Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw

Abstract

The production of cellulose and hemicellulose-degrading enzymes by cultivation of Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, Schizophyllum commune ATCC 38548, and Trichoderma reesei Rut-C30 was studied. Wet-oxidised wheat straw suspension supplemented with NH₄NO₃, MgSO₄, and KH₂PO₄ was used as cultivation medium aiming to obtain an enzyme mixture optimal for enzymatic hydrolysis of wet-oxidised wheat straw. The cultivations with B. cinerea and P. brasilianum gave the highest endoglucanase (EC 3.2.1.4) and β-glucosidase (EC 3.2.1.21) activities, in contrast to the other fungi where lower activities were found. The culture filtrates were concentrated by ammonium sulphate precipitation. After enzyme concentration, the highest enzyme activities (1.34 FPU/ml) were found in the culture broth originating from P. brasilianum. Enzymatic hydrolysis of filter cake from wet-oxidised wheat straw for 48 h with an enzyme loading of 5 FPU/g biomass resulted in glucose yields from cellulose of 58% (w/w) and 39% (w/w) using enzymes produced by P. brasilianum and a commercial enzyme mixture, respectively. At higher enzyme loading (25 FPU/g biomass) using either enzyme mixtures the glucose yield from cellulose was in the range of 77–79% (w/w).

Introduction

In many countries, wheat straw is an abundant lignocellulosic by-product from farming, consisting of cellulose (35–40% w/w) and hemicellulose (25–30% w/w) in close association with lignin (10–15% w/w). The polysaccharides in lignocellulosic materials can be used for bioethanol production and efficient pre-treatment and fermentation technologies for that purpose are being developed [1]. The utilisation of both the cellulose and hemicellulose fraction is required in an economically feasible bioethanol production. The cellulose cannot be enzymatically hydrolysed to glucose without a physical and chemical pre-treatment to break down the lignin and overcome the resistance of cellulose to hydrolytic cleavage due to its partly crystalline structure [2].

The pre-treatment processes normally applied on wheat straw are acidic hydrolysis, steam explosion and wet oxidation [3]. During wet oxidation of wheat straw, a reaction involving O₂ at elevated temperature and pressure, 50% (w/w) of the lignin is decomposed to low molecular weight carboxylic acids, phenolic compounds, CO₂, and H₂O [4], [5]. Most of the hemicellulose (80% w/w) is dissolved and oxidised to carboxylic acids, CO₂, and H₂O leaving a solid fraction rich of cellulose [6]. Degradation products that could have inhibitory action in later fermentation steps are avoided during pre-treatment by wet oxidation [5]. The soluble sugars produced by wet oxidation can be utilised for enzyme production by cultivation of Aspergillus niger and for ethanol fermentation by Thermoanaerobacter mathranii [7] and Saccharomyces cerevisiae [8] fermenting the pentoses and hexoses, respectively. However, low contents of hemicellulose and lignin remain insoluble after wet oxidation [6] so in addition to cellulose-degrading enzymes, both hemicellulose and lignin degrading enzymes might be required for complete hydrolysis of the solid fraction.

Cellulose-degrading enzymes are commercially available, but still too expensive for production of fuel ethanol. Another problem is that these enzymes are not developed for hydrolysis of lignocellulose [3], and usually produced using glucose as carbon source. Since wheat straw is an agricultural waste product the use of it in cultivations will lower the raw material cost and thereby the enzyme production cost. However, research and development is needed in order to make the enzyme production feasible, since cellulose is a less simple carbon source than glucose. For complete hydrolysis of cellulose, the cellulose chains are cut at random positions by endoglucanase (EC 3.2.1.4) and at the chain ends by exoglucanase (EC 3.2.1.91) producing cellobiose [9]. When commercial Celluclast is utilised for enzymatic hydrolysis of cellulose, extra β-glucosidase (EC 3.2.1.21) must be added for hydrolysis of the produced cellobiose. Hemicellulose is a heterogeneous branched polymer in which the composition and structure depend on the raw material source. In wheat straw, hemicellulose consists of mainly arabinoxylan (81% w/w) [2] and enzymes such as endoxylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37) are required for hydrolysis.

Many bacteria and filamentous fungi can produce cellulose-degrading enzymes. Most bacteria cannot utilise crystalline cellulose, which can be done by many filamentous fungi [3]. In this study, an enzyme mixture suitable for hydrolysis of wet-oxidised wheat straw was approached by enzyme production with four filamentous fungi, Aspergillus niger ATCC 9029, Botrytis cinerea ATCC 28466, Penicillium brasilianum IBT 20888, and Schizophyllum commune ATCC 38548. The enzyme production was compared to the frequently used Trichoderma reesei Rut-C30. Wet-oxidised wheat straw was the only carbon source and only the most essential nutrients were added. The cell mass could not be followed by optical density (OD) measurements due to interference caused by solid particles (wet-oxidised wheat straw). The cell mass was instead followed by measuring the carbon dioxide development and the composition of the cultivation solid residue. The enzyme activity of endoglucanase, β-glucosidase, endoxylanase, and β-xylosidase was determined as a function of time. Finally, a comparative study of the hydrolytic effect was made in which the produced enzymes and Celluclast were compared.