Adaptive Laboratory Evolution of a Microbial Consortium Enhancing Non-Protein Nitrogen Assimilation for Feed Protein Production

Abstract

The increasing global demand for protein underscores the necessity for sustainable alternatives to soybean-based animal feed, which poses a challenge to human food security. Thus, the search for sustainable, alternative protein sources is transforming the feed industry in its effort to sustainable operations. In this study, a microbial consortium was subjected to adaptive laboratory evolution using non-protein nitrogen (NPN) and wheat straw as the sole carbon source. The evolved microbial consortium was subsequently utilized to perform solid-state fermentation on wheat straw and NPN to produce feed protein. After 20 generations, the microbial consortium demonstrated tolerance to 5 g/L NPN, including ammonium sulfate, ammonium chloride, and urea, which represents a fivefold increase compared to the original microbial consortium. Among the three NPNs tested, the evolved microbial consortium exhibited optimal growth performance with ammonium sulfate. Subsequently, the evolved microbial consortium was employed for the solid-state fermentation (SSF) of wheat straw, and the fermentation conditions were optimized. It was found that the true protein content of wheat straw could be increased from 2.74% to 10.42% under specific conditions: ammoniated wheat straw (15% w/w), non-sterilization of the substrate, an inoculation amount of 15% (v/w), nitrogen addition amount of 0.5% (w/w), an initial moisture content of 70%, a fermentation temperature of 30 °C, and a fermentation duration of 10 days. Finally, the SSF process for wheat straw was successfully scaled up from 0.04 to 2.5 kg, resulting in an increased true protein content of 9.84%. This study provides a promising approach for the production of feed protein from straw and NPN through microbial fermentation, addressing protein resource shortages in animal feed and improving the value of waste straw.

Keywords: adaptive laboratory evolution; microbial consortium; non-protein nitrogen assimilation; solid-state fermentation; wheat straw.

Figure 1

Adaptive laboratory evolution of the microbial consortium to enhance its NPN assimilation ability. The growth of the microbial consortium from different adaptation generations under different nitrogen sources (urea, ammonium chloride, and ammonium sulfate) (a–c). The nitrogen utilization efficiency in the medium for all the generations under different nitrogen sources (urea, ammonium chloride, and ammonium sulfate) (d–f). The nitrogen concentration in a medium for all the generations under different nitrogen sources (urea, ammonium chloride, and ammonium sulfate) (g–i).

Figure 2

The growth of the evolved microbial consortium in medium with different concentrations of urea (a), ammonium chloride (b), and ammonium sulfate (c) as the sole nitrogen source and wheat straw as the sole carbon source. The data represent the averages of biological triplicates, and the scale bar represents the standard deviation (SD).

Figure 3

The NPN assimilation capacity of the 0th, 5th, 8th, 15th, and 20th generations of microbial consortium in liquid medium with NPN (5 g/L) as the sole nitrogen source and wheat straw as the sole carbon source for 72 h. The growth of the original and evolved microbial consortium under different nitrogen sources (urea, ammonium chloride, and ammonium sulfate) (a–c). The biomass yield of the original and evolved microbial consortium under different nitrogen sources (urea, ammonium chloride, and ammonium sulfate) (d–f). The nitrogen utilization of the original and evolved microbial consortium under different nitrogen sources (urea, ammonium chloride, and ammonium sulfate) (g–i). The data represent the averages of biological triplicates, and the scale bar represents the standard deviation (SD).

Figure 4

The true protein of the wheat straw fermented by using the original and the evolved microbial consortium with the addition of ammonium sulfate as a nitrogen source. The data represent the averages of biological triplicates, the scale bar represents the standard deviation (SD), and different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 5

The changes in the true protein content and lignocellulose content (lignin, cellulose, and hemicellulose) during long-term SSF of wheat straw pretreated with different concentrations of ammonia and NaOH. True protein content (a,c); lignocellulose content (lignin, cellulose, and hemicellulose) (b,d). The data represent the averages of biological triplicates, the scale bar represents the standard deviation (SD), and different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 6

Protein content of SSF of ammoniated wheat straw by the evolved microbial consortium with different fermentation times (a), inoculation amounts (b), nitrogen source addition amounts (c), moisture contents (d), temperatures (e), and sterilized conditions (f). The data represent the averages of biological triplicates, the scale bar represents the standard deviation (SD), and different lowercase letters indicate significant differences between treatments (p < 0.05).

Figure 7

True protein and lignocellulose content of the wheat straw with the 2.5 kg SSF process. Protein content (a); lignocellulose content (lignin, cellulose, and hemicellulose) (b). The data represent the averages of biological triplicates, and the scale bar represents the standard deviation (SD).