Enrichment of Anaerobic Microbial Communities from Midgut and Hindgut of Sun Beetle Larvae (Pachnoda marginata) on Wheat Straw: Effect of Inoculum Preparation

Abstract

The Pachnoda marginata larva have complex gut microbiota capable of the effective conversion of lignocellulosic biomass. Biotechnological utilization of these microorganisms in an engineered system can be achieved by establishing enrichment cultures using a lignocellulosic substrate. We established enrichment cultures from contents of the midgut and hindgut of the beetle larva using wheat straw in an alkaline medium at mesophilic conditions. Two different inoculation preparations were used: procedure 1 (P1) was performed in a sterile bench under oxic conditions using 0.4% inoculum and small gauge needles. Procedure 2 (P2) was carried out under anoxic conditions using more inoculum (4%) and bigger gauge needles. Higher methane production was achieved with P2, while the highest acetic acid concentrations were observed with P1. In the enrichment cultures, the most abundant bacterial families were Dysgonomonadaceae, Heliobacteriaceae, Ruminococcaceae, and Marinilabiliaceae. Further, the most abundant methanogenic genera were Methanobrevibacter, Methanoculleus, and Methanosarcina. Our observations suggest that in samples processed with P1, the volatile fatty acids were not completely converted to methane. This is supported by the finding that enrichment cultures obtained with P2 included acetoclastic methanogens, which might have prevented the accumulation of acetic acid. We conclude that differences in the inoculum preparation may have a major influence on the outcome of enrichment cultures from the P. marginata larvae gut.

Effect of Alkaline and Mechanical Pretreatment of Wheat Straw on Enrichment Cultures from Pachnoda marginata Larva Gut

“…The TS and vs. were 94.1% fresh mass and 91.8% TS , respectively. Enrichment cultures from the midgut and hindgut of the beetle larvae Pachnoda marginata, fourth transfer [37] and an enrichment culture from the Szarvas Hungarian soda lake, seventy-fifth transfer [57] were used as inoculum. The calcium hydroxide (Ca(OH) 2 ) wheat straw pretreatment was carried out directly inside the culture bottles.…”

“…Subsequently, the food enters the midgut, which has a high pH as its main characteristic [36] and exerts an effective natural alkaline pretreatment. In addition, the enrichment cultures from the Pachnoda marginata midgut content in lignocellulosic biomass showed that bacteria capable of converting cellulose and hemicellulose into VFAs are present in this larva gut section and they are able to survive in alkaline artificial systems; however, hydrolysis remained to be the limiting factor for total biomass conversion [37]. The hindgut is known to harbor a great diversity of microorganisms, among them archaea, capable of producing methane [37] and bacteria such as Promicromonospora pachnodae, able to produce endoglucanases and xylanases [38], and Dysgonomonas sp., able to convert carbohydrates into different acids [37].…”

“…Some characteristics related to the inoculum seem to play a key role in the performance of anaerobic digestion of lignocellulose-rich biomass [39]. The inoculum source [40][41][42] combined with the substrate/inoculum ratio [37,[43][44][45] are parameters often cited in the literature as one of the main characteristics shaping the anaerobic digestion process. Inoculum from ruminant animal manure and anaerobic sludge from different sources are the most commonly used in the anaerobic digestion of LCB [46][47][48][49][50].…”

“…Insects that feed on LCB have been shown to harbor a rich microbiota [51] with potential for application in biorefineries [52]. Although the microbiomes from insect larvae gut or insect gut have been studied [12,37,[53][54][55][56], they are not applied in the anaerobic digestion of lignocellulosic biomass on a large scale or studied using different types of pretreated biomasses.…”

“…Enrichment cultures from the insect gut microbiota selecting features similar to those found in insect gut, when applied to engineered systems, can improve LCB degradation and increase production of desired compounds [1,53,56]. Therefore, in the present study, we aimed to assess the effect of alkaline and mechanical pretreatment of wheat straw on the microbial community, volatile fatty acids, and methane production of two larva gut enrichment cultures from a former study [37] and one enrichment culture derived from a Hungarian soda lake sediment [57] as candidate.…”