/kg DM in soybeans). This makes the organic protein product produced very promising as a feed ingredient for organic farming of monogastric animals in Europe.

β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis–Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3 U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4 °C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.

Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this review, we discuss the important role beta-glucosidases play in complex biomass hydrolysis and how they create a bottleneck in industrial use of lignocellulosic materials. An efficient beta-glucosidase facilitates hydrolysis at specified process conditions, and key points to consider in this respect are hydrolysis rate, inhibitors, and stability. Product inhibition impairing yields, thermal inactivation of enzymes, and the high cost of enzyme production are the main obstacles to commercial cellulose hydrolysis. Therefore, this sets the stage in the search for better alternatives to the currently available enzyme preparations either by improving known or screening for new beta-glucosidases.

Aspergillus carbonarius exhibits excellent abilities to utilize a wide range of carbon sources and to produce various organic acids. In this study, wheat straw hydrolysate containing high concentrations of glucose and xylose was used for organic acid production by A. carbonarius. The results indicated that A. carbonarius efficiently co-consumed glucose and xylose and produced various types of organic acids in hydrolysate adjusted to pH 7. The inhibitor tolerance of A. carbonarius to the hydrolysate at different pH values was investigated and compared using spores and recycled mycelia. This comparison showed a slight difference in the inhibitor tolerance of the spores and the recycled mycelia based on their growth patterns. Moreover, the wild-type and a glucose oxidase deficient (Δgox) mutant were compared for their abilities to produce organic acids using the hydrolysate and a defined medium. The two strains showed a different pattern of organic acid production in the hydrolysate where the Δgox mutant produced more oxalic acid but less citric acid than the wild-type, which was different from the results obtained in the defined medium This study demonstrates the feasibility of using lignocellulosic biomass for the organic acid production by A. carbonarius.

Nutrient recovery from organic green biomass after processing in a green biorefinery concept for the production of protein feed, biogas and fertilizer for organic farming was investigated. Mass balances in terms of wet weight, TS, VS, C, N, P, K and S were evaluated for processing red clover and clover grass into protein concentrate, press cake and brown juice according to the green biorefinery concept. Depending on the biomass, between 60 and 79% of C and between 52 and 63% of N, P and S in the fresh biomass were found in the press cake, while only 6–10% of C, 8–15% of N, but up to 27% of P and up to 26% of S were found in the brown juice. In contrast, less than 45% of K was transferred to the press cake and 19–31% of K ended up in the brown juice. Moreover, nutrient recovery in the digestate after anaerobic co-digestion of press cake and brown juice produced from clover grass in a pilot-scale trial was assessed in a bench-scale biogas reactor. The analysis of the digestate from the AD process revealed that 56% of the influent total-C was converted into biogas and that the share of ammonia-N of total-N was increased from 9.4 to 43% during the biogas process. Therefore, the proportion of plant available N was improved in the digestate. The digestate to be applied as organic fertilizer presented a C:N ratio of 7 and a nutrients N:P:K ratio of 4:1:12.

AbstractThere is an increasing interest in green biorefineries for the utilization of green biomass to provide a variety of valuable products. Proteins can be extracted from green biomasses such as alfalfa, clover, or grass and separated into protein‐rich concentrates suitable for feeding to monogastric animals. Press cake suitable for feeding ruminant animals is also produced. Full implementation of such a green biorefinery requires knowledge and experience of production at demonstration scale. Moreover, product evaluation in animal‐feeding trials requires a large amount of product. There has been collaboration in Denmark between several partners from industry and academia to assess the practicality and scalability of a green biorefining plant producing protein concentrate and press cake silage from organic grass clover at the scale of ca. 10 tons per hour fresh biomass input. Lactic acid fermentation of the pressed green juice was successfully performed to precipitate proteins and stabilize the juice. Despite minor difficulties, the demonstration‐scale biorefining plant was operative for four days, processed 444 tons of grass clover, and resulted in the overall production of 1 ton of dried protein concentrate, 7 tons of protein paste, and 223 tons of press cake silage. The protein concentrate was rich in essential amino acids and was tested in feeding trials with poultry; the press cake silage, rich in processed fibers and fiber‐associated proteins, was tested in feeding trials with dairy cows. © 2018 Society of Chemical Industry and John Wiley & Sons, Ltd